Research ArticleNovel Epoxy Activated Hydrogels for Solving Lactose Intolerance

Magdy M M Elnashar123 and Mohamed E Hassan14

1 Center of Excellence Encapsulation amp Nanobiotechnology Group National Research Center El-Behouth Street Cairo 12311 Egypt2 Polymers Department National Research Center El-Behouth Street Cairo 12311 Egypt3 Biochemistry Department Taif University Taif Saudi Arabia4Chemistry of Natural and Microbial Products Department National Research Center El-Behouth Street Cairo 12311 Egypt

Correspondence should be addressed to Magdy M M Elnashar magmelgmailcom

Received 28 March 2014 Revised 10 May 2014 Accepted 13 May 2014 Published 11 June 2014

Academic Editor Shirui Mao

Copyright copy 2014 M M M Elnashar and M E Hassan This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

ldquoLactose intolerancerdquo is a medical problem for almost 70 of the world population Milk and dairy products contain 5ndash10wvlactose Hydrolysis of lactose by immobilized lactase is an industrial solution In this work we succeeded to increase the lactaseloading capacity tomore than 3-fold to 363Ug gel using epoxy activated hydrogels compared to 11 Ug gel using aldehyde activatedcarrageenan The hydrogelrsquos mode of interaction was proven by FTIR DSC and TGA The high activity of the epoxy group wasregarded to its ability to attach to the enzymersquos ndashSH ndashNH and ndashOH groups whereas the aldehyde group could only bind to theenzymersquos ndashNH

2group The optimum conditions for immobilization such as epoxy chain length and enzyme concentration have

been studied Furthermore the optimum enzyme conditions were also deliberated and showed better stability for the immobilizedenzyme and the Michaelis constants 119870

119898and 119881max were doubled Results revealed also that both free and immobilized enzymes

reached their maximum rate of lactose conversion after 2 h albeit the aldehyde activated hydrogel could only reach 63 of thefree enzyme In brief the epoxy activated hydrogels are more efficient in immobilizing more enzymes than the aldehyde activatedhydrogel

1 Introduction

Lactases (120573-galactosidases) are indeed important enzymesin food industry and have found significant applications inenhancing sweetness solubility flavor and digestibility ofdairy products [1] A major application of 120573-galactosidaseis lactose hydrolysis a process that results in the formationof glucose and galactose Lactose is the major sugar (4-5) present in milk The consumption of foods with a highcontent of lactose is causing a medical problem for almost70 of the world population especially in the developingcountries as the naturally present enzyme in the humanintestine loses its activity during lifetime so its hydrolysismakes milk fit for consumption of lactose intolerant people[2] Hydrolysis of lactose present in milk using enzymes suchas lactases will produce lactose-free milk and lactose-freedairy products [3]

In industries immobilized enzymes are preferred overthe free ones The immobilization technique would enable

the reusability of enzymes for tens of times reducing theenzyme and product cost significantly Unfortunately effi-cient commercial carriers suitable for immobilization ofenzymes are relatively expensive [4 5] Understandably forfood pharmaceutical medical and agricultural applicationsnontoxicity and biocompatibility of the materials are alsorequired Among many carriers that have been consideredand studied for immobilizing enzymes organic or inorganicnatural or synthetic chitosan and carrageenan are of interestin that they offer most of the above characteristics and areavailable at a reasonable cost [3]

Chitosan is a cationic naturally occurring polymer obtai-ned from the deacetylation of chitin which is the secondmostabundant polymer in nature after cellulose [6 7] Chitosanhas an abundance of amino groups (70ndash95) that makechitosan a cationic polyelectrolyte (pKa asymp 65) and one ofthe few found in nature This basicity gives chitosan singularproperties chitosan is soluble in aqueous acidic media at

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 817985 9 pageshttpdxdoiorg1011552014817985

2 BioMed Research International

pH lt 65 and when dissolved possesses high positive chargeon NH

3

+ groups The protonated (NH3

+) could adhere tonegatively charged surfaces such as polyanionic compounds[8 9]

Carrageenan is a naturally occurring anionic polysac-charide isolated from the seaweeds According to the studymade by Chao et al 1986 [10] to harden the carrageenangels using different amine compounds they concluded thatonly polyamines such as chitosan substantially improvedthe carrageenan gels thermal stability via polyelectrolytersquosinteractions Unfortunately the carrageenan-polyelectrolytesystems were limited to the entrapment of enzymes [11ndash13] which have the major problem of enzyme leakage Forexample Boadi and Neufeld 2001 [14] used alginate andcarrageenan to entrap tannase and then crosslinked thegel beads with chitosan followed by glutaraldehyde Theentrapment technique limits their industrial use as supportsfor enzyme immobilization due to enzyme leakage So effortsto immobilize enzymes on newer type of carriers especiallywith covalent bonds are still underway in many laboratories[15ndash18]

To our knowledge previous reports have not any greaterextent that dealt with carrageenan-chitosan polyelectrolyteas a use for the covalent immobilization of enzymes withan exception of our recent work [19 20] In that work weimmobilized 120573-galactosidase covalently via its amino groupsto carrageenan treated with chitosan and glutaraldehydeAlthough the linkage was covalent however the enzymeloading capacity was limited to up to 11 Ug gel beadsThis could be regarded to the free aldehyde groups on thecarrageenan treated chitosan that could only react with thefree amino groups (NH

2) on the enzyme via Schiff rsquos base

formation ndashC=NHndash [19 20]Thus the purpose of this research was to modify the

carrageenan with chitosan and a more efficient functionalgroup epoxy group which imparts three extra benefits tocarrageenan

(i) the first is improvement of the carrageenan gelrsquos ther-mal stability by forming a polyelectrolyte complex(PEC) between the carrageenan ndashOSO

3

minus and thechitosan ndashNH

3

+(ii) the second is creation of a new functionality free

chitosan amino groups (NH2)

(iii) the third is activation of the free chitosan aminogroups (ndashNH

2) with 14-butanediol diglycidyl ether

to impart free epoxy groups to covalently immobilize120573-galactosidase via the ndashSH ndashOH and ndashNH of theiramino acids

Accordingly we expect more enzymes to bind tothe epoxy activated carrier via three groups on the enzyme(ndashSH ndashOH and ndashNH) whereas the free aldehyde groupscould only bind to the ndashNH

2of the amino acids To our

knowledge there are no reports on the use of carrageenanfor the immobilization of 120573-galactosidase using covalenttechnique via chitosan and 14-butanediol diglycidyl etherThe novel gel formulation was prepared in beads shape usingthe Encapsulator to enable gel beads production on the

semipilot scale and to increase the gelrsquos surface area Thegrafted formulationwas illustrated using a schematic diagramand the chemical and thermal modification was proved usingthe FTIR and DSC techniques respectively On the otherhand the enzyme loading capacity was optimized by usingshort and long chain of the epoxy activated carrier and usingdifferent concentrations of the enzyme Finally the free andimmobilized enzymes were characterized for their activitiesat different pHs and temperatures and theMichaelis constantswere studied as well as the lactose hydrolysis using free andimmobilized enzyme over time

2 Materials and Methods

As a general rule all experimentswere carried out in triplicateand data are means plusmn SD (119899 = 3)The abbreviations section islisting all the abbreviations used in the formulations samples

21 Materials 120581-Carrageenan (MW 154000) sulfate ester sim25 and chitosanwere supplied by Fluka120573-Galactosidase (EC32123) was obtained fromAspergillus oryzae and 118Umgwas obtained from Sigma-Fluka-Aldrich All other chemicalswere of pure grades (Analar or equivalent quality) TheEncapsulator model IE-50 was purchased from InnotechEncapsulator in Switzerland The gel disks dimensions weremeasured using a micrometer (Micro 2000 0ndash25mm)

22 Determination of 120573-Galactosidase Activity 120573-Galacto-sidase activity was determined by the rate of glucose forma-tion in the reaction medium Known amount of immobilizedor free enzyme was incubated into 10mL of 200mM lactosesolution in 100mM citrate phosphate buffer at pH 45 for 3 hat 37∘C and 100 rpm At the end of the time 50 120583L of reactionmixture was added to 950 120583L buffer and boiled for 10min toinactivate the enzyme and analyzed for glucose content usingthe glucose test One enzyme unit (IU) was defined as theamount of enzyme that catalyzes the formation of 1 120583mol ofglucose per minute under the specified conditions

Equations (1) (2) and (3) show the hydrolysis of lac-tose by 120573-galactosidase and glucose determination using amixture of enzymes glucose oxidase (GOD) and peroxidase(POD)

Lactose120573-galactosidase997888997888997888997888997888997888997888997888997888997888rarr Glucose + Galactose (1)

Glucose +O2+H2O GOD997888997888997888997888rarr Glucolactone +H

2O2

(2)

H2O2+Hydroxybenoate-Na-4-aminoantipyrine

POD997888997888997888rarr Quinon complex +H

2O

(3)

Glucose concentration was measured spectrophotomet-rically with a glucose test based on the Trinder reagentGlucose is transformed to gluconic acid and hydrogen per-oxide by glucose oxidase (GOD) The hydrogen peroxideformed reacts in the presence of peroxidase (POD) with 4-aminoantipyrine and p-hydroxybenzene sulfonate to form aquinoneimine dye as shown in Equations (1) (2) and (3)

BioMed Research International 3

The intensity of the color produced is directly propor-tional to the glucose concentration in the sample The assaywas performed by mixing 30 120583L of a sample of unknownconcentration and 3mL of Trinder reagent the reaction wasallowed to proceed for 20min at room temperature and theabsorbance of the unknown concentrationwas read at 510 nm[19]

23 Preparation of Gel Beads 120581-Carrageenan gel was pre-pared by dissolving 25 (wv) carrageenan in distilled waterat 70∘C using an overhead mechanical stirrer until completedissolution had occurred The Carrageenan gel solution wasdropped through a nozzle of 300 120583m using the InnotechEncapsulator in a hardening solution of 03M KCl Thenbeads were hardened using 03M KCl for 3 h

24Modification of Gel BeadsUsingChitosan andEpoxy Twoactivated epoxy hydrogels were prepared the short and thelong chain as follows

(a) TheShort Chain Carr-Ch-Epo beads were soaked ina solution of 075 chitosan previously prepared in 1(vv) acetic acidThen they were suspended in 75mL05M NaOH containing 150mg sodium cyanoboro-hydride under stirring Slowly 75mL 1 4-butanedioldiglycidyl ether was added with constant stirring andthe reaction was left at room temperature overnightFinally the activated gel beads were extensivelywashed with water to remove excess reagent

(b) TheLong Chain Carr-Ch-Epo-Ch-Epo formula (a)was furthermodified with chitosan and then epoxy asshown above

25 Elucidation of the Modified Gel Using Fourier Trans-form Infrared Spectroscopy The infrared spectra of all for-mulations were recorded with Fourier transform infraredspectroscopy (FTIR-8300 Shimadzu Japan) FTIR spectrawere taken in the wavelength region 4000 to 400 cmminus1 atambient temperature The FTIR spectrophotometer (FTIR-8300 Shimadzu Japan) was used to prove the presenceof the new functional group epoxy groups in both formsof the modified gels Five samples were used for this testcarrageenan (Carr) carrageenan coatedwith chitosan (Carr-Ch) carrageenan coated with chitosan followed by epoxy(short chain epoxy activated hydrogel Carr-Ch-Epo) car-rageenan coated with chitosan followed by epoxy followedby chitosan (Carr-Ch-Epo-Ch) and finally carrageenancoated with chitosan followed by epoxy followed by chitosanfollowed by epoxy (long chain epoxy activated hydrogelCarr-Ch-Epo-Ch-Epo)

A total of 2 (ww) of the sample with respect to thepotassium bromide (KBr S D Fine Chem Ltd) disk wasmixed with dry KBr The mixture was ground into a finepowder using an agatemortar before it was compressed into aKBr disk under a hydraulic press at 10000 psi Each KBr diskwas scanned 16 times at 4mms at a resolution of 2 cmminus1 overa wavenumber range of 400ndash4000 cmminus1 using Happ-Genzelapodization

26 Differential Scanning Calorimetry andThermal Gravimet-ric Analysis Differential scanning calorimetry (DSC) andthermal gravimetric analysis (TGAA) were performed toprove the formation of a strong polyelectrolyte complexbetween carrageenan and chitosan followed by di-epoxyThethermal behavior of five gel formulations was performedCarr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo The differential scanning calorimetry wasstudied using DSC (SDT 600 TA Instruments USA)Approximately 3 to 6mg of the dried gels was weighed intoan alumina panThe samples were heated from40∘C to 340∘Cat a heating rate of 10∘Cmin The thermal behavior of thedifferent gel formulations was characterized for their TGA(SDT 600 TA Instruments USA) Alumina pans were usedand approximately 3 to 6mg of the dried gels were weighedThe samples were heated from 50 to 300∘C at a heating rateof 10∘Cmin

27 Immobilization of 120573-galactosidase 120573-Galactosidase wasimmobilized onto the short and long chain of the epoxyactivated hydrogels as follows One gram of the activatedgel beads was washed thoroughly with distilled water andwas incubated into 10mL of enzyme solution (30UmL)prepared in 100mM citrate-phosphate buffer at pH 45 for16 h The immobilized enzyme was washed thoroughly withthe buffer solution containing Tris-HCl to block any freealdehyde group and to remove any unbound enzyme Theimmobilized enzyme was stored at 4∘C for further mea-surements Two parameters were used to reach the enzymersquosmaximum loading capacity (ELC) and epoxy activated longand short chains as well as the enzyme concentration

The ELC or the amount of enzymesrsquo units immobilizedonto and into gel beads was calculated as follows

ELC =(119872119900minus119872119891)

119882 (4)

where119872119900is the initial enzyme activity (U)119872

119891is the enzyme

activity of the filtrate (U) after immobilization and119882 is theweight of gel beads (g)

28 Optimization and Evaluation of the Free and Immobilized120573-Galactosidase

281 Temperature and pH Profiles for the Free andImmobilized 120573-Galactosidase The free and immobilized 120573-galactosidases were incubated into 10mL of 200mM lactoseat temperatures from 30∘C to 70∘C for 3 hrs The enzymeactivity has been determined according to Section 22 Theoptimum temperature has been chosen to study the effect ofpH where the free and immobilized 120573-galactosidases wereincubated into 10mL of 200mM lactose at pH 30ndash90 at37∘C for 3 hrs

282 119870119898

and 119881max of the Free and Immobilized 120573-Galactosidase The Michaelis-Menten kinetic models ade-quate for the description of the hydrolysis of lactose by thefree and the immobilized enzyme apparent 119870

119898and 119881max

4 BioMed Research International

(a)

(b)

(c)

ChitosanCarrageenan Carr Ch

NH2 NH2

+++ +

+

minusminusminusminusminusminusminusminus

minusminusminusminusminus

minusminusminusminusminusminusminusminus

minusminusminusminusminus

14-Butanediol diglycidyl etherCarr Ch Epoxy activated carrageenan

NH2 ndashO(CH2)4OCH2CH2OO+

+

+minusminusminusminusminusminusminusminus

minusminusminusminusminus

ndashO(CH2)4OCH2O

NHndashCH2ndashCHndashCH2

++minusminusminusminusminusminusminusminus

minusminusminusminusminus

NHndash ndashCHOH

EnzymeEpoxy activated Carr

NHndashCH2ndashCH CHndashCH ndashNHndashEndashSHOH

H2NndashEndashSH

CarrndashChndashEpondashenzyme

CH2 CH2

ndash ndash

O +++

minusminusminusminusminusminusminusminus

minusminusminusminusminus

++

minusminusminusminusminusminusminusminus

minusminusminusminusminus

Scheme 1 Grafted carrageenan gel beads with epoxy groups and immobilization of enzymes (a) Modification of carrageenan beads withchitosan via ionic interaction (b) Incorporation of epoxy groups to the carrageenan-chitosan (c) Immobilization of enzymes to the graftedcarrageenan-epoxy via ndashSH ndashOH or ndashNH

2

of free and immobilized 120573-galactosidase were determinedfor lactose using the Hanes-Woolf plot method Free andimmobilized 120573-galactosidases were incubated into 10mL of25 to 200mM at 37∘C and pH 45 for 3 hrs under standardassay conditions

283 Lactose Hydrolysis Using Immobilized 120573-GalactosidaseTo evaluate the efficiency of the immobilized enzyme it wasused for lactose hydrolysis using the optimum conditionsobtained from above optimization for the free and immobi-lized enzymes

3 Results and Discussion

31 GraftedAlginate Elucidation Structure Protonated aminogroups (ndashNH

3

+) of Ch formed a polyelectrolyte complexwiththe ndashOSO

3

minus of the Carr gel and incorporated free aminogroups to Carr [19] The free amino groups (ndashNH

2) of Ch

were used to covalently immobilize 120573-galactosidase via thedi-epoxy groups as a mediator beside its main role as a cross-linkerThe enzyme could be bound to the carrierrsquos free epoxygroups via its ndashSH ndashOH and ndashNH groups However asshown in Scheme 1 we represented that one uses the free ndashNH groups as an example to follow the ndashOH and ndashSH groups

The FTIR bands of Carr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo were shown in Figure 1Spectrums of the three compounds Carr Carr-Ch Carr-Ch-Epo revealed a new and strong band at 870 cmminus1 whichappears only for the modified gel spectrum with epoxygroupsThis band proved the presence of a new gel functionalgroup epoxy group which is in agreement with the authorrsquosprevious work [20] This band disappeared after treatment

75015002250300037504500

T (

)

3460

30

1120

64

3479

58

1126

43

1066

64

3458

37 11

110

0

3435

22 11

225

710

666

4

3419

79

1112

93

(cmminus1)CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 1 FTIR of five formulations of carrageenan and modifiedcarrageenan with chitosan and epoxy groups Carr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo

of the Carr-Ch-Epo with Ch and reappeared after furthertreatment with epoxy (Carr-Ch-Epo-Ch-Epo) at 880 cmminus1The FTIR bands also revealed a decrease in intensity and a

BioMed Research International 5

Table 1 Values ofDSC andTGAA thermograms of carrageenan andmodified carrageenan with chitosan and epoxy groups

Number Formula DSC temp ∘C TGA temp ∘C1 Carr 220 2002 Carr-Ch 230 2103 Carr-Ch-Epo 250 amp 320 2504 Carr-Ch-Epo-Ch 260 2405 Carr-Ch-Epo-Ch-Epo 260 amp 330 270

0123456789

10

0 50 100 150 200 250 300 350

Hea

t flow

exo

up

(mW

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 2 DSC thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

shift of the ndashOSO3

minus absorption band of Carr from 1446 cmminus1to 1390 cmminus1after reaction with the chitosan This ionicinteraction between the carrageenan and the chitosan evi-denced the formation of strong polyelectrolyte complexes[21]

Table 1 is tabulating the main characteristic values of theDSC and TGAA thermograms as shown in Figures 2 and 3respectively

The treatment of carrageenan with chitosan and epoxyhas shown gradual and obvious improvement in their DSCand TGA The DSC exothermic effect has been shifted tohigher temperatures from formula numbers 1ndash5 that is from220∘C to 320∘C The Carr has shown an exothermic band at220∘C which has been shifted to 230∘C after treatment withCh This improvement could be attributed to the formationof a complex network between the Carr and the Ch Similarbehavior has been attained when the di-epoxy has beensubstituted with glutaraldehyde [3] Further treatment withdi-epoxy Carr-Ch-Epo gradually increased the temperatureto two peaks at 250 and 230∘C This could be attributed toextra crosslinking with the di-epoxy and the two bands couldbe referred to the Ch and Epo however we could not tell atthis stage which is which However by addition of Ch Carr-Ch-Epo-Ch the temperature increased to a single band at260∘C which should be regarded to the Ch That means thatfor the short chain Carr-Ch-Epo the two peaks should be forCh and Epo respectively Finally by adding more di-epoxyCarr-Ch-Epo-Ch-Epo (long chain) two bands appeared for

60

65

70

75

80

85

90

95

100

0 50 100 150 200 250 300 350

Wei

ght (

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 3 TGA thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

the Ch and Epo at the highest temperatures 260 and 330∘Crespectively

On the other hand the TGA of the modified Carrformula numbers 2-5 showed a better stability against degra-dation as shown in Figure 3 andTable 1 For example unmod-ified Carr had a sudden decomposition at 200∘C This valuehas been increased to 210∘C after its treatment with Ch withretention of the sudden decomposition behavior The gelsrsquothermal improvement could be explained by the formation ofpolyelectrolyte interaction between the polyanions (ndashOSO

3

minus)of the Carr and the polycations (NH

3

+) of the Ch Furtherhardening of the gel beads using Epo showed a much higherincrease in the TGAA Carr-Ch-Epo (short chain) to 250∘Cand the decomposition behavior was slower and gradualThe improvement in the TGA could be attributed to extracrosslinking between the free Chrsquos amino groups and theEpo Further treatment of the short chain with Ch does notincrease its TGA however it was more or less the same at240∘C Finally for the long chain Carr-Ch-Epo-Ch-Epo theTGA increased to its maximum of 270∘C which could beregarded to further crosslinking It is worth noting that theshort and long chains have the highest TGA values of 250 and270∘Cwith slower and gradual decomposition rate comparedto other formulations

32 Optimization of Enzymersquos Loading Capacity Two factorshave been studied to optimize the loading capacity of 120573-galactosidases onto treated carrageenan gel beads

321 Effect of Epoxy Chain Length In this experiment shortchain of epoxy modified gel beads Carr-Ch-Epo and longchain Carr-Ch-Epo-Ch-Epo were examined to assess chainlength efficiency for immobilization of the enzyme Accord-ing to Sung and Bae 2003 [22] the effect of the chain lengthcould have a positive effect on the immobilization loadingefficiency till certain length and then it declines afterwards Inour case the short chain immobilizedmore enzymes than thelong chain Results as shown in Figure 4 showed that the short

6 BioMed Research International

0

5

10

15

20

25238

182

(Ug

bea

ds)

CarrndashChndashEpo CarrndashChndashEpondashChndashEpo

Figure 4 Effect of epoxy chain length on 120573-galactosidase loadingcapacity

chain immobilized 238 Ug gel beds compared to 182 Uggel beads for the long chain These results are in accordancewith that of Gancarz et al 2003 [23] who observed that anincrease in surface epoxy groups led to an increase in quantityof immobilized enzyme but a decrease in retained enzymeactivity

To understand this phenomenon we calculated from thesupernatants the expected amounts of immobilized enzymeson the short and long chains formulas and they were foundto be 18 and 21Ug respectively These results were in favorof the long chain formula however the retained activity ofthe immobilized enzymes was in favor of the short chainwhere 238Ug were immobilized showing 133 retention ofactivity This increase in the enzyme activity after immobi-lization could be regarded to the hydrogen bond interactionsbetween the modified gel (polysaccharide) containing ndashOHndashOSO

3H ndashC=O ndashNH

2 and the lactose substrate containing

ndashOH ndashC=O groups These H-bonding interactions couldalso increase the lactose concentration surrounding the gelsurface more than the bulk solution and thus the activity ofthe immobilized enzyme increases till reaching saturation ofthe gel surface with lactase [24]

On the other hand the long chain formulation wasexpected to immobilize 21Ug and in practice it showedonly 182Ug which revealed 86 retention of activity ofthe immobilized enzyme This could be regarded to the longchain havingmultipoint attachment orandmultilayers of theimmobilized enzymesteric hindrance that resulted in lossof the enzymersquos 3D structure and consequently its activityAccordingly for further experiments the short chain wasused

322 Effect of 120573-Galactosidase Concentration 120573-Galacto-sidase was immobilized onto gel beads treated with shortchain epoxy activated carrageenan Carr-Ch-Epo as shownin Figure 5

Results revealed that by increasing the concentrationof 120573-galactosidase from 10U to 60U the ELC increasedgradually till it reached its maximum of 36Ug gel beadsusing 50U of free enzyme after which any more addedenzyme has almost no effect on the ELC This could beregarded to all free epoxy groups that have been engagedwith the enzymes [25] However we have chosen for furtheroptimization the ELC of 36Ug gel beads as it shows better

05

10152025303540

10 20 30 40 50 60

(Ug)

8958854327 1239676625 2492457653 3101527564 3602115799 362855251

(Ug

bea

ds)

Figure 5 pH profile of the free and immobilized 120573-galactosidase

0

20

40

60

80

100

120

20 30 40 50 60 70 80

Rela

tive a

ctiv

ity (

)

FreeImmo

Temperature (∘C)

Figure 6 Michaelis constants of free and immobilized 120573-galactosidase

enzyme loading efficiency of 49 which is more economicas it saves unloaded enzyme from being wasted

33 Evaluation of Catalytic Activity of Free and Immobilized120573-Galactosidase At this stage five experiments were studiedFirstly the optimum reaction temperature pH and substrateconcentrations were examined for both the free and immo-bilized enzyme Secondly the best results from the first stepwere used to obtain themaximum substrate hydrolysis as wellas the operational stability of the immobilized enzyme

331 Optimum Temperature for the Free and Immobilized 120573-Galactosidase The optimum temperature for the free andimmobilized enzyme was examined Results as shown inFigure 6 revealed that the optimum temperature for theimmobilized enzyme was found to be at a slightly highertemperature (37ndash40∘C) compared to the free enzyme (30ndash37∘C)

The shift of the optimum temperature towards highertemperatures when the biocatalyst is immobilized indicatesthat the enzyme structure is strengthened by the immobiliza-tion process and the formation of a molecular cage aroundthe protein molecule (enzyme) was found to enhance theenzyme thermal stability The increase of the immobilized

BioMed Research International 7

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10

Rela

tive a

ctiv

ity (

)

pH

ImmoFree

Figure 7 Lactose hydrolysis using free and immobilized 120573-galactosidase

enzyme temperature tolerance may also be due to diffusionaleffects where the reaction velocity is more likely to bediffusion limited so that improvements in thermal diffusionwould correspondingly result in proportionally higher reac-tion rates [3]

332 pH Profile Figure 7 illustrates the pH activity profile ofthe free and immobilized 120573-galactosidase The optimum pHvalues for free and immobilized enzyme were 45ndash5 and 4-6 respectively which showed that the immobilized enzymewas more stable at higher and wider range of pH [25] Theseproperties could be very useful for lactolysis in sweet wheypermeate which has a pH range of 55ndash6 Moreover at pH4 the immobilized enzyme retained more than 95 of itsrelative activity compared to only 56 for the free enzyme

The shift in the pH activity profile of the immobilized 120573-galactosidase and the better pH stability may be attributedto the partition effects that were arising from differentconcentrations of charged species in the microenvironmentof the immobilized enzyme and in the domain of the bulksolution [3]

333 Determination of Kinetic Parameters of Free and Immo-bilized 120573-Galactosidase The kinetic constants of free andimmobilized 120573-galactosidase as shown in Figure 8 weretabulated in Table 2

The apparent 119870119898

after immobilization 1314mM ishigher than that of the free enzyme 589mMwhich indicatesthat a higher concentration of substrate 2-fold is needed forthe immobilized enzyme compared to the free enzyme Nev-ertheless higher 119870

119898values for immobilized 120573-galactosidase

have been reported by other authors with increases from 12-fold up to 54-fold [26] These results are most likely dueto the fact that the immobilized enzyme surfaces are notaccessible to all the reacting species However no substrate orproduct inhibition by the increase of substrate concentrationup to 200mM could be observed during our experiment as

020406080

100120140160180

minus150 minus100 minus50 0 50 100 150 200 250[S] (mM)

ImmoFree

y = 0481x + 63219

R2 = 09332

y = 05566x + 32786

R2 = 09889

[S]V

Figure 8 Reusability of the immobilized 120573-galactosidase

Table 2 Michaelis-Menten constants and maximal reaction ratevalues for free and immobilized lactase

120573-Galactosidase form Kinetic constants119870119898(mM) 119881max (120583molsdotminminus1)

Free 589 327Immobilized 1314 632

shown by the straight line of the Hanes-Wolf representation(Figure 8)

On the other hand the maximum reaction velocity119881maxvalues for the immobilized enzyme were remarkable it wasfound to double that of the free enzyme that is it increasedfrom 327 to 632 120583molsdotminminus1This result is in agreement withthe speculation that the improvement in the immobilizedenzyme thermal stability as in Section 331 could result in ahigher reaction velocity It is worth noting that the increasein the reaction velocity is generally favored in industries

334 Lactose Hydrolysis Using Free and Immobilized 120573-Galactosidase This experiment has been carried out so thatthe immobilized enzyme could attain its maximum efficiencyand act with its highest velocity using almost double the 119870

119898

concentration of substrate and using the enzymersquos optimumconditions A high concentration of substrate 200mM atpH 45 and 37∘C was used in this study as this enzymewas supposed to be suitable for hydrolysis of higher lactoseconcentrations found in mammal milk (88ndash234mM lactose)and whey permeate (85 lactose) [25]

The results as shown in Figure 9 revealed that for the firsthour the rate of conversion of the free enzyme was higherthan that of the immobilized one This could be attributedto the fact that the gel needed longer time to reach itsmaximum swelling This swelling will allow more substratesto penetrate into the pores and consequently decrease thediffusion limitation However at 90min both enzyme formsfollowed the same trend and the same speed till they reachedmaximum relative conversion at 120min It is worth notingthat at 90min the gel beads carrying the enzyme reached its

8 BioMed Research International

0

20

40

60

80

100

120

0 50 100 150 200 250

Hyd

roly

sis (

)

Time (min)

ImmoFree

Figure 9 Effect of 120573-galactosidase concentration on the enzymersquosloading capacity

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Rela

tive a

ctiv

ity (

)

Cycle number

Figure 10 Temperature profile of free and immobilized enzyme

maximum swelling overcame its substrateproduct diffusionlimitation and followed the same trend as the free enzymewhich is advantageous in industries That means that thesmall gel beads used in this work could overcome the problemthe authors previously had when they used big gel disks asthe enzyme suffered fromdiffusion limitation and hydrolyzedonly 63 of the free enzyme [3]

335 Reusability of Immobilized Enzyme To evaluate thereusability of the immobilized enzyme the beadswere soakedin 200mM lactose for 120min till full conversion of lactoseto glucose and galactose The gel beads were removed fromthe product washed with buffer solution after use and thenresuspended in a fresh aliquot of a substrate to measure theenzymatic activity

This procedure was repeated until the enzyme lost itsactivity The turn over number of the enzyme catalyzed pro-cess was calculated As shown in Figure 10 the immobilizedenzyme retained 60 of its relative activity by the seconduse and 21 by the 3rd use Nevertheless these results werein agreement with those obtained by other authors usingthe commercial carrier Novozym 435 as the immobilizedactivity decreased to 23 after the second use and to 37 by

the third use [27] The loss in activity was attributed by otherauthors to inactivation of enzyme due to continuous use [28]Although our carrier has shown better performance than thatof Novozyme 435 we think that the modified gel with epoxycould be further modified for future work

4 Conclusion

Novel biopolymer based on epoxy activated carrageenan wasprepared for immobilization of lactase as an example ofmedical enzyme The results were compared to those of ourprevious work using aldehyde activated carrageenan Theepoxy formula showed far better immobilization efficiencythat was triple that shown using the aldehyde one Thatcould be regarded to that the epoxy group is more activethan the aldehyde group The aldehyde group could onlybind to the enzymersquos free amino groups whereas the epoxygroup could bind to three groups ndashSH ndashNH

2 and ndashOH

The results showed in Figure 9 hydrolysis of lactose usingfree and immobilized lactase revealed that the immobilizedenzyme could attain its maximum efficiency and act withits highest velocity as fast as the free enzyme That wasregarded to the gel beads carrying the enzyme that reachedits maximum swelling and overcame its substrateproductdiffusion limitation and followed the same trend as thefree enzyme which is advantageous in industries The highactivity of the epoxy formulation is highly recommended tobe used for immobilization of other enzymesproteins andordrug delivery systems

Abbreviations

Carr CarrageenanCarr-Ch Carrageenan-chitosanCarr-Ch-Epo Carrageenan-chitosan-epoxyCarr-Ch-Epo-Ch Carrageenan-chitosan-epoxy-

chitosanCarr-Ch-Epo-Ch-Epo Carrageenan-chitosan-epoxy-

chitosan-epoxy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q Husain ldquo120573-Galactosidases and their potential applications areviewrdquo Critical Reviews in Biotechnology vol 30 no 1 pp 41ndash62 2010

[2] MM Elnashar G E Awad M E Hassan M S Mohy Eldin BM Haroun and A I El-Diwany ldquoOptimal immobilization of120573-galactosidase onto 120581-Carrageenan gel beads using responsesurface methodology and its applicationsrdquoThe Scientific WorldJournal vol 2014 Article ID 571682 7 pages 2014

[3] M M M Elnashar and M A Yassin ldquoLactose hydrolysisby 120573-galactosidase covalently immobilized to thermally stablebiopolymersrdquo Applied Biochemistry and Biotechnology vol 159no 2 pp 426ndash437 2009

BioMed Research International 9

[4] G F Bickerstaf ldquoImpact of genetic technology on enzymetechnologyrdquo The Genetic Engineer and Biotechnologist vol 15pp 13ndash30 1995

[5] M M Elnashar ldquoThe art of immobilization using biopoly-mers biomaterials and nanobiotechnologyrdquo in Biotechnology ofBiopolymers pp 1ndash30 Intech 2011

[6] G Roberts ldquoStructure of chitin and chitosanrdquo in Chitin Chem-istry G A F Roberts Ed pp 1ndash53 MacMillan HoundmillsUK 1992

[7] R Hejazi andM Amiji ldquoChitosan-based gastrointestinal deliv-ery systemsrdquo Journal of Controlled Release vol 89 no 2 pp 151ndash165 2003

[8] B Krajewska ldquoApplication of chitin- and chitosan-based mate-rials for enzyme immobilizations a reviewrdquo Enzyme andMicro-bial Technology vol 35 no 2-3 pp 126ndash139 2004

[9] M M Elnashar O A Ali and H A Ragaa ldquoImmobilizedpenicillin G acylase onto grafted k-carrageenan hypothesis onthe effect of pH on the gel-enzyme interactionrdquoArabian Journalof Chemistry In press

[10] K C Chao M M Haugen and G P Royer ldquoStabiliza-tion of kappa-carrageenan gel with polymeric amines useof immobilized cells as biocatalysts at elevated temperaturesrdquoBiotechnology and Bioengineering vol 28 no 9 pp 1289ndash12931986

[11] J S Chang C Chou and S Y Chen ldquoDecolorization of azo dyeswith immobilized Pseudomonas luteolardquo Process Biochemistryvol 36 no 8-9 pp 757ndash763 2001

[12] S H Moon and S J Parulekar ldquoCharacterization of 120581-carrageenan gels used for immobilization of Bacillus firmusrdquoBiotechnology Progress vol 7 no 6 pp 516ndash525 1991

[13] E N Danial M M M Elnashar and G E A Awad ldquoImmobi-lized inulinase on grafted alginate beads prepared by the one-step and the two-steps methodsrdquo Industrial and EngineeringChemistry Research vol 49 no 7 pp 3120ndash3125 2010

[14] D K Boadi and R J Neufeld ldquoEncapsulation of tannase for thehydrolysis of tea tanninsrdquo Enzyme and Microbial Technologyvol 28 no 7-8 pp 590ndash595 2001

[15] A M Eberhardt V Pedroni M Volpe and M L FerreiraldquoImmobilization of catalase from Aspergillus niger on inorganicand biopolymeric supports for H

2O2decompositionrdquo Applied

Catalysis B Environmental vol 47 no 3 pp 153ndash163 2004[16] M M Elnashar ldquoReview article immobilized molecules using

biomaterials and nanobiotechnologyrdquo Journal of Biomaterialsand Nanobiotechnology vol 1 pp 61ndash76 2010

[17] EMagnan I Catarino D Paolucci-Jeanjean L Preziosi-Belloyand M P Belleville ldquoImmobilization of lipase on a ceramicmembrane activity and stabilityrdquo Journal of Membrane Sciencevol 241 no 1 pp 161ndash166 2004

[18] S Rocchietti A S V Urrutia M Pregnolato et al ldquoInfluenceof the enzyme derivative preparation and substrate structureon the enantioselectivity of penicillin G acylaserdquo Enzyme andMicrobial Technology vol 31 no 1-2 pp 88ndash93 2002

[19] MMM Elnashar andM A Yassin ldquoCovalent immobilizationof 120573-galactosidase on carrageenan coated with chitosanrdquo Jour-nal of Applied Polymer Science vol 114 no 1 pp 17ndash24 2009

[20] A A El-Sanabary M M Elnashar A A Magda and B MBadran ldquoPreparation and evaluation of some new corrosioninhibitors in varnishesrdquo Anti-Corrosion Methods and Materialsvol 48 no 1 pp 47ndash58 2001

[21] C Tapia Z Escobar E Costa et al ldquoComparative studies onpolyelectrolyte complexes and mixtures of chitosan-alginate

and chitosan-carrageenan as prolonged diltiazem clorhydraterelease systemsrdquo European Journal of Pharmaceutics and Bio-pharmaceutics vol 57 no 1 pp 65ndash75 2004

[22] W J Sung and Y H Bae ldquoA glucose oxidase electrode basedonpolypyrrolewith polyanionPEGenzyme conjugate dopantrdquoBiosensors and Bioelectronics vol 18 no 10 pp 1231ndash1239 2003

[23] I Gancarz J Bryjak M Bryjak G Pozniak and W TylusldquoPlasma modified polymers as a support for enzyme immobi-lization 1 Allyl alcohol plasmardquo European Polymer Journal vol39 no 8 pp 1615ndash1622 2003

[24] MMM ElnasharM A Yassin and T Kahil ldquoNovel thermallyandmechanically stable hydrogel for enzyme immobilization ofpenicillin G acylase via covalent techniquerdquo Journal of AppliedPolymer Science vol 109 no 6 pp 4105ndash4111 2008

[25] A Tanriseven and S Dogan ldquoA novel method for the immobi-lization of 120573-galactosidaserdquo Process Biochemistry vol 38 no 1pp 27ndash30 2002

[26] Q Z K Zhou and X Dong Chen ldquoImmobilization of 120573-galactosidase on graphite surface by glutaraldehyderdquo Journal ofFood Engineering vol 48 no 1 pp 69ndash74 2001

[27] B Chen J Hu E M Miller W Xie M Cai and R AGross ldquoCandida antarctica Lipase B chemically immobilized onepoxy-activated micro- and nanobeads catalysts for polyestersynthesisrdquo Biomacromolecules vol 9 no 2 pp 463ndash471 2008

[28] K Nakane T Ogihara N Ogata and Y Kurokawa ldquoEntrap-immobilization of invertase on composite gel fiber of celluloseacetate and zirconium alkoxide by sol-gel processrdquo Journal ofApplied Polymer Science vol 81 no 9 pp 2084ndash2088 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

pH lt 65 and when dissolved possesses high positive chargeon NH

3

+ groups The protonated (NH3

+) could adhere tonegatively charged surfaces such as polyanionic compounds[8 9]

Carrageenan is a naturally occurring anionic polysac-charide isolated from the seaweeds According to the studymade by Chao et al 1986 [10] to harden the carrageenangels using different amine compounds they concluded thatonly polyamines such as chitosan substantially improvedthe carrageenan gels thermal stability via polyelectrolytersquosinteractions Unfortunately the carrageenan-polyelectrolytesystems were limited to the entrapment of enzymes [11ndash13] which have the major problem of enzyme leakage Forexample Boadi and Neufeld 2001 [14] used alginate andcarrageenan to entrap tannase and then crosslinked thegel beads with chitosan followed by glutaraldehyde Theentrapment technique limits their industrial use as supportsfor enzyme immobilization due to enzyme leakage So effortsto immobilize enzymes on newer type of carriers especiallywith covalent bonds are still underway in many laboratories[15ndash18]

To our knowledge previous reports have not any greaterextent that dealt with carrageenan-chitosan polyelectrolyteas a use for the covalent immobilization of enzymes withan exception of our recent work [19 20] In that work weimmobilized 120573-galactosidase covalently via its amino groupsto carrageenan treated with chitosan and glutaraldehydeAlthough the linkage was covalent however the enzymeloading capacity was limited to up to 11 Ug gel beadsThis could be regarded to the free aldehyde groups on thecarrageenan treated chitosan that could only react with thefree amino groups (NH

2) on the enzyme via Schiff rsquos base

formation ndashC=NHndash [19 20]Thus the purpose of this research was to modify the

carrageenan with chitosan and a more efficient functionalgroup epoxy group which imparts three extra benefits tocarrageenan

(i) the first is improvement of the carrageenan gelrsquos ther-mal stability by forming a polyelectrolyte complex(PEC) between the carrageenan ndashOSO

3

minus and thechitosan ndashNH

3

+(ii) the second is creation of a new functionality free

chitosan amino groups (NH2)

(iii) the third is activation of the free chitosan aminogroups (ndashNH

2) with 14-butanediol diglycidyl ether

to impart free epoxy groups to covalently immobilize120573-galactosidase via the ndashSH ndashOH and ndashNH of theiramino acids

Accordingly we expect more enzymes to bind tothe epoxy activated carrier via three groups on the enzyme(ndashSH ndashOH and ndashNH) whereas the free aldehyde groupscould only bind to the ndashNH

2of the amino acids To our

knowledge there are no reports on the use of carrageenanfor the immobilization of 120573-galactosidase using covalenttechnique via chitosan and 14-butanediol diglycidyl etherThe novel gel formulation was prepared in beads shape usingthe Encapsulator to enable gel beads production on the

semipilot scale and to increase the gelrsquos surface area Thegrafted formulationwas illustrated using a schematic diagramand the chemical and thermal modification was proved usingthe FTIR and DSC techniques respectively On the otherhand the enzyme loading capacity was optimized by usingshort and long chain of the epoxy activated carrier and usingdifferent concentrations of the enzyme Finally the free andimmobilized enzymes were characterized for their activitiesat different pHs and temperatures and theMichaelis constantswere studied as well as the lactose hydrolysis using free andimmobilized enzyme over time

2 Materials and Methods

As a general rule all experimentswere carried out in triplicateand data are means plusmn SD (119899 = 3)The abbreviations section islisting all the abbreviations used in the formulations samples

21 Materials 120581-Carrageenan (MW 154000) sulfate ester sim25 and chitosanwere supplied by Fluka120573-Galactosidase (EC32123) was obtained fromAspergillus oryzae and 118Umgwas obtained from Sigma-Fluka-Aldrich All other chemicalswere of pure grades (Analar or equivalent quality) TheEncapsulator model IE-50 was purchased from InnotechEncapsulator in Switzerland The gel disks dimensions weremeasured using a micrometer (Micro 2000 0ndash25mm)

22 Determination of 120573-Galactosidase Activity 120573-Galacto-sidase activity was determined by the rate of glucose forma-tion in the reaction medium Known amount of immobilizedor free enzyme was incubated into 10mL of 200mM lactosesolution in 100mM citrate phosphate buffer at pH 45 for 3 hat 37∘C and 100 rpm At the end of the time 50 120583L of reactionmixture was added to 950 120583L buffer and boiled for 10min toinactivate the enzyme and analyzed for glucose content usingthe glucose test One enzyme unit (IU) was defined as theamount of enzyme that catalyzes the formation of 1 120583mol ofglucose per minute under the specified conditions

Equations (1) (2) and (3) show the hydrolysis of lac-tose by 120573-galactosidase and glucose determination using amixture of enzymes glucose oxidase (GOD) and peroxidase(POD)

Lactose120573-galactosidase997888997888997888997888997888997888997888997888997888997888rarr Glucose + Galactose (1)

Glucose +O2+H2O GOD997888997888997888997888rarr Glucolactone +H

2O2

(2)

H2O2+Hydroxybenoate-Na-4-aminoantipyrine

POD997888997888997888rarr Quinon complex +H

2O

(3)

Glucose concentration was measured spectrophotomet-rically with a glucose test based on the Trinder reagentGlucose is transformed to gluconic acid and hydrogen per-oxide by glucose oxidase (GOD) The hydrogen peroxideformed reacts in the presence of peroxidase (POD) with 4-aminoantipyrine and p-hydroxybenzene sulfonate to form aquinoneimine dye as shown in Equations (1) (2) and (3)

BioMed Research International 3

The intensity of the color produced is directly propor-tional to the glucose concentration in the sample The assaywas performed by mixing 30 120583L of a sample of unknownconcentration and 3mL of Trinder reagent the reaction wasallowed to proceed for 20min at room temperature and theabsorbance of the unknown concentrationwas read at 510 nm[19]

23 Preparation of Gel Beads 120581-Carrageenan gel was pre-pared by dissolving 25 (wv) carrageenan in distilled waterat 70∘C using an overhead mechanical stirrer until completedissolution had occurred The Carrageenan gel solution wasdropped through a nozzle of 300 120583m using the InnotechEncapsulator in a hardening solution of 03M KCl Thenbeads were hardened using 03M KCl for 3 h

24Modification of Gel BeadsUsingChitosan andEpoxy Twoactivated epoxy hydrogels were prepared the short and thelong chain as follows

(a) TheShort Chain Carr-Ch-Epo beads were soaked ina solution of 075 chitosan previously prepared in 1(vv) acetic acidThen they were suspended in 75mL05M NaOH containing 150mg sodium cyanoboro-hydride under stirring Slowly 75mL 1 4-butanedioldiglycidyl ether was added with constant stirring andthe reaction was left at room temperature overnightFinally the activated gel beads were extensivelywashed with water to remove excess reagent

(b) TheLong Chain Carr-Ch-Epo-Ch-Epo formula (a)was furthermodified with chitosan and then epoxy asshown above

25 Elucidation of the Modified Gel Using Fourier Trans-form Infrared Spectroscopy The infrared spectra of all for-mulations were recorded with Fourier transform infraredspectroscopy (FTIR-8300 Shimadzu Japan) FTIR spectrawere taken in the wavelength region 4000 to 400 cmminus1 atambient temperature The FTIR spectrophotometer (FTIR-8300 Shimadzu Japan) was used to prove the presenceof the new functional group epoxy groups in both formsof the modified gels Five samples were used for this testcarrageenan (Carr) carrageenan coatedwith chitosan (Carr-Ch) carrageenan coated with chitosan followed by epoxy(short chain epoxy activated hydrogel Carr-Ch-Epo) car-rageenan coated with chitosan followed by epoxy followedby chitosan (Carr-Ch-Epo-Ch) and finally carrageenancoated with chitosan followed by epoxy followed by chitosanfollowed by epoxy (long chain epoxy activated hydrogelCarr-Ch-Epo-Ch-Epo)

A total of 2 (ww) of the sample with respect to thepotassium bromide (KBr S D Fine Chem Ltd) disk wasmixed with dry KBr The mixture was ground into a finepowder using an agatemortar before it was compressed into aKBr disk under a hydraulic press at 10000 psi Each KBr diskwas scanned 16 times at 4mms at a resolution of 2 cmminus1 overa wavenumber range of 400ndash4000 cmminus1 using Happ-Genzelapodization

26 Differential Scanning Calorimetry andThermal Gravimet-ric Analysis Differential scanning calorimetry (DSC) andthermal gravimetric analysis (TGAA) were performed toprove the formation of a strong polyelectrolyte complexbetween carrageenan and chitosan followed by di-epoxyThethermal behavior of five gel formulations was performedCarr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo The differential scanning calorimetry wasstudied using DSC (SDT 600 TA Instruments USA)Approximately 3 to 6mg of the dried gels was weighed intoan alumina panThe samples were heated from40∘C to 340∘Cat a heating rate of 10∘Cmin The thermal behavior of thedifferent gel formulations was characterized for their TGA(SDT 600 TA Instruments USA) Alumina pans were usedand approximately 3 to 6mg of the dried gels were weighedThe samples were heated from 50 to 300∘C at a heating rateof 10∘Cmin

27 Immobilization of 120573-galactosidase 120573-Galactosidase wasimmobilized onto the short and long chain of the epoxyactivated hydrogels as follows One gram of the activatedgel beads was washed thoroughly with distilled water andwas incubated into 10mL of enzyme solution (30UmL)prepared in 100mM citrate-phosphate buffer at pH 45 for16 h The immobilized enzyme was washed thoroughly withthe buffer solution containing Tris-HCl to block any freealdehyde group and to remove any unbound enzyme Theimmobilized enzyme was stored at 4∘C for further mea-surements Two parameters were used to reach the enzymersquosmaximum loading capacity (ELC) and epoxy activated longand short chains as well as the enzyme concentration

The ELC or the amount of enzymesrsquo units immobilizedonto and into gel beads was calculated as follows

ELC =(119872119900minus119872119891)

119882 (4)

where119872119900is the initial enzyme activity (U)119872

119891is the enzyme

activity of the filtrate (U) after immobilization and119882 is theweight of gel beads (g)

28 Optimization and Evaluation of the Free and Immobilized120573-Galactosidase

281 Temperature and pH Profiles for the Free andImmobilized 120573-Galactosidase The free and immobilized 120573-galactosidases were incubated into 10mL of 200mM lactoseat temperatures from 30∘C to 70∘C for 3 hrs The enzymeactivity has been determined according to Section 22 Theoptimum temperature has been chosen to study the effect ofpH where the free and immobilized 120573-galactosidases wereincubated into 10mL of 200mM lactose at pH 30ndash90 at37∘C for 3 hrs

282 119870119898

and 119881max of the Free and Immobilized 120573-Galactosidase The Michaelis-Menten kinetic models ade-quate for the description of the hydrolysis of lactose by thefree and the immobilized enzyme apparent 119870

119898and 119881max

4 BioMed Research International

(a)

(b)

(c)

ChitosanCarrageenan Carr Ch

NH2 NH2

+++ +

+

minusminusminusminusminusminusminusminus

minusminusminusminusminus

minusminusminusminusminusminusminusminus

minusminusminusminusminus

14-Butanediol diglycidyl etherCarr Ch Epoxy activated carrageenan

NH2 ndashO(CH2)4OCH2CH2OO+

+

+minusminusminusminusminusminusminusminus

minusminusminusminusminus

ndashO(CH2)4OCH2O

NHndashCH2ndashCHndashCH2

++minusminusminusminusminusminusminusminus

minusminusminusminusminus

NHndash ndashCHOH

EnzymeEpoxy activated Carr

NHndashCH2ndashCH CHndashCH ndashNHndashEndashSHOH

H2NndashEndashSH

CarrndashChndashEpondashenzyme

CH2 CH2

ndash ndash

O +++

minusminusminusminusminusminusminusminus

minusminusminusminusminus

++

minusminusminusminusminusminusminusminus

minusminusminusminusminus

Scheme 1 Grafted carrageenan gel beads with epoxy groups and immobilization of enzymes (a) Modification of carrageenan beads withchitosan via ionic interaction (b) Incorporation of epoxy groups to the carrageenan-chitosan (c) Immobilization of enzymes to the graftedcarrageenan-epoxy via ndashSH ndashOH or ndashNH

2

of free and immobilized 120573-galactosidase were determinedfor lactose using the Hanes-Woolf plot method Free andimmobilized 120573-galactosidases were incubated into 10mL of25 to 200mM at 37∘C and pH 45 for 3 hrs under standardassay conditions

283 Lactose Hydrolysis Using Immobilized 120573-GalactosidaseTo evaluate the efficiency of the immobilized enzyme it wasused for lactose hydrolysis using the optimum conditionsobtained from above optimization for the free and immobi-lized enzymes

3 Results and Discussion

31 GraftedAlginate Elucidation Structure Protonated aminogroups (ndashNH

3

+) of Ch formed a polyelectrolyte complexwiththe ndashOSO

3

minus of the Carr gel and incorporated free aminogroups to Carr [19] The free amino groups (ndashNH

2) of Ch

were used to covalently immobilize 120573-galactosidase via thedi-epoxy groups as a mediator beside its main role as a cross-linkerThe enzyme could be bound to the carrierrsquos free epoxygroups via its ndashSH ndashOH and ndashNH groups However asshown in Scheme 1 we represented that one uses the free ndashNH groups as an example to follow the ndashOH and ndashSH groups

The FTIR bands of Carr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo were shown in Figure 1Spectrums of the three compounds Carr Carr-Ch Carr-Ch-Epo revealed a new and strong band at 870 cmminus1 whichappears only for the modified gel spectrum with epoxygroupsThis band proved the presence of a new gel functionalgroup epoxy group which is in agreement with the authorrsquosprevious work [20] This band disappeared after treatment

75015002250300037504500

T (

)

3460

30

1120

64

3479

58

1126

43

1066

64

3458

37 11

110

0

3435

22 11

225

710

666

4

3419

79

1112

93

(cmminus1)CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 1 FTIR of five formulations of carrageenan and modifiedcarrageenan with chitosan and epoxy groups Carr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo

of the Carr-Ch-Epo with Ch and reappeared after furthertreatment with epoxy (Carr-Ch-Epo-Ch-Epo) at 880 cmminus1The FTIR bands also revealed a decrease in intensity and a

BioMed Research International 5

Table 1 Values ofDSC andTGAA thermograms of carrageenan andmodified carrageenan with chitosan and epoxy groups

Number Formula DSC temp ∘C TGA temp ∘C1 Carr 220 2002 Carr-Ch 230 2103 Carr-Ch-Epo 250 amp 320 2504 Carr-Ch-Epo-Ch 260 2405 Carr-Ch-Epo-Ch-Epo 260 amp 330 270

0123456789

10

0 50 100 150 200 250 300 350

Hea

t flow

exo

up

(mW

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 2 DSC thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

shift of the ndashOSO3

minus absorption band of Carr from 1446 cmminus1to 1390 cmminus1after reaction with the chitosan This ionicinteraction between the carrageenan and the chitosan evi-denced the formation of strong polyelectrolyte complexes[21]

Table 1 is tabulating the main characteristic values of theDSC and TGAA thermograms as shown in Figures 2 and 3respectively

The treatment of carrageenan with chitosan and epoxyhas shown gradual and obvious improvement in their DSCand TGA The DSC exothermic effect has been shifted tohigher temperatures from formula numbers 1ndash5 that is from220∘C to 320∘C The Carr has shown an exothermic band at220∘C which has been shifted to 230∘C after treatment withCh This improvement could be attributed to the formationof a complex network between the Carr and the Ch Similarbehavior has been attained when the di-epoxy has beensubstituted with glutaraldehyde [3] Further treatment withdi-epoxy Carr-Ch-Epo gradually increased the temperatureto two peaks at 250 and 230∘C This could be attributed toextra crosslinking with the di-epoxy and the two bands couldbe referred to the Ch and Epo however we could not tell atthis stage which is which However by addition of Ch Carr-Ch-Epo-Ch the temperature increased to a single band at260∘C which should be regarded to the Ch That means thatfor the short chain Carr-Ch-Epo the two peaks should be forCh and Epo respectively Finally by adding more di-epoxyCarr-Ch-Epo-Ch-Epo (long chain) two bands appeared for

60

65

70

75

80

85

90

95

100

0 50 100 150 200 250 300 350

Wei

ght (

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 3 TGA thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

the Ch and Epo at the highest temperatures 260 and 330∘Crespectively

On the other hand the TGA of the modified Carrformula numbers 2-5 showed a better stability against degra-dation as shown in Figure 3 andTable 1 For example unmod-ified Carr had a sudden decomposition at 200∘C This valuehas been increased to 210∘C after its treatment with Ch withretention of the sudden decomposition behavior The gelsrsquothermal improvement could be explained by the formation ofpolyelectrolyte interaction between the polyanions (ndashOSO

3

minus)of the Carr and the polycations (NH

3

+) of the Ch Furtherhardening of the gel beads using Epo showed a much higherincrease in the TGAA Carr-Ch-Epo (short chain) to 250∘Cand the decomposition behavior was slower and gradualThe improvement in the TGA could be attributed to extracrosslinking between the free Chrsquos amino groups and theEpo Further treatment of the short chain with Ch does notincrease its TGA however it was more or less the same at240∘C Finally for the long chain Carr-Ch-Epo-Ch-Epo theTGA increased to its maximum of 270∘C which could beregarded to further crosslinking It is worth noting that theshort and long chains have the highest TGA values of 250 and270∘Cwith slower and gradual decomposition rate comparedto other formulations

32 Optimization of Enzymersquos Loading Capacity Two factorshave been studied to optimize the loading capacity of 120573-galactosidases onto treated carrageenan gel beads

321 Effect of Epoxy Chain Length In this experiment shortchain of epoxy modified gel beads Carr-Ch-Epo and longchain Carr-Ch-Epo-Ch-Epo were examined to assess chainlength efficiency for immobilization of the enzyme Accord-ing to Sung and Bae 2003 [22] the effect of the chain lengthcould have a positive effect on the immobilization loadingefficiency till certain length and then it declines afterwards Inour case the short chain immobilizedmore enzymes than thelong chain Results as shown in Figure 4 showed that the short

6 BioMed Research International

0

5

10

15

20

25238

182

(Ug

bea

ds)

CarrndashChndashEpo CarrndashChndashEpondashChndashEpo

Figure 4 Effect of epoxy chain length on 120573-galactosidase loadingcapacity

chain immobilized 238 Ug gel beds compared to 182 Uggel beads for the long chain These results are in accordancewith that of Gancarz et al 2003 [23] who observed that anincrease in surface epoxy groups led to an increase in quantityof immobilized enzyme but a decrease in retained enzymeactivity

To understand this phenomenon we calculated from thesupernatants the expected amounts of immobilized enzymeson the short and long chains formulas and they were foundto be 18 and 21Ug respectively These results were in favorof the long chain formula however the retained activity ofthe immobilized enzymes was in favor of the short chainwhere 238Ug were immobilized showing 133 retention ofactivity This increase in the enzyme activity after immobi-lization could be regarded to the hydrogen bond interactionsbetween the modified gel (polysaccharide) containing ndashOHndashOSO

3H ndashC=O ndashNH

2 and the lactose substrate containing

ndashOH ndashC=O groups These H-bonding interactions couldalso increase the lactose concentration surrounding the gelsurface more than the bulk solution and thus the activity ofthe immobilized enzyme increases till reaching saturation ofthe gel surface with lactase [24]

On the other hand the long chain formulation wasexpected to immobilize 21Ug and in practice it showedonly 182Ug which revealed 86 retention of activity ofthe immobilized enzyme This could be regarded to the longchain havingmultipoint attachment orandmultilayers of theimmobilized enzymesteric hindrance that resulted in lossof the enzymersquos 3D structure and consequently its activityAccordingly for further experiments the short chain wasused

322 Effect of 120573-Galactosidase Concentration 120573-Galacto-sidase was immobilized onto gel beads treated with shortchain epoxy activated carrageenan Carr-Ch-Epo as shownin Figure 5

Results revealed that by increasing the concentrationof 120573-galactosidase from 10U to 60U the ELC increasedgradually till it reached its maximum of 36Ug gel beadsusing 50U of free enzyme after which any more addedenzyme has almost no effect on the ELC This could beregarded to all free epoxy groups that have been engagedwith the enzymes [25] However we have chosen for furtheroptimization the ELC of 36Ug gel beads as it shows better

05

10152025303540

10 20 30 40 50 60

(Ug)

8958854327 1239676625 2492457653 3101527564 3602115799 362855251

(Ug

bea

ds)

Figure 5 pH profile of the free and immobilized 120573-galactosidase

0

20

40

60

80

100

120

20 30 40 50 60 70 80

Rela

tive a

ctiv

ity (

)

FreeImmo

Temperature (∘C)

Figure 6 Michaelis constants of free and immobilized 120573-galactosidase

enzyme loading efficiency of 49 which is more economicas it saves unloaded enzyme from being wasted

33 Evaluation of Catalytic Activity of Free and Immobilized120573-Galactosidase At this stage five experiments were studiedFirstly the optimum reaction temperature pH and substrateconcentrations were examined for both the free and immo-bilized enzyme Secondly the best results from the first stepwere used to obtain themaximum substrate hydrolysis as wellas the operational stability of the immobilized enzyme

331 Optimum Temperature for the Free and Immobilized 120573-Galactosidase The optimum temperature for the free andimmobilized enzyme was examined Results as shown inFigure 6 revealed that the optimum temperature for theimmobilized enzyme was found to be at a slightly highertemperature (37ndash40∘C) compared to the free enzyme (30ndash37∘C)

The shift of the optimum temperature towards highertemperatures when the biocatalyst is immobilized indicatesthat the enzyme structure is strengthened by the immobiliza-tion process and the formation of a molecular cage aroundthe protein molecule (enzyme) was found to enhance theenzyme thermal stability The increase of the immobilized

BioMed Research International 7

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10

Rela

tive a

ctiv

ity (

)

pH

ImmoFree

Figure 7 Lactose hydrolysis using free and immobilized 120573-galactosidase

enzyme temperature tolerance may also be due to diffusionaleffects where the reaction velocity is more likely to bediffusion limited so that improvements in thermal diffusionwould correspondingly result in proportionally higher reac-tion rates [3]

332 pH Profile Figure 7 illustrates the pH activity profile ofthe free and immobilized 120573-galactosidase The optimum pHvalues for free and immobilized enzyme were 45ndash5 and 4-6 respectively which showed that the immobilized enzymewas more stable at higher and wider range of pH [25] Theseproperties could be very useful for lactolysis in sweet wheypermeate which has a pH range of 55ndash6 Moreover at pH4 the immobilized enzyme retained more than 95 of itsrelative activity compared to only 56 for the free enzyme

The shift in the pH activity profile of the immobilized 120573-galactosidase and the better pH stability may be attributedto the partition effects that were arising from differentconcentrations of charged species in the microenvironmentof the immobilized enzyme and in the domain of the bulksolution [3]

333 Determination of Kinetic Parameters of Free and Immo-bilized 120573-Galactosidase The kinetic constants of free andimmobilized 120573-galactosidase as shown in Figure 8 weretabulated in Table 2

The apparent 119870119898

after immobilization 1314mM ishigher than that of the free enzyme 589mMwhich indicatesthat a higher concentration of substrate 2-fold is needed forthe immobilized enzyme compared to the free enzyme Nev-ertheless higher 119870

119898values for immobilized 120573-galactosidase

have been reported by other authors with increases from 12-fold up to 54-fold [26] These results are most likely dueto the fact that the immobilized enzyme surfaces are notaccessible to all the reacting species However no substrate orproduct inhibition by the increase of substrate concentrationup to 200mM could be observed during our experiment as

020406080

100120140160180

minus150 minus100 minus50 0 50 100 150 200 250[S] (mM)

ImmoFree

y = 0481x + 63219

R2 = 09332

y = 05566x + 32786

R2 = 09889

[S]V

Figure 8 Reusability of the immobilized 120573-galactosidase

Table 2 Michaelis-Menten constants and maximal reaction ratevalues for free and immobilized lactase

120573-Galactosidase form Kinetic constants119870119898(mM) 119881max (120583molsdotminminus1)

Free 589 327Immobilized 1314 632

shown by the straight line of the Hanes-Wolf representation(Figure 8)

On the other hand the maximum reaction velocity119881maxvalues for the immobilized enzyme were remarkable it wasfound to double that of the free enzyme that is it increasedfrom 327 to 632 120583molsdotminminus1This result is in agreement withthe speculation that the improvement in the immobilizedenzyme thermal stability as in Section 331 could result in ahigher reaction velocity It is worth noting that the increasein the reaction velocity is generally favored in industries

334 Lactose Hydrolysis Using Free and Immobilized 120573-Galactosidase This experiment has been carried out so thatthe immobilized enzyme could attain its maximum efficiencyand act with its highest velocity using almost double the 119870

119898

concentration of substrate and using the enzymersquos optimumconditions A high concentration of substrate 200mM atpH 45 and 37∘C was used in this study as this enzymewas supposed to be suitable for hydrolysis of higher lactoseconcentrations found in mammal milk (88ndash234mM lactose)and whey permeate (85 lactose) [25]

The results as shown in Figure 9 revealed that for the firsthour the rate of conversion of the free enzyme was higherthan that of the immobilized one This could be attributedto the fact that the gel needed longer time to reach itsmaximum swelling This swelling will allow more substratesto penetrate into the pores and consequently decrease thediffusion limitation However at 90min both enzyme formsfollowed the same trend and the same speed till they reachedmaximum relative conversion at 120min It is worth notingthat at 90min the gel beads carrying the enzyme reached its

8 BioMed Research International

0

20

40

60

80

100

120

0 50 100 150 200 250

Hyd

roly

sis (

)

Time (min)

ImmoFree

Figure 9 Effect of 120573-galactosidase concentration on the enzymersquosloading capacity

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Rela

tive a

ctiv

ity (

)

Cycle number

Figure 10 Temperature profile of free and immobilized enzyme

maximum swelling overcame its substrateproduct diffusionlimitation and followed the same trend as the free enzymewhich is advantageous in industries That means that thesmall gel beads used in this work could overcome the problemthe authors previously had when they used big gel disks asthe enzyme suffered fromdiffusion limitation and hydrolyzedonly 63 of the free enzyme [3]

335 Reusability of Immobilized Enzyme To evaluate thereusability of the immobilized enzyme the beadswere soakedin 200mM lactose for 120min till full conversion of lactoseto glucose and galactose The gel beads were removed fromthe product washed with buffer solution after use and thenresuspended in a fresh aliquot of a substrate to measure theenzymatic activity

This procedure was repeated until the enzyme lost itsactivity The turn over number of the enzyme catalyzed pro-cess was calculated As shown in Figure 10 the immobilizedenzyme retained 60 of its relative activity by the seconduse and 21 by the 3rd use Nevertheless these results werein agreement with those obtained by other authors usingthe commercial carrier Novozym 435 as the immobilizedactivity decreased to 23 after the second use and to 37 by

the third use [27] The loss in activity was attributed by otherauthors to inactivation of enzyme due to continuous use [28]Although our carrier has shown better performance than thatof Novozyme 435 we think that the modified gel with epoxycould be further modified for future work

4 Conclusion

Novel biopolymer based on epoxy activated carrageenan wasprepared for immobilization of lactase as an example ofmedical enzyme The results were compared to those of ourprevious work using aldehyde activated carrageenan Theepoxy formula showed far better immobilization efficiencythat was triple that shown using the aldehyde one Thatcould be regarded to that the epoxy group is more activethan the aldehyde group The aldehyde group could onlybind to the enzymersquos free amino groups whereas the epoxygroup could bind to three groups ndashSH ndashNH

2 and ndashOH

The results showed in Figure 9 hydrolysis of lactose usingfree and immobilized lactase revealed that the immobilizedenzyme could attain its maximum efficiency and act withits highest velocity as fast as the free enzyme That wasregarded to the gel beads carrying the enzyme that reachedits maximum swelling and overcame its substrateproductdiffusion limitation and followed the same trend as thefree enzyme which is advantageous in industries The highactivity of the epoxy formulation is highly recommended tobe used for immobilization of other enzymesproteins andordrug delivery systems

Abbreviations

Carr CarrageenanCarr-Ch Carrageenan-chitosanCarr-Ch-Epo Carrageenan-chitosan-epoxyCarr-Ch-Epo-Ch Carrageenan-chitosan-epoxy-

chitosanCarr-Ch-Epo-Ch-Epo Carrageenan-chitosan-epoxy-

chitosan-epoxy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q Husain ldquo120573-Galactosidases and their potential applications areviewrdquo Critical Reviews in Biotechnology vol 30 no 1 pp 41ndash62 2010

[2] MM Elnashar G E Awad M E Hassan M S Mohy Eldin BM Haroun and A I El-Diwany ldquoOptimal immobilization of120573-galactosidase onto 120581-Carrageenan gel beads using responsesurface methodology and its applicationsrdquoThe Scientific WorldJournal vol 2014 Article ID 571682 7 pages 2014

[3] M M M Elnashar and M A Yassin ldquoLactose hydrolysisby 120573-galactosidase covalently immobilized to thermally stablebiopolymersrdquo Applied Biochemistry and Biotechnology vol 159no 2 pp 426ndash437 2009

BioMed Research International 9

[4] G F Bickerstaf ldquoImpact of genetic technology on enzymetechnologyrdquo The Genetic Engineer and Biotechnologist vol 15pp 13ndash30 1995

[5] M M Elnashar ldquoThe art of immobilization using biopoly-mers biomaterials and nanobiotechnologyrdquo in Biotechnology ofBiopolymers pp 1ndash30 Intech 2011

[6] G Roberts ldquoStructure of chitin and chitosanrdquo in Chitin Chem-istry G A F Roberts Ed pp 1ndash53 MacMillan HoundmillsUK 1992

[7] R Hejazi andM Amiji ldquoChitosan-based gastrointestinal deliv-ery systemsrdquo Journal of Controlled Release vol 89 no 2 pp 151ndash165 2003

[8] B Krajewska ldquoApplication of chitin- and chitosan-based mate-rials for enzyme immobilizations a reviewrdquo Enzyme andMicro-bial Technology vol 35 no 2-3 pp 126ndash139 2004

[9] M M Elnashar O A Ali and H A Ragaa ldquoImmobilizedpenicillin G acylase onto grafted k-carrageenan hypothesis onthe effect of pH on the gel-enzyme interactionrdquoArabian Journalof Chemistry In press

[10] K C Chao M M Haugen and G P Royer ldquoStabiliza-tion of kappa-carrageenan gel with polymeric amines useof immobilized cells as biocatalysts at elevated temperaturesrdquoBiotechnology and Bioengineering vol 28 no 9 pp 1289ndash12931986

[11] J S Chang C Chou and S Y Chen ldquoDecolorization of azo dyeswith immobilized Pseudomonas luteolardquo Process Biochemistryvol 36 no 8-9 pp 757ndash763 2001

[12] S H Moon and S J Parulekar ldquoCharacterization of 120581-carrageenan gels used for immobilization of Bacillus firmusrdquoBiotechnology Progress vol 7 no 6 pp 516ndash525 1991

[13] E N Danial M M M Elnashar and G E A Awad ldquoImmobi-lized inulinase on grafted alginate beads prepared by the one-step and the two-steps methodsrdquo Industrial and EngineeringChemistry Research vol 49 no 7 pp 3120ndash3125 2010

[14] D K Boadi and R J Neufeld ldquoEncapsulation of tannase for thehydrolysis of tea tanninsrdquo Enzyme and Microbial Technologyvol 28 no 7-8 pp 590ndash595 2001

[15] A M Eberhardt V Pedroni M Volpe and M L FerreiraldquoImmobilization of catalase from Aspergillus niger on inorganicand biopolymeric supports for H

2O2decompositionrdquo Applied

Catalysis B Environmental vol 47 no 3 pp 153ndash163 2004[16] M M Elnashar ldquoReview article immobilized molecules using

biomaterials and nanobiotechnologyrdquo Journal of Biomaterialsand Nanobiotechnology vol 1 pp 61ndash76 2010

[17] EMagnan I Catarino D Paolucci-Jeanjean L Preziosi-Belloyand M P Belleville ldquoImmobilization of lipase on a ceramicmembrane activity and stabilityrdquo Journal of Membrane Sciencevol 241 no 1 pp 161ndash166 2004

[18] S Rocchietti A S V Urrutia M Pregnolato et al ldquoInfluenceof the enzyme derivative preparation and substrate structureon the enantioselectivity of penicillin G acylaserdquo Enzyme andMicrobial Technology vol 31 no 1-2 pp 88ndash93 2002

[19] MMM Elnashar andM A Yassin ldquoCovalent immobilizationof 120573-galactosidase on carrageenan coated with chitosanrdquo Jour-nal of Applied Polymer Science vol 114 no 1 pp 17ndash24 2009

[20] A A El-Sanabary M M Elnashar A A Magda and B MBadran ldquoPreparation and evaluation of some new corrosioninhibitors in varnishesrdquo Anti-Corrosion Methods and Materialsvol 48 no 1 pp 47ndash58 2001

[21] C Tapia Z Escobar E Costa et al ldquoComparative studies onpolyelectrolyte complexes and mixtures of chitosan-alginate

and chitosan-carrageenan as prolonged diltiazem clorhydraterelease systemsrdquo European Journal of Pharmaceutics and Bio-pharmaceutics vol 57 no 1 pp 65ndash75 2004

[22] W J Sung and Y H Bae ldquoA glucose oxidase electrode basedonpolypyrrolewith polyanionPEGenzyme conjugate dopantrdquoBiosensors and Bioelectronics vol 18 no 10 pp 1231ndash1239 2003

[23] I Gancarz J Bryjak M Bryjak G Pozniak and W TylusldquoPlasma modified polymers as a support for enzyme immobi-lization 1 Allyl alcohol plasmardquo European Polymer Journal vol39 no 8 pp 1615ndash1622 2003

[24] MMM ElnasharM A Yassin and T Kahil ldquoNovel thermallyandmechanically stable hydrogel for enzyme immobilization ofpenicillin G acylase via covalent techniquerdquo Journal of AppliedPolymer Science vol 109 no 6 pp 4105ndash4111 2008

[25] A Tanriseven and S Dogan ldquoA novel method for the immobi-lization of 120573-galactosidaserdquo Process Biochemistry vol 38 no 1pp 27ndash30 2002

[26] Q Z K Zhou and X Dong Chen ldquoImmobilization of 120573-galactosidase on graphite surface by glutaraldehyderdquo Journal ofFood Engineering vol 48 no 1 pp 69ndash74 2001

[27] B Chen J Hu E M Miller W Xie M Cai and R AGross ldquoCandida antarctica Lipase B chemically immobilized onepoxy-activated micro- and nanobeads catalysts for polyestersynthesisrdquo Biomacromolecules vol 9 no 2 pp 463ndash471 2008

[28] K Nakane T Ogihara N Ogata and Y Kurokawa ldquoEntrap-immobilization of invertase on composite gel fiber of celluloseacetate and zirconium alkoxide by sol-gel processrdquo Journal ofApplied Polymer Science vol 81 no 9 pp 2084ndash2088 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

The intensity of the color produced is directly propor-tional to the glucose concentration in the sample The assaywas performed by mixing 30 120583L of a sample of unknownconcentration and 3mL of Trinder reagent the reaction wasallowed to proceed for 20min at room temperature and theabsorbance of the unknown concentrationwas read at 510 nm[19]

23 Preparation of Gel Beads 120581-Carrageenan gel was pre-pared by dissolving 25 (wv) carrageenan in distilled waterat 70∘C using an overhead mechanical stirrer until completedissolution had occurred The Carrageenan gel solution wasdropped through a nozzle of 300 120583m using the InnotechEncapsulator in a hardening solution of 03M KCl Thenbeads were hardened using 03M KCl for 3 h

24Modification of Gel BeadsUsingChitosan andEpoxy Twoactivated epoxy hydrogels were prepared the short and thelong chain as follows

(a) TheShort Chain Carr-Ch-Epo beads were soaked ina solution of 075 chitosan previously prepared in 1(vv) acetic acidThen they were suspended in 75mL05M NaOH containing 150mg sodium cyanoboro-hydride under stirring Slowly 75mL 1 4-butanedioldiglycidyl ether was added with constant stirring andthe reaction was left at room temperature overnightFinally the activated gel beads were extensivelywashed with water to remove excess reagent

(b) TheLong Chain Carr-Ch-Epo-Ch-Epo formula (a)was furthermodified with chitosan and then epoxy asshown above

25 Elucidation of the Modified Gel Using Fourier Trans-form Infrared Spectroscopy The infrared spectra of all for-mulations were recorded with Fourier transform infraredspectroscopy (FTIR-8300 Shimadzu Japan) FTIR spectrawere taken in the wavelength region 4000 to 400 cmminus1 atambient temperature The FTIR spectrophotometer (FTIR-8300 Shimadzu Japan) was used to prove the presenceof the new functional group epoxy groups in both formsof the modified gels Five samples were used for this testcarrageenan (Carr) carrageenan coatedwith chitosan (Carr-Ch) carrageenan coated with chitosan followed by epoxy(short chain epoxy activated hydrogel Carr-Ch-Epo) car-rageenan coated with chitosan followed by epoxy followedby chitosan (Carr-Ch-Epo-Ch) and finally carrageenancoated with chitosan followed by epoxy followed by chitosanfollowed by epoxy (long chain epoxy activated hydrogelCarr-Ch-Epo-Ch-Epo)

A total of 2 (ww) of the sample with respect to thepotassium bromide (KBr S D Fine Chem Ltd) disk wasmixed with dry KBr The mixture was ground into a finepowder using an agatemortar before it was compressed into aKBr disk under a hydraulic press at 10000 psi Each KBr diskwas scanned 16 times at 4mms at a resolution of 2 cmminus1 overa wavenumber range of 400ndash4000 cmminus1 using Happ-Genzelapodization

26 Differential Scanning Calorimetry andThermal Gravimet-ric Analysis Differential scanning calorimetry (DSC) andthermal gravimetric analysis (TGAA) were performed toprove the formation of a strong polyelectrolyte complexbetween carrageenan and chitosan followed by di-epoxyThethermal behavior of five gel formulations was performedCarr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo The differential scanning calorimetry wasstudied using DSC (SDT 600 TA Instruments USA)Approximately 3 to 6mg of the dried gels was weighed intoan alumina panThe samples were heated from40∘C to 340∘Cat a heating rate of 10∘Cmin The thermal behavior of thedifferent gel formulations was characterized for their TGA(SDT 600 TA Instruments USA) Alumina pans were usedand approximately 3 to 6mg of the dried gels were weighedThe samples were heated from 50 to 300∘C at a heating rateof 10∘Cmin

27 Immobilization of 120573-galactosidase 120573-Galactosidase wasimmobilized onto the short and long chain of the epoxyactivated hydrogels as follows One gram of the activatedgel beads was washed thoroughly with distilled water andwas incubated into 10mL of enzyme solution (30UmL)prepared in 100mM citrate-phosphate buffer at pH 45 for16 h The immobilized enzyme was washed thoroughly withthe buffer solution containing Tris-HCl to block any freealdehyde group and to remove any unbound enzyme Theimmobilized enzyme was stored at 4∘C for further mea-surements Two parameters were used to reach the enzymersquosmaximum loading capacity (ELC) and epoxy activated longand short chains as well as the enzyme concentration

The ELC or the amount of enzymesrsquo units immobilizedonto and into gel beads was calculated as follows

ELC =(119872119900minus119872119891)

119882 (4)

where119872119900is the initial enzyme activity (U)119872

119891is the enzyme

activity of the filtrate (U) after immobilization and119882 is theweight of gel beads (g)

28 Optimization and Evaluation of the Free and Immobilized120573-Galactosidase

281 Temperature and pH Profiles for the Free andImmobilized 120573-Galactosidase The free and immobilized 120573-galactosidases were incubated into 10mL of 200mM lactoseat temperatures from 30∘C to 70∘C for 3 hrs The enzymeactivity has been determined according to Section 22 Theoptimum temperature has been chosen to study the effect ofpH where the free and immobilized 120573-galactosidases wereincubated into 10mL of 200mM lactose at pH 30ndash90 at37∘C for 3 hrs

282 119870119898

and 119881max of the Free and Immobilized 120573-Galactosidase The Michaelis-Menten kinetic models ade-quate for the description of the hydrolysis of lactose by thefree and the immobilized enzyme apparent 119870

119898and 119881max

4 BioMed Research International

(a)

(b)

(c)

ChitosanCarrageenan Carr Ch

NH2 NH2

+++ +

+

minusminusminusminusminusminusminusminus

minusminusminusminusminus

minusminusminusminusminusminusminusminus

minusminusminusminusminus

14-Butanediol diglycidyl etherCarr Ch Epoxy activated carrageenan

NH2 ndashO(CH2)4OCH2CH2OO+

+

+minusminusminusminusminusminusminusminus

minusminusminusminusminus

ndashO(CH2)4OCH2O

NHndashCH2ndashCHndashCH2

++minusminusminusminusminusminusminusminus

minusminusminusminusminus

NHndash ndashCHOH

EnzymeEpoxy activated Carr

NHndashCH2ndashCH CHndashCH ndashNHndashEndashSHOH

H2NndashEndashSH

CarrndashChndashEpondashenzyme

CH2 CH2

ndash ndash

O +++

minusminusminusminusminusminusminusminus

minusminusminusminusminus

++

minusminusminusminusminusminusminusminus

minusminusminusminusminus

Scheme 1 Grafted carrageenan gel beads with epoxy groups and immobilization of enzymes (a) Modification of carrageenan beads withchitosan via ionic interaction (b) Incorporation of epoxy groups to the carrageenan-chitosan (c) Immobilization of enzymes to the graftedcarrageenan-epoxy via ndashSH ndashOH or ndashNH

2

of free and immobilized 120573-galactosidase were determinedfor lactose using the Hanes-Woolf plot method Free andimmobilized 120573-galactosidases were incubated into 10mL of25 to 200mM at 37∘C and pH 45 for 3 hrs under standardassay conditions

283 Lactose Hydrolysis Using Immobilized 120573-GalactosidaseTo evaluate the efficiency of the immobilized enzyme it wasused for lactose hydrolysis using the optimum conditionsobtained from above optimization for the free and immobi-lized enzymes

3 Results and Discussion

31 GraftedAlginate Elucidation Structure Protonated aminogroups (ndashNH

3

+) of Ch formed a polyelectrolyte complexwiththe ndashOSO

3

minus of the Carr gel and incorporated free aminogroups to Carr [19] The free amino groups (ndashNH

2) of Ch

were used to covalently immobilize 120573-galactosidase via thedi-epoxy groups as a mediator beside its main role as a cross-linkerThe enzyme could be bound to the carrierrsquos free epoxygroups via its ndashSH ndashOH and ndashNH groups However asshown in Scheme 1 we represented that one uses the free ndashNH groups as an example to follow the ndashOH and ndashSH groups

The FTIR bands of Carr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo were shown in Figure 1Spectrums of the three compounds Carr Carr-Ch Carr-Ch-Epo revealed a new and strong band at 870 cmminus1 whichappears only for the modified gel spectrum with epoxygroupsThis band proved the presence of a new gel functionalgroup epoxy group which is in agreement with the authorrsquosprevious work [20] This band disappeared after treatment

75015002250300037504500

T (

)

3460

30

1120

64

3479

58

1126

43

1066

64

3458

37 11

110

0

3435

22 11

225

710

666

4

3419

79

1112

93

(cmminus1)CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 1 FTIR of five formulations of carrageenan and modifiedcarrageenan with chitosan and epoxy groups Carr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo

of the Carr-Ch-Epo with Ch and reappeared after furthertreatment with epoxy (Carr-Ch-Epo-Ch-Epo) at 880 cmminus1The FTIR bands also revealed a decrease in intensity and a

BioMed Research International 5

Table 1 Values ofDSC andTGAA thermograms of carrageenan andmodified carrageenan with chitosan and epoxy groups

Number Formula DSC temp ∘C TGA temp ∘C1 Carr 220 2002 Carr-Ch 230 2103 Carr-Ch-Epo 250 amp 320 2504 Carr-Ch-Epo-Ch 260 2405 Carr-Ch-Epo-Ch-Epo 260 amp 330 270

0123456789

10

0 50 100 150 200 250 300 350

Hea

t flow

exo

up

(mW

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 2 DSC thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

shift of the ndashOSO3

minus absorption band of Carr from 1446 cmminus1to 1390 cmminus1after reaction with the chitosan This ionicinteraction between the carrageenan and the chitosan evi-denced the formation of strong polyelectrolyte complexes[21]

Table 1 is tabulating the main characteristic values of theDSC and TGAA thermograms as shown in Figures 2 and 3respectively

The treatment of carrageenan with chitosan and epoxyhas shown gradual and obvious improvement in their DSCand TGA The DSC exothermic effect has been shifted tohigher temperatures from formula numbers 1ndash5 that is from220∘C to 320∘C The Carr has shown an exothermic band at220∘C which has been shifted to 230∘C after treatment withCh This improvement could be attributed to the formationof a complex network between the Carr and the Ch Similarbehavior has been attained when the di-epoxy has beensubstituted with glutaraldehyde [3] Further treatment withdi-epoxy Carr-Ch-Epo gradually increased the temperatureto two peaks at 250 and 230∘C This could be attributed toextra crosslinking with the di-epoxy and the two bands couldbe referred to the Ch and Epo however we could not tell atthis stage which is which However by addition of Ch Carr-Ch-Epo-Ch the temperature increased to a single band at260∘C which should be regarded to the Ch That means thatfor the short chain Carr-Ch-Epo the two peaks should be forCh and Epo respectively Finally by adding more di-epoxyCarr-Ch-Epo-Ch-Epo (long chain) two bands appeared for

60

65

70

75

80

85

90

95

100

0 50 100 150 200 250 300 350

Wei

ght (

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 3 TGA thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

the Ch and Epo at the highest temperatures 260 and 330∘Crespectively

On the other hand the TGA of the modified Carrformula numbers 2-5 showed a better stability against degra-dation as shown in Figure 3 andTable 1 For example unmod-ified Carr had a sudden decomposition at 200∘C This valuehas been increased to 210∘C after its treatment with Ch withretention of the sudden decomposition behavior The gelsrsquothermal improvement could be explained by the formation ofpolyelectrolyte interaction between the polyanions (ndashOSO

3

minus)of the Carr and the polycations (NH

3

+) of the Ch Furtherhardening of the gel beads using Epo showed a much higherincrease in the TGAA Carr-Ch-Epo (short chain) to 250∘Cand the decomposition behavior was slower and gradualThe improvement in the TGA could be attributed to extracrosslinking between the free Chrsquos amino groups and theEpo Further treatment of the short chain with Ch does notincrease its TGA however it was more or less the same at240∘C Finally for the long chain Carr-Ch-Epo-Ch-Epo theTGA increased to its maximum of 270∘C which could beregarded to further crosslinking It is worth noting that theshort and long chains have the highest TGA values of 250 and270∘Cwith slower and gradual decomposition rate comparedto other formulations

32 Optimization of Enzymersquos Loading Capacity Two factorshave been studied to optimize the loading capacity of 120573-galactosidases onto treated carrageenan gel beads

321 Effect of Epoxy Chain Length In this experiment shortchain of epoxy modified gel beads Carr-Ch-Epo and longchain Carr-Ch-Epo-Ch-Epo were examined to assess chainlength efficiency for immobilization of the enzyme Accord-ing to Sung and Bae 2003 [22] the effect of the chain lengthcould have a positive effect on the immobilization loadingefficiency till certain length and then it declines afterwards Inour case the short chain immobilizedmore enzymes than thelong chain Results as shown in Figure 4 showed that the short

6 BioMed Research International

0

5

10

15

20

25238

182

(Ug

bea

ds)

CarrndashChndashEpo CarrndashChndashEpondashChndashEpo

Figure 4 Effect of epoxy chain length on 120573-galactosidase loadingcapacity

chain immobilized 238 Ug gel beds compared to 182 Uggel beads for the long chain These results are in accordancewith that of Gancarz et al 2003 [23] who observed that anincrease in surface epoxy groups led to an increase in quantityof immobilized enzyme but a decrease in retained enzymeactivity

To understand this phenomenon we calculated from thesupernatants the expected amounts of immobilized enzymeson the short and long chains formulas and they were foundto be 18 and 21Ug respectively These results were in favorof the long chain formula however the retained activity ofthe immobilized enzymes was in favor of the short chainwhere 238Ug were immobilized showing 133 retention ofactivity This increase in the enzyme activity after immobi-lization could be regarded to the hydrogen bond interactionsbetween the modified gel (polysaccharide) containing ndashOHndashOSO

3H ndashC=O ndashNH

2 and the lactose substrate containing

ndashOH ndashC=O groups These H-bonding interactions couldalso increase the lactose concentration surrounding the gelsurface more than the bulk solution and thus the activity ofthe immobilized enzyme increases till reaching saturation ofthe gel surface with lactase [24]

On the other hand the long chain formulation wasexpected to immobilize 21Ug and in practice it showedonly 182Ug which revealed 86 retention of activity ofthe immobilized enzyme This could be regarded to the longchain havingmultipoint attachment orandmultilayers of theimmobilized enzymesteric hindrance that resulted in lossof the enzymersquos 3D structure and consequently its activityAccordingly for further experiments the short chain wasused

322 Effect of 120573-Galactosidase Concentration 120573-Galacto-sidase was immobilized onto gel beads treated with shortchain epoxy activated carrageenan Carr-Ch-Epo as shownin Figure 5

Results revealed that by increasing the concentrationof 120573-galactosidase from 10U to 60U the ELC increasedgradually till it reached its maximum of 36Ug gel beadsusing 50U of free enzyme after which any more addedenzyme has almost no effect on the ELC This could beregarded to all free epoxy groups that have been engagedwith the enzymes [25] However we have chosen for furtheroptimization the ELC of 36Ug gel beads as it shows better

05

10152025303540

10 20 30 40 50 60

(Ug)

8958854327 1239676625 2492457653 3101527564 3602115799 362855251

(Ug

bea

ds)

Figure 5 pH profile of the free and immobilized 120573-galactosidase

0

20

40

60

80

100

120

20 30 40 50 60 70 80

Rela

tive a

ctiv

ity (

)

FreeImmo

Temperature (∘C)

Figure 6 Michaelis constants of free and immobilized 120573-galactosidase

enzyme loading efficiency of 49 which is more economicas it saves unloaded enzyme from being wasted

33 Evaluation of Catalytic Activity of Free and Immobilized120573-Galactosidase At this stage five experiments were studiedFirstly the optimum reaction temperature pH and substrateconcentrations were examined for both the free and immo-bilized enzyme Secondly the best results from the first stepwere used to obtain themaximum substrate hydrolysis as wellas the operational stability of the immobilized enzyme

331 Optimum Temperature for the Free and Immobilized 120573-Galactosidase The optimum temperature for the free andimmobilized enzyme was examined Results as shown inFigure 6 revealed that the optimum temperature for theimmobilized enzyme was found to be at a slightly highertemperature (37ndash40∘C) compared to the free enzyme (30ndash37∘C)

The shift of the optimum temperature towards highertemperatures when the biocatalyst is immobilized indicatesthat the enzyme structure is strengthened by the immobiliza-tion process and the formation of a molecular cage aroundthe protein molecule (enzyme) was found to enhance theenzyme thermal stability The increase of the immobilized

BioMed Research International 7

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10

Rela

tive a

ctiv

ity (

)

pH

ImmoFree

Figure 7 Lactose hydrolysis using free and immobilized 120573-galactosidase

enzyme temperature tolerance may also be due to diffusionaleffects where the reaction velocity is more likely to bediffusion limited so that improvements in thermal diffusionwould correspondingly result in proportionally higher reac-tion rates [3]

332 pH Profile Figure 7 illustrates the pH activity profile ofthe free and immobilized 120573-galactosidase The optimum pHvalues for free and immobilized enzyme were 45ndash5 and 4-6 respectively which showed that the immobilized enzymewas more stable at higher and wider range of pH [25] Theseproperties could be very useful for lactolysis in sweet wheypermeate which has a pH range of 55ndash6 Moreover at pH4 the immobilized enzyme retained more than 95 of itsrelative activity compared to only 56 for the free enzyme

The shift in the pH activity profile of the immobilized 120573-galactosidase and the better pH stability may be attributedto the partition effects that were arising from differentconcentrations of charged species in the microenvironmentof the immobilized enzyme and in the domain of the bulksolution [3]

333 Determination of Kinetic Parameters of Free and Immo-bilized 120573-Galactosidase The kinetic constants of free andimmobilized 120573-galactosidase as shown in Figure 8 weretabulated in Table 2

The apparent 119870119898

after immobilization 1314mM ishigher than that of the free enzyme 589mMwhich indicatesthat a higher concentration of substrate 2-fold is needed forthe immobilized enzyme compared to the free enzyme Nev-ertheless higher 119870

119898values for immobilized 120573-galactosidase

have been reported by other authors with increases from 12-fold up to 54-fold [26] These results are most likely dueto the fact that the immobilized enzyme surfaces are notaccessible to all the reacting species However no substrate orproduct inhibition by the increase of substrate concentrationup to 200mM could be observed during our experiment as

020406080

100120140160180

minus150 minus100 minus50 0 50 100 150 200 250[S] (mM)

ImmoFree

y = 0481x + 63219

R2 = 09332

y = 05566x + 32786

R2 = 09889

[S]V

Figure 8 Reusability of the immobilized 120573-galactosidase

Table 2 Michaelis-Menten constants and maximal reaction ratevalues for free and immobilized lactase

120573-Galactosidase form Kinetic constants119870119898(mM) 119881max (120583molsdotminminus1)

Free 589 327Immobilized 1314 632

shown by the straight line of the Hanes-Wolf representation(Figure 8)

On the other hand the maximum reaction velocity119881maxvalues for the immobilized enzyme were remarkable it wasfound to double that of the free enzyme that is it increasedfrom 327 to 632 120583molsdotminminus1This result is in agreement withthe speculation that the improvement in the immobilizedenzyme thermal stability as in Section 331 could result in ahigher reaction velocity It is worth noting that the increasein the reaction velocity is generally favored in industries

334 Lactose Hydrolysis Using Free and Immobilized 120573-Galactosidase This experiment has been carried out so thatthe immobilized enzyme could attain its maximum efficiencyand act with its highest velocity using almost double the 119870

119898

concentration of substrate and using the enzymersquos optimumconditions A high concentration of substrate 200mM atpH 45 and 37∘C was used in this study as this enzymewas supposed to be suitable for hydrolysis of higher lactoseconcentrations found in mammal milk (88ndash234mM lactose)and whey permeate (85 lactose) [25]

The results as shown in Figure 9 revealed that for the firsthour the rate of conversion of the free enzyme was higherthan that of the immobilized one This could be attributedto the fact that the gel needed longer time to reach itsmaximum swelling This swelling will allow more substratesto penetrate into the pores and consequently decrease thediffusion limitation However at 90min both enzyme formsfollowed the same trend and the same speed till they reachedmaximum relative conversion at 120min It is worth notingthat at 90min the gel beads carrying the enzyme reached its

8 BioMed Research International

0

20

40

60

80

100

120

0 50 100 150 200 250

Hyd

roly

sis (

)

Time (min)

ImmoFree

Figure 9 Effect of 120573-galactosidase concentration on the enzymersquosloading capacity

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Rela

tive a

ctiv

ity (

)

Cycle number

Figure 10 Temperature profile of free and immobilized enzyme

maximum swelling overcame its substrateproduct diffusionlimitation and followed the same trend as the free enzymewhich is advantageous in industries That means that thesmall gel beads used in this work could overcome the problemthe authors previously had when they used big gel disks asthe enzyme suffered fromdiffusion limitation and hydrolyzedonly 63 of the free enzyme [3]

335 Reusability of Immobilized Enzyme To evaluate thereusability of the immobilized enzyme the beadswere soakedin 200mM lactose for 120min till full conversion of lactoseto glucose and galactose The gel beads were removed fromthe product washed with buffer solution after use and thenresuspended in a fresh aliquot of a substrate to measure theenzymatic activity

This procedure was repeated until the enzyme lost itsactivity The turn over number of the enzyme catalyzed pro-cess was calculated As shown in Figure 10 the immobilizedenzyme retained 60 of its relative activity by the seconduse and 21 by the 3rd use Nevertheless these results werein agreement with those obtained by other authors usingthe commercial carrier Novozym 435 as the immobilizedactivity decreased to 23 after the second use and to 37 by

the third use [27] The loss in activity was attributed by otherauthors to inactivation of enzyme due to continuous use [28]Although our carrier has shown better performance than thatof Novozyme 435 we think that the modified gel with epoxycould be further modified for future work

4 Conclusion

Novel biopolymer based on epoxy activated carrageenan wasprepared for immobilization of lactase as an example ofmedical enzyme The results were compared to those of ourprevious work using aldehyde activated carrageenan Theepoxy formula showed far better immobilization efficiencythat was triple that shown using the aldehyde one Thatcould be regarded to that the epoxy group is more activethan the aldehyde group The aldehyde group could onlybind to the enzymersquos free amino groups whereas the epoxygroup could bind to three groups ndashSH ndashNH

2 and ndashOH

The results showed in Figure 9 hydrolysis of lactose usingfree and immobilized lactase revealed that the immobilizedenzyme could attain its maximum efficiency and act withits highest velocity as fast as the free enzyme That wasregarded to the gel beads carrying the enzyme that reachedits maximum swelling and overcame its substrateproductdiffusion limitation and followed the same trend as thefree enzyme which is advantageous in industries The highactivity of the epoxy formulation is highly recommended tobe used for immobilization of other enzymesproteins andordrug delivery systems

Abbreviations

Carr CarrageenanCarr-Ch Carrageenan-chitosanCarr-Ch-Epo Carrageenan-chitosan-epoxyCarr-Ch-Epo-Ch Carrageenan-chitosan-epoxy-

chitosanCarr-Ch-Epo-Ch-Epo Carrageenan-chitosan-epoxy-

chitosan-epoxy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q Husain ldquo120573-Galactosidases and their potential applications areviewrdquo Critical Reviews in Biotechnology vol 30 no 1 pp 41ndash62 2010

[2] MM Elnashar G E Awad M E Hassan M S Mohy Eldin BM Haroun and A I El-Diwany ldquoOptimal immobilization of120573-galactosidase onto 120581-Carrageenan gel beads using responsesurface methodology and its applicationsrdquoThe Scientific WorldJournal vol 2014 Article ID 571682 7 pages 2014

[3] M M M Elnashar and M A Yassin ldquoLactose hydrolysisby 120573-galactosidase covalently immobilized to thermally stablebiopolymersrdquo Applied Biochemistry and Biotechnology vol 159no 2 pp 426ndash437 2009

BioMed Research International 9

[4] G F Bickerstaf ldquoImpact of genetic technology on enzymetechnologyrdquo The Genetic Engineer and Biotechnologist vol 15pp 13ndash30 1995

[5] M M Elnashar ldquoThe art of immobilization using biopoly-mers biomaterials and nanobiotechnologyrdquo in Biotechnology ofBiopolymers pp 1ndash30 Intech 2011

[6] G Roberts ldquoStructure of chitin and chitosanrdquo in Chitin Chem-istry G A F Roberts Ed pp 1ndash53 MacMillan HoundmillsUK 1992

[7] R Hejazi andM Amiji ldquoChitosan-based gastrointestinal deliv-ery systemsrdquo Journal of Controlled Release vol 89 no 2 pp 151ndash165 2003

[8] B Krajewska ldquoApplication of chitin- and chitosan-based mate-rials for enzyme immobilizations a reviewrdquo Enzyme andMicro-bial Technology vol 35 no 2-3 pp 126ndash139 2004

[9] M M Elnashar O A Ali and H A Ragaa ldquoImmobilizedpenicillin G acylase onto grafted k-carrageenan hypothesis onthe effect of pH on the gel-enzyme interactionrdquoArabian Journalof Chemistry In press

[10] K C Chao M M Haugen and G P Royer ldquoStabiliza-tion of kappa-carrageenan gel with polymeric amines useof immobilized cells as biocatalysts at elevated temperaturesrdquoBiotechnology and Bioengineering vol 28 no 9 pp 1289ndash12931986

[11] J S Chang C Chou and S Y Chen ldquoDecolorization of azo dyeswith immobilized Pseudomonas luteolardquo Process Biochemistryvol 36 no 8-9 pp 757ndash763 2001

[12] S H Moon and S J Parulekar ldquoCharacterization of 120581-carrageenan gels used for immobilization of Bacillus firmusrdquoBiotechnology Progress vol 7 no 6 pp 516ndash525 1991

[13] E N Danial M M M Elnashar and G E A Awad ldquoImmobi-lized inulinase on grafted alginate beads prepared by the one-step and the two-steps methodsrdquo Industrial and EngineeringChemistry Research vol 49 no 7 pp 3120ndash3125 2010

[14] D K Boadi and R J Neufeld ldquoEncapsulation of tannase for thehydrolysis of tea tanninsrdquo Enzyme and Microbial Technologyvol 28 no 7-8 pp 590ndash595 2001

[15] A M Eberhardt V Pedroni M Volpe and M L FerreiraldquoImmobilization of catalase from Aspergillus niger on inorganicand biopolymeric supports for H

2O2decompositionrdquo Applied

Catalysis B Environmental vol 47 no 3 pp 153ndash163 2004[16] M M Elnashar ldquoReview article immobilized molecules using

biomaterials and nanobiotechnologyrdquo Journal of Biomaterialsand Nanobiotechnology vol 1 pp 61ndash76 2010

[17] EMagnan I Catarino D Paolucci-Jeanjean L Preziosi-Belloyand M P Belleville ldquoImmobilization of lipase on a ceramicmembrane activity and stabilityrdquo Journal of Membrane Sciencevol 241 no 1 pp 161ndash166 2004

[18] S Rocchietti A S V Urrutia M Pregnolato et al ldquoInfluenceof the enzyme derivative preparation and substrate structureon the enantioselectivity of penicillin G acylaserdquo Enzyme andMicrobial Technology vol 31 no 1-2 pp 88ndash93 2002

[19] MMM Elnashar andM A Yassin ldquoCovalent immobilizationof 120573-galactosidase on carrageenan coated with chitosanrdquo Jour-nal of Applied Polymer Science vol 114 no 1 pp 17ndash24 2009

[20] A A El-Sanabary M M Elnashar A A Magda and B MBadran ldquoPreparation and evaluation of some new corrosioninhibitors in varnishesrdquo Anti-Corrosion Methods and Materialsvol 48 no 1 pp 47ndash58 2001

[21] C Tapia Z Escobar E Costa et al ldquoComparative studies onpolyelectrolyte complexes and mixtures of chitosan-alginate

and chitosan-carrageenan as prolonged diltiazem clorhydraterelease systemsrdquo European Journal of Pharmaceutics and Bio-pharmaceutics vol 57 no 1 pp 65ndash75 2004

[22] W J Sung and Y H Bae ldquoA glucose oxidase electrode basedonpolypyrrolewith polyanionPEGenzyme conjugate dopantrdquoBiosensors and Bioelectronics vol 18 no 10 pp 1231ndash1239 2003

[23] I Gancarz J Bryjak M Bryjak G Pozniak and W TylusldquoPlasma modified polymers as a support for enzyme immobi-lization 1 Allyl alcohol plasmardquo European Polymer Journal vol39 no 8 pp 1615ndash1622 2003

[24] MMM ElnasharM A Yassin and T Kahil ldquoNovel thermallyandmechanically stable hydrogel for enzyme immobilization ofpenicillin G acylase via covalent techniquerdquo Journal of AppliedPolymer Science vol 109 no 6 pp 4105ndash4111 2008

[25] A Tanriseven and S Dogan ldquoA novel method for the immobi-lization of 120573-galactosidaserdquo Process Biochemistry vol 38 no 1pp 27ndash30 2002

[26] Q Z K Zhou and X Dong Chen ldquoImmobilization of 120573-galactosidase on graphite surface by glutaraldehyderdquo Journal ofFood Engineering vol 48 no 1 pp 69ndash74 2001

[27] B Chen J Hu E M Miller W Xie M Cai and R AGross ldquoCandida antarctica Lipase B chemically immobilized onepoxy-activated micro- and nanobeads catalysts for polyestersynthesisrdquo Biomacromolecules vol 9 no 2 pp 463ndash471 2008

[28] K Nakane T Ogihara N Ogata and Y Kurokawa ldquoEntrap-immobilization of invertase on composite gel fiber of celluloseacetate and zirconium alkoxide by sol-gel processrdquo Journal ofApplied Polymer Science vol 81 no 9 pp 2084ndash2088 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

(a)

(b)

(c)

ChitosanCarrageenan Carr Ch

NH2 NH2

+++ +

+

minusminusminusminusminusminusminusminus

minusminusminusminusminus

minusminusminusminusminusminusminusminus

minusminusminusminusminus

14-Butanediol diglycidyl etherCarr Ch Epoxy activated carrageenan

NH2 ndashO(CH2)4OCH2CH2OO+

+

+minusminusminusminusminusminusminusminus

minusminusminusminusminus

ndashO(CH2)4OCH2O

NHndashCH2ndashCHndashCH2

++minusminusminusminusminusminusminusminus

minusminusminusminusminus

NHndash ndashCHOH

EnzymeEpoxy activated Carr

NHndashCH2ndashCH CHndashCH ndashNHndashEndashSHOH

H2NndashEndashSH

CarrndashChndashEpondashenzyme

CH2 CH2

ndash ndash

O +++

minusminusminusminusminusminusminusminus

minusminusminusminusminus

++

minusminusminusminusminusminusminusminus

minusminusminusminusminus

Scheme 1 Grafted carrageenan gel beads with epoxy groups and immobilization of enzymes (a) Modification of carrageenan beads withchitosan via ionic interaction (b) Incorporation of epoxy groups to the carrageenan-chitosan (c) Immobilization of enzymes to the graftedcarrageenan-epoxy via ndashSH ndashOH or ndashNH

2

of free and immobilized 120573-galactosidase were determinedfor lactose using the Hanes-Woolf plot method Free andimmobilized 120573-galactosidases were incubated into 10mL of25 to 200mM at 37∘C and pH 45 for 3 hrs under standardassay conditions

283 Lactose Hydrolysis Using Immobilized 120573-GalactosidaseTo evaluate the efficiency of the immobilized enzyme it wasused for lactose hydrolysis using the optimum conditionsobtained from above optimization for the free and immobi-lized enzymes

3 Results and Discussion

31 GraftedAlginate Elucidation Structure Protonated aminogroups (ndashNH

3

+) of Ch formed a polyelectrolyte complexwiththe ndashOSO

3

minus of the Carr gel and incorporated free aminogroups to Carr [19] The free amino groups (ndashNH

2) of Ch

were used to covalently immobilize 120573-galactosidase via thedi-epoxy groups as a mediator beside its main role as a cross-linkerThe enzyme could be bound to the carrierrsquos free epoxygroups via its ndashSH ndashOH and ndashNH groups However asshown in Scheme 1 we represented that one uses the free ndashNH groups as an example to follow the ndashOH and ndashSH groups

The FTIR bands of Carr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo were shown in Figure 1Spectrums of the three compounds Carr Carr-Ch Carr-Ch-Epo revealed a new and strong band at 870 cmminus1 whichappears only for the modified gel spectrum with epoxygroupsThis band proved the presence of a new gel functionalgroup epoxy group which is in agreement with the authorrsquosprevious work [20] This band disappeared after treatment

75015002250300037504500

T (

)

3460

30

1120

64

3479

58

1126

43

1066

64

3458

37 11

110

0

3435

22 11

225

710

666

4

3419

79

1112

93

(cmminus1)CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 1 FTIR of five formulations of carrageenan and modifiedcarrageenan with chitosan and epoxy groups Carr Carr-Ch Carr-Ch-Epo Carr-Ch-Epo-Ch Carr-Ch-Epo-Ch-Epo

of the Carr-Ch-Epo with Ch and reappeared after furthertreatment with epoxy (Carr-Ch-Epo-Ch-Epo) at 880 cmminus1The FTIR bands also revealed a decrease in intensity and a

BioMed Research International 5

Table 1 Values ofDSC andTGAA thermograms of carrageenan andmodified carrageenan with chitosan and epoxy groups

Number Formula DSC temp ∘C TGA temp ∘C1 Carr 220 2002 Carr-Ch 230 2103 Carr-Ch-Epo 250 amp 320 2504 Carr-Ch-Epo-Ch 260 2405 Carr-Ch-Epo-Ch-Epo 260 amp 330 270

0123456789

10

0 50 100 150 200 250 300 350

Hea

t flow

exo

up

(mW

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 2 DSC thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

shift of the ndashOSO3

minus absorption band of Carr from 1446 cmminus1to 1390 cmminus1after reaction with the chitosan This ionicinteraction between the carrageenan and the chitosan evi-denced the formation of strong polyelectrolyte complexes[21]

Table 1 is tabulating the main characteristic values of theDSC and TGAA thermograms as shown in Figures 2 and 3respectively

The treatment of carrageenan with chitosan and epoxyhas shown gradual and obvious improvement in their DSCand TGA The DSC exothermic effect has been shifted tohigher temperatures from formula numbers 1ndash5 that is from220∘C to 320∘C The Carr has shown an exothermic band at220∘C which has been shifted to 230∘C after treatment withCh This improvement could be attributed to the formationof a complex network between the Carr and the Ch Similarbehavior has been attained when the di-epoxy has beensubstituted with glutaraldehyde [3] Further treatment withdi-epoxy Carr-Ch-Epo gradually increased the temperatureto two peaks at 250 and 230∘C This could be attributed toextra crosslinking with the di-epoxy and the two bands couldbe referred to the Ch and Epo however we could not tell atthis stage which is which However by addition of Ch Carr-Ch-Epo-Ch the temperature increased to a single band at260∘C which should be regarded to the Ch That means thatfor the short chain Carr-Ch-Epo the two peaks should be forCh and Epo respectively Finally by adding more di-epoxyCarr-Ch-Epo-Ch-Epo (long chain) two bands appeared for

60

65

70

75

80

85

90

95

100

0 50 100 150 200 250 300 350

Wei

ght (

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 3 TGA thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

the Ch and Epo at the highest temperatures 260 and 330∘Crespectively

On the other hand the TGA of the modified Carrformula numbers 2-5 showed a better stability against degra-dation as shown in Figure 3 andTable 1 For example unmod-ified Carr had a sudden decomposition at 200∘C This valuehas been increased to 210∘C after its treatment with Ch withretention of the sudden decomposition behavior The gelsrsquothermal improvement could be explained by the formation ofpolyelectrolyte interaction between the polyanions (ndashOSO

3

minus)of the Carr and the polycations (NH

3

+) of the Ch Furtherhardening of the gel beads using Epo showed a much higherincrease in the TGAA Carr-Ch-Epo (short chain) to 250∘Cand the decomposition behavior was slower and gradualThe improvement in the TGA could be attributed to extracrosslinking between the free Chrsquos amino groups and theEpo Further treatment of the short chain with Ch does notincrease its TGA however it was more or less the same at240∘C Finally for the long chain Carr-Ch-Epo-Ch-Epo theTGA increased to its maximum of 270∘C which could beregarded to further crosslinking It is worth noting that theshort and long chains have the highest TGA values of 250 and270∘Cwith slower and gradual decomposition rate comparedto other formulations

32 Optimization of Enzymersquos Loading Capacity Two factorshave been studied to optimize the loading capacity of 120573-galactosidases onto treated carrageenan gel beads

321 Effect of Epoxy Chain Length In this experiment shortchain of epoxy modified gel beads Carr-Ch-Epo and longchain Carr-Ch-Epo-Ch-Epo were examined to assess chainlength efficiency for immobilization of the enzyme Accord-ing to Sung and Bae 2003 [22] the effect of the chain lengthcould have a positive effect on the immobilization loadingefficiency till certain length and then it declines afterwards Inour case the short chain immobilizedmore enzymes than thelong chain Results as shown in Figure 4 showed that the short

6 BioMed Research International

0

5

10

15

20

25238

182

(Ug

bea

ds)

CarrndashChndashEpo CarrndashChndashEpondashChndashEpo

Figure 4 Effect of epoxy chain length on 120573-galactosidase loadingcapacity

chain immobilized 238 Ug gel beds compared to 182 Uggel beads for the long chain These results are in accordancewith that of Gancarz et al 2003 [23] who observed that anincrease in surface epoxy groups led to an increase in quantityof immobilized enzyme but a decrease in retained enzymeactivity

To understand this phenomenon we calculated from thesupernatants the expected amounts of immobilized enzymeson the short and long chains formulas and they were foundto be 18 and 21Ug respectively These results were in favorof the long chain formula however the retained activity ofthe immobilized enzymes was in favor of the short chainwhere 238Ug were immobilized showing 133 retention ofactivity This increase in the enzyme activity after immobi-lization could be regarded to the hydrogen bond interactionsbetween the modified gel (polysaccharide) containing ndashOHndashOSO

3H ndashC=O ndashNH

2 and the lactose substrate containing

ndashOH ndashC=O groups These H-bonding interactions couldalso increase the lactose concentration surrounding the gelsurface more than the bulk solution and thus the activity ofthe immobilized enzyme increases till reaching saturation ofthe gel surface with lactase [24]

On the other hand the long chain formulation wasexpected to immobilize 21Ug and in practice it showedonly 182Ug which revealed 86 retention of activity ofthe immobilized enzyme This could be regarded to the longchain havingmultipoint attachment orandmultilayers of theimmobilized enzymesteric hindrance that resulted in lossof the enzymersquos 3D structure and consequently its activityAccordingly for further experiments the short chain wasused

322 Effect of 120573-Galactosidase Concentration 120573-Galacto-sidase was immobilized onto gel beads treated with shortchain epoxy activated carrageenan Carr-Ch-Epo as shownin Figure 5

Results revealed that by increasing the concentrationof 120573-galactosidase from 10U to 60U the ELC increasedgradually till it reached its maximum of 36Ug gel beadsusing 50U of free enzyme after which any more addedenzyme has almost no effect on the ELC This could beregarded to all free epoxy groups that have been engagedwith the enzymes [25] However we have chosen for furtheroptimization the ELC of 36Ug gel beads as it shows better

05

10152025303540

10 20 30 40 50 60

(Ug)

8958854327 1239676625 2492457653 3101527564 3602115799 362855251

(Ug

bea

ds)

Figure 5 pH profile of the free and immobilized 120573-galactosidase

0

20

40

60

80

100

120

20 30 40 50 60 70 80

Rela

tive a

ctiv

ity (

)

FreeImmo

Temperature (∘C)

Figure 6 Michaelis constants of free and immobilized 120573-galactosidase

enzyme loading efficiency of 49 which is more economicas it saves unloaded enzyme from being wasted

33 Evaluation of Catalytic Activity of Free and Immobilized120573-Galactosidase At this stage five experiments were studiedFirstly the optimum reaction temperature pH and substrateconcentrations were examined for both the free and immo-bilized enzyme Secondly the best results from the first stepwere used to obtain themaximum substrate hydrolysis as wellas the operational stability of the immobilized enzyme

331 Optimum Temperature for the Free and Immobilized 120573-Galactosidase The optimum temperature for the free andimmobilized enzyme was examined Results as shown inFigure 6 revealed that the optimum temperature for theimmobilized enzyme was found to be at a slightly highertemperature (37ndash40∘C) compared to the free enzyme (30ndash37∘C)

The shift of the optimum temperature towards highertemperatures when the biocatalyst is immobilized indicatesthat the enzyme structure is strengthened by the immobiliza-tion process and the formation of a molecular cage aroundthe protein molecule (enzyme) was found to enhance theenzyme thermal stability The increase of the immobilized

BioMed Research International 7

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10

Rela

tive a

ctiv

ity (

)

pH

ImmoFree

Figure 7 Lactose hydrolysis using free and immobilized 120573-galactosidase

enzyme temperature tolerance may also be due to diffusionaleffects where the reaction velocity is more likely to bediffusion limited so that improvements in thermal diffusionwould correspondingly result in proportionally higher reac-tion rates [3]

332 pH Profile Figure 7 illustrates the pH activity profile ofthe free and immobilized 120573-galactosidase The optimum pHvalues for free and immobilized enzyme were 45ndash5 and 4-6 respectively which showed that the immobilized enzymewas more stable at higher and wider range of pH [25] Theseproperties could be very useful for lactolysis in sweet wheypermeate which has a pH range of 55ndash6 Moreover at pH4 the immobilized enzyme retained more than 95 of itsrelative activity compared to only 56 for the free enzyme

The shift in the pH activity profile of the immobilized 120573-galactosidase and the better pH stability may be attributedto the partition effects that were arising from differentconcentrations of charged species in the microenvironmentof the immobilized enzyme and in the domain of the bulksolution [3]

333 Determination of Kinetic Parameters of Free and Immo-bilized 120573-Galactosidase The kinetic constants of free andimmobilized 120573-galactosidase as shown in Figure 8 weretabulated in Table 2

The apparent 119870119898

after immobilization 1314mM ishigher than that of the free enzyme 589mMwhich indicatesthat a higher concentration of substrate 2-fold is needed forthe immobilized enzyme compared to the free enzyme Nev-ertheless higher 119870

119898values for immobilized 120573-galactosidase

have been reported by other authors with increases from 12-fold up to 54-fold [26] These results are most likely dueto the fact that the immobilized enzyme surfaces are notaccessible to all the reacting species However no substrate orproduct inhibition by the increase of substrate concentrationup to 200mM could be observed during our experiment as

020406080

100120140160180

minus150 minus100 minus50 0 50 100 150 200 250[S] (mM)

ImmoFree

y = 0481x + 63219

R2 = 09332

y = 05566x + 32786

R2 = 09889

[S]V

Figure 8 Reusability of the immobilized 120573-galactosidase

Table 2 Michaelis-Menten constants and maximal reaction ratevalues for free and immobilized lactase

120573-Galactosidase form Kinetic constants119870119898(mM) 119881max (120583molsdotminminus1)

Free 589 327Immobilized 1314 632

shown by the straight line of the Hanes-Wolf representation(Figure 8)

On the other hand the maximum reaction velocity119881maxvalues for the immobilized enzyme were remarkable it wasfound to double that of the free enzyme that is it increasedfrom 327 to 632 120583molsdotminminus1This result is in agreement withthe speculation that the improvement in the immobilizedenzyme thermal stability as in Section 331 could result in ahigher reaction velocity It is worth noting that the increasein the reaction velocity is generally favored in industries

334 Lactose Hydrolysis Using Free and Immobilized 120573-Galactosidase This experiment has been carried out so thatthe immobilized enzyme could attain its maximum efficiencyand act with its highest velocity using almost double the 119870

119898

concentration of substrate and using the enzymersquos optimumconditions A high concentration of substrate 200mM atpH 45 and 37∘C was used in this study as this enzymewas supposed to be suitable for hydrolysis of higher lactoseconcentrations found in mammal milk (88ndash234mM lactose)and whey permeate (85 lactose) [25]

The results as shown in Figure 9 revealed that for the firsthour the rate of conversion of the free enzyme was higherthan that of the immobilized one This could be attributedto the fact that the gel needed longer time to reach itsmaximum swelling This swelling will allow more substratesto penetrate into the pores and consequently decrease thediffusion limitation However at 90min both enzyme formsfollowed the same trend and the same speed till they reachedmaximum relative conversion at 120min It is worth notingthat at 90min the gel beads carrying the enzyme reached its

8 BioMed Research International

0

20

40

60

80

100

120

0 50 100 150 200 250

Hyd

roly

sis (

)

Time (min)

ImmoFree

Figure 9 Effect of 120573-galactosidase concentration on the enzymersquosloading capacity

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Rela

tive a

ctiv

ity (

)

Cycle number

Figure 10 Temperature profile of free and immobilized enzyme

maximum swelling overcame its substrateproduct diffusionlimitation and followed the same trend as the free enzymewhich is advantageous in industries That means that thesmall gel beads used in this work could overcome the problemthe authors previously had when they used big gel disks asthe enzyme suffered fromdiffusion limitation and hydrolyzedonly 63 of the free enzyme [3]

335 Reusability of Immobilized Enzyme To evaluate thereusability of the immobilized enzyme the beadswere soakedin 200mM lactose for 120min till full conversion of lactoseto glucose and galactose The gel beads were removed fromthe product washed with buffer solution after use and thenresuspended in a fresh aliquot of a substrate to measure theenzymatic activity

This procedure was repeated until the enzyme lost itsactivity The turn over number of the enzyme catalyzed pro-cess was calculated As shown in Figure 10 the immobilizedenzyme retained 60 of its relative activity by the seconduse and 21 by the 3rd use Nevertheless these results werein agreement with those obtained by other authors usingthe commercial carrier Novozym 435 as the immobilizedactivity decreased to 23 after the second use and to 37 by

the third use [27] The loss in activity was attributed by otherauthors to inactivation of enzyme due to continuous use [28]Although our carrier has shown better performance than thatof Novozyme 435 we think that the modified gel with epoxycould be further modified for future work

4 Conclusion

Novel biopolymer based on epoxy activated carrageenan wasprepared for immobilization of lactase as an example ofmedical enzyme The results were compared to those of ourprevious work using aldehyde activated carrageenan Theepoxy formula showed far better immobilization efficiencythat was triple that shown using the aldehyde one Thatcould be regarded to that the epoxy group is more activethan the aldehyde group The aldehyde group could onlybind to the enzymersquos free amino groups whereas the epoxygroup could bind to three groups ndashSH ndashNH

2 and ndashOH

The results showed in Figure 9 hydrolysis of lactose usingfree and immobilized lactase revealed that the immobilizedenzyme could attain its maximum efficiency and act withits highest velocity as fast as the free enzyme That wasregarded to the gel beads carrying the enzyme that reachedits maximum swelling and overcame its substrateproductdiffusion limitation and followed the same trend as thefree enzyme which is advantageous in industries The highactivity of the epoxy formulation is highly recommended tobe used for immobilization of other enzymesproteins andordrug delivery systems

Abbreviations

Carr CarrageenanCarr-Ch Carrageenan-chitosanCarr-Ch-Epo Carrageenan-chitosan-epoxyCarr-Ch-Epo-Ch Carrageenan-chitosan-epoxy-

chitosanCarr-Ch-Epo-Ch-Epo Carrageenan-chitosan-epoxy-

chitosan-epoxy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q Husain ldquo120573-Galactosidases and their potential applications areviewrdquo Critical Reviews in Biotechnology vol 30 no 1 pp 41ndash62 2010

[2] MM Elnashar G E Awad M E Hassan M S Mohy Eldin BM Haroun and A I El-Diwany ldquoOptimal immobilization of120573-galactosidase onto 120581-Carrageenan gel beads using responsesurface methodology and its applicationsrdquoThe Scientific WorldJournal vol 2014 Article ID 571682 7 pages 2014

[3] M M M Elnashar and M A Yassin ldquoLactose hydrolysisby 120573-galactosidase covalently immobilized to thermally stablebiopolymersrdquo Applied Biochemistry and Biotechnology vol 159no 2 pp 426ndash437 2009

BioMed Research International 9

[4] G F Bickerstaf ldquoImpact of genetic technology on enzymetechnologyrdquo The Genetic Engineer and Biotechnologist vol 15pp 13ndash30 1995

[5] M M Elnashar ldquoThe art of immobilization using biopoly-mers biomaterials and nanobiotechnologyrdquo in Biotechnology ofBiopolymers pp 1ndash30 Intech 2011

[6] G Roberts ldquoStructure of chitin and chitosanrdquo in Chitin Chem-istry G A F Roberts Ed pp 1ndash53 MacMillan HoundmillsUK 1992

[7] R Hejazi andM Amiji ldquoChitosan-based gastrointestinal deliv-ery systemsrdquo Journal of Controlled Release vol 89 no 2 pp 151ndash165 2003

[8] B Krajewska ldquoApplication of chitin- and chitosan-based mate-rials for enzyme immobilizations a reviewrdquo Enzyme andMicro-bial Technology vol 35 no 2-3 pp 126ndash139 2004

[9] M M Elnashar O A Ali and H A Ragaa ldquoImmobilizedpenicillin G acylase onto grafted k-carrageenan hypothesis onthe effect of pH on the gel-enzyme interactionrdquoArabian Journalof Chemistry In press

[10] K C Chao M M Haugen and G P Royer ldquoStabiliza-tion of kappa-carrageenan gel with polymeric amines useof immobilized cells as biocatalysts at elevated temperaturesrdquoBiotechnology and Bioengineering vol 28 no 9 pp 1289ndash12931986

[11] J S Chang C Chou and S Y Chen ldquoDecolorization of azo dyeswith immobilized Pseudomonas luteolardquo Process Biochemistryvol 36 no 8-9 pp 757ndash763 2001

[12] S H Moon and S J Parulekar ldquoCharacterization of 120581-carrageenan gels used for immobilization of Bacillus firmusrdquoBiotechnology Progress vol 7 no 6 pp 516ndash525 1991

[13] E N Danial M M M Elnashar and G E A Awad ldquoImmobi-lized inulinase on grafted alginate beads prepared by the one-step and the two-steps methodsrdquo Industrial and EngineeringChemistry Research vol 49 no 7 pp 3120ndash3125 2010

[14] D K Boadi and R J Neufeld ldquoEncapsulation of tannase for thehydrolysis of tea tanninsrdquo Enzyme and Microbial Technologyvol 28 no 7-8 pp 590ndash595 2001

[15] A M Eberhardt V Pedroni M Volpe and M L FerreiraldquoImmobilization of catalase from Aspergillus niger on inorganicand biopolymeric supports for H

2O2decompositionrdquo Applied

Catalysis B Environmental vol 47 no 3 pp 153ndash163 2004[16] M M Elnashar ldquoReview article immobilized molecules using

biomaterials and nanobiotechnologyrdquo Journal of Biomaterialsand Nanobiotechnology vol 1 pp 61ndash76 2010

[17] EMagnan I Catarino D Paolucci-Jeanjean L Preziosi-Belloyand M P Belleville ldquoImmobilization of lipase on a ceramicmembrane activity and stabilityrdquo Journal of Membrane Sciencevol 241 no 1 pp 161ndash166 2004

[18] S Rocchietti A S V Urrutia M Pregnolato et al ldquoInfluenceof the enzyme derivative preparation and substrate structureon the enantioselectivity of penicillin G acylaserdquo Enzyme andMicrobial Technology vol 31 no 1-2 pp 88ndash93 2002

[19] MMM Elnashar andM A Yassin ldquoCovalent immobilizationof 120573-galactosidase on carrageenan coated with chitosanrdquo Jour-nal of Applied Polymer Science vol 114 no 1 pp 17ndash24 2009

[20] A A El-Sanabary M M Elnashar A A Magda and B MBadran ldquoPreparation and evaluation of some new corrosioninhibitors in varnishesrdquo Anti-Corrosion Methods and Materialsvol 48 no 1 pp 47ndash58 2001

[21] C Tapia Z Escobar E Costa et al ldquoComparative studies onpolyelectrolyte complexes and mixtures of chitosan-alginate

and chitosan-carrageenan as prolonged diltiazem clorhydraterelease systemsrdquo European Journal of Pharmaceutics and Bio-pharmaceutics vol 57 no 1 pp 65ndash75 2004

[22] W J Sung and Y H Bae ldquoA glucose oxidase electrode basedonpolypyrrolewith polyanionPEGenzyme conjugate dopantrdquoBiosensors and Bioelectronics vol 18 no 10 pp 1231ndash1239 2003

[23] I Gancarz J Bryjak M Bryjak G Pozniak and W TylusldquoPlasma modified polymers as a support for enzyme immobi-lization 1 Allyl alcohol plasmardquo European Polymer Journal vol39 no 8 pp 1615ndash1622 2003

[24] MMM ElnasharM A Yassin and T Kahil ldquoNovel thermallyandmechanically stable hydrogel for enzyme immobilization ofpenicillin G acylase via covalent techniquerdquo Journal of AppliedPolymer Science vol 109 no 6 pp 4105ndash4111 2008

[25] A Tanriseven and S Dogan ldquoA novel method for the immobi-lization of 120573-galactosidaserdquo Process Biochemistry vol 38 no 1pp 27ndash30 2002

[26] Q Z K Zhou and X Dong Chen ldquoImmobilization of 120573-galactosidase on graphite surface by glutaraldehyderdquo Journal ofFood Engineering vol 48 no 1 pp 69ndash74 2001

[27] B Chen J Hu E M Miller W Xie M Cai and R AGross ldquoCandida antarctica Lipase B chemically immobilized onepoxy-activated micro- and nanobeads catalysts for polyestersynthesisrdquo Biomacromolecules vol 9 no 2 pp 463ndash471 2008

[28] K Nakane T Ogihara N Ogata and Y Kurokawa ldquoEntrap-immobilization of invertase on composite gel fiber of celluloseacetate and zirconium alkoxide by sol-gel processrdquo Journal ofApplied Polymer Science vol 81 no 9 pp 2084ndash2088 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Table 1 Values ofDSC andTGAA thermograms of carrageenan andmodified carrageenan with chitosan and epoxy groups

Number Formula DSC temp ∘C TGA temp ∘C1 Carr 220 2002 Carr-Ch 230 2103 Carr-Ch-Epo 250 amp 320 2504 Carr-Ch-Epo-Ch 260 2405 Carr-Ch-Epo-Ch-Epo 260 amp 330 270

0123456789

10

0 50 100 150 200 250 300 350

Hea

t flow

exo

up

(mW

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 2 DSC thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

shift of the ndashOSO3

minus absorption band of Carr from 1446 cmminus1to 1390 cmminus1after reaction with the chitosan This ionicinteraction between the carrageenan and the chitosan evi-denced the formation of strong polyelectrolyte complexes[21]

Table 1 is tabulating the main characteristic values of theDSC and TGAA thermograms as shown in Figures 2 and 3respectively

The treatment of carrageenan with chitosan and epoxyhas shown gradual and obvious improvement in their DSCand TGA The DSC exothermic effect has been shifted tohigher temperatures from formula numbers 1ndash5 that is from220∘C to 320∘C The Carr has shown an exothermic band at220∘C which has been shifted to 230∘C after treatment withCh This improvement could be attributed to the formationof a complex network between the Carr and the Ch Similarbehavior has been attained when the di-epoxy has beensubstituted with glutaraldehyde [3] Further treatment withdi-epoxy Carr-Ch-Epo gradually increased the temperatureto two peaks at 250 and 230∘C This could be attributed toextra crosslinking with the di-epoxy and the two bands couldbe referred to the Ch and Epo however we could not tell atthis stage which is which However by addition of Ch Carr-Ch-Epo-Ch the temperature increased to a single band at260∘C which should be regarded to the Ch That means thatfor the short chain Carr-Ch-Epo the two peaks should be forCh and Epo respectively Finally by adding more di-epoxyCarr-Ch-Epo-Ch-Epo (long chain) two bands appeared for

60

65

70

75

80

85

90

95

100

0 50 100 150 200 250 300 350

Wei

ght (

)

Temperature (∘C)

CarrCarrndashChCarrndashChndashEpo

CarrndashChndashEpondashChCarrndashChndashEpondashChndashEpo

Figure 3 TGA thermogram of carrageenan and modified car-rageenan with chitosan and epoxy groups

the Ch and Epo at the highest temperatures 260 and 330∘Crespectively

On the other hand the TGA of the modified Carrformula numbers 2-5 showed a better stability against degra-dation as shown in Figure 3 andTable 1 For example unmod-ified Carr had a sudden decomposition at 200∘C This valuehas been increased to 210∘C after its treatment with Ch withretention of the sudden decomposition behavior The gelsrsquothermal improvement could be explained by the formation ofpolyelectrolyte interaction between the polyanions (ndashOSO

3

minus)of the Carr and the polycations (NH

3

+) of the Ch Furtherhardening of the gel beads using Epo showed a much higherincrease in the TGAA Carr-Ch-Epo (short chain) to 250∘Cand the decomposition behavior was slower and gradualThe improvement in the TGA could be attributed to extracrosslinking between the free Chrsquos amino groups and theEpo Further treatment of the short chain with Ch does notincrease its TGA however it was more or less the same at240∘C Finally for the long chain Carr-Ch-Epo-Ch-Epo theTGA increased to its maximum of 270∘C which could beregarded to further crosslinking It is worth noting that theshort and long chains have the highest TGA values of 250 and270∘Cwith slower and gradual decomposition rate comparedto other formulations

32 Optimization of Enzymersquos Loading Capacity Two factorshave been studied to optimize the loading capacity of 120573-galactosidases onto treated carrageenan gel beads

321 Effect of Epoxy Chain Length In this experiment shortchain of epoxy modified gel beads Carr-Ch-Epo and longchain Carr-Ch-Epo-Ch-Epo were examined to assess chainlength efficiency for immobilization of the enzyme Accord-ing to Sung and Bae 2003 [22] the effect of the chain lengthcould have a positive effect on the immobilization loadingefficiency till certain length and then it declines afterwards Inour case the short chain immobilizedmore enzymes than thelong chain Results as shown in Figure 4 showed that the short

6 BioMed Research International

0

5

10

15

20

25238

182

(Ug

bea

ds)

CarrndashChndashEpo CarrndashChndashEpondashChndashEpo

Figure 4 Effect of epoxy chain length on 120573-galactosidase loadingcapacity

chain immobilized 238 Ug gel beds compared to 182 Uggel beads for the long chain These results are in accordancewith that of Gancarz et al 2003 [23] who observed that anincrease in surface epoxy groups led to an increase in quantityof immobilized enzyme but a decrease in retained enzymeactivity

To understand this phenomenon we calculated from thesupernatants the expected amounts of immobilized enzymeson the short and long chains formulas and they were foundto be 18 and 21Ug respectively These results were in favorof the long chain formula however the retained activity ofthe immobilized enzymes was in favor of the short chainwhere 238Ug were immobilized showing 133 retention ofactivity This increase in the enzyme activity after immobi-lization could be regarded to the hydrogen bond interactionsbetween the modified gel (polysaccharide) containing ndashOHndashOSO

3H ndashC=O ndashNH

2 and the lactose substrate containing

ndashOH ndashC=O groups These H-bonding interactions couldalso increase the lactose concentration surrounding the gelsurface more than the bulk solution and thus the activity ofthe immobilized enzyme increases till reaching saturation ofthe gel surface with lactase [24]

On the other hand the long chain formulation wasexpected to immobilize 21Ug and in practice it showedonly 182Ug which revealed 86 retention of activity ofthe immobilized enzyme This could be regarded to the longchain havingmultipoint attachment orandmultilayers of theimmobilized enzymesteric hindrance that resulted in lossof the enzymersquos 3D structure and consequently its activityAccordingly for further experiments the short chain wasused

322 Effect of 120573-Galactosidase Concentration 120573-Galacto-sidase was immobilized onto gel beads treated with shortchain epoxy activated carrageenan Carr-Ch-Epo as shownin Figure 5

Results revealed that by increasing the concentrationof 120573-galactosidase from 10U to 60U the ELC increasedgradually till it reached its maximum of 36Ug gel beadsusing 50U of free enzyme after which any more addedenzyme has almost no effect on the ELC This could beregarded to all free epoxy groups that have been engagedwith the enzymes [25] However we have chosen for furtheroptimization the ELC of 36Ug gel beads as it shows better

05

10152025303540

10 20 30 40 50 60

(Ug)

8958854327 1239676625 2492457653 3101527564 3602115799 362855251

(Ug

bea

ds)

Figure 5 pH profile of the free and immobilized 120573-galactosidase

0

20

40

60

80

100

120

20 30 40 50 60 70 80

Rela

tive a

ctiv

ity (

)

FreeImmo

Temperature (∘C)

Figure 6 Michaelis constants of free and immobilized 120573-galactosidase

enzyme loading efficiency of 49 which is more economicas it saves unloaded enzyme from being wasted

33 Evaluation of Catalytic Activity of Free and Immobilized120573-Galactosidase At this stage five experiments were studiedFirstly the optimum reaction temperature pH and substrateconcentrations were examined for both the free and immo-bilized enzyme Secondly the best results from the first stepwere used to obtain themaximum substrate hydrolysis as wellas the operational stability of the immobilized enzyme

331 Optimum Temperature for the Free and Immobilized 120573-Galactosidase The optimum temperature for the free andimmobilized enzyme was examined Results as shown inFigure 6 revealed that the optimum temperature for theimmobilized enzyme was found to be at a slightly highertemperature (37ndash40∘C) compared to the free enzyme (30ndash37∘C)

The shift of the optimum temperature towards highertemperatures when the biocatalyst is immobilized indicatesthat the enzyme structure is strengthened by the immobiliza-tion process and the formation of a molecular cage aroundthe protein molecule (enzyme) was found to enhance theenzyme thermal stability The increase of the immobilized

BioMed Research International 7

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10

Rela

tive a

ctiv

ity (

)

pH

ImmoFree

Figure 7 Lactose hydrolysis using free and immobilized 120573-galactosidase

enzyme temperature tolerance may also be due to diffusionaleffects where the reaction velocity is more likely to bediffusion limited so that improvements in thermal diffusionwould correspondingly result in proportionally higher reac-tion rates [3]

332 pH Profile Figure 7 illustrates the pH activity profile ofthe free and immobilized 120573-galactosidase The optimum pHvalues for free and immobilized enzyme were 45ndash5 and 4-6 respectively which showed that the immobilized enzymewas more stable at higher and wider range of pH [25] Theseproperties could be very useful for lactolysis in sweet wheypermeate which has a pH range of 55ndash6 Moreover at pH4 the immobilized enzyme retained more than 95 of itsrelative activity compared to only 56 for the free enzyme

The shift in the pH activity profile of the immobilized 120573-galactosidase and the better pH stability may be attributedto the partition effects that were arising from differentconcentrations of charged species in the microenvironmentof the immobilized enzyme and in the domain of the bulksolution [3]

333 Determination of Kinetic Parameters of Free and Immo-bilized 120573-Galactosidase The kinetic constants of free andimmobilized 120573-galactosidase as shown in Figure 8 weretabulated in Table 2

The apparent 119870119898

after immobilization 1314mM ishigher than that of the free enzyme 589mMwhich indicatesthat a higher concentration of substrate 2-fold is needed forthe immobilized enzyme compared to the free enzyme Nev-ertheless higher 119870

119898values for immobilized 120573-galactosidase

have been reported by other authors with increases from 12-fold up to 54-fold [26] These results are most likely dueto the fact that the immobilized enzyme surfaces are notaccessible to all the reacting species However no substrate orproduct inhibition by the increase of substrate concentrationup to 200mM could be observed during our experiment as

020406080

100120140160180

minus150 minus100 minus50 0 50 100 150 200 250[S] (mM)

ImmoFree

y = 0481x + 63219

R2 = 09332

y = 05566x + 32786

R2 = 09889

[S]V

Figure 8 Reusability of the immobilized 120573-galactosidase

Table 2 Michaelis-Menten constants and maximal reaction ratevalues for free and immobilized lactase

120573-Galactosidase form Kinetic constants119870119898(mM) 119881max (120583molsdotminminus1)

Free 589 327Immobilized 1314 632

shown by the straight line of the Hanes-Wolf representation(Figure 8)

On the other hand the maximum reaction velocity119881maxvalues for the immobilized enzyme were remarkable it wasfound to double that of the free enzyme that is it increasedfrom 327 to 632 120583molsdotminminus1This result is in agreement withthe speculation that the improvement in the immobilizedenzyme thermal stability as in Section 331 could result in ahigher reaction velocity It is worth noting that the increasein the reaction velocity is generally favored in industries

334 Lactose Hydrolysis Using Free and Immobilized 120573-Galactosidase This experiment has been carried out so thatthe immobilized enzyme could attain its maximum efficiencyand act with its highest velocity using almost double the 119870

119898

concentration of substrate and using the enzymersquos optimumconditions A high concentration of substrate 200mM atpH 45 and 37∘C was used in this study as this enzymewas supposed to be suitable for hydrolysis of higher lactoseconcentrations found in mammal milk (88ndash234mM lactose)and whey permeate (85 lactose) [25]

The results as shown in Figure 9 revealed that for the firsthour the rate of conversion of the free enzyme was higherthan that of the immobilized one This could be attributedto the fact that the gel needed longer time to reach itsmaximum swelling This swelling will allow more substratesto penetrate into the pores and consequently decrease thediffusion limitation However at 90min both enzyme formsfollowed the same trend and the same speed till they reachedmaximum relative conversion at 120min It is worth notingthat at 90min the gel beads carrying the enzyme reached its

8 BioMed Research International

0

20

40

60

80

100

120

0 50 100 150 200 250

Hyd

roly

sis (

)

Time (min)

ImmoFree

Figure 9 Effect of 120573-galactosidase concentration on the enzymersquosloading capacity

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Rela

tive a

ctiv

ity (

)

Cycle number

Figure 10 Temperature profile of free and immobilized enzyme

maximum swelling overcame its substrateproduct diffusionlimitation and followed the same trend as the free enzymewhich is advantageous in industries That means that thesmall gel beads used in this work could overcome the problemthe authors previously had when they used big gel disks asthe enzyme suffered fromdiffusion limitation and hydrolyzedonly 63 of the free enzyme [3]

335 Reusability of Immobilized Enzyme To evaluate thereusability of the immobilized enzyme the beadswere soakedin 200mM lactose for 120min till full conversion of lactoseto glucose and galactose The gel beads were removed fromthe product washed with buffer solution after use and thenresuspended in a fresh aliquot of a substrate to measure theenzymatic activity

This procedure was repeated until the enzyme lost itsactivity The turn over number of the enzyme catalyzed pro-cess was calculated As shown in Figure 10 the immobilizedenzyme retained 60 of its relative activity by the seconduse and 21 by the 3rd use Nevertheless these results werein agreement with those obtained by other authors usingthe commercial carrier Novozym 435 as the immobilizedactivity decreased to 23 after the second use and to 37 by

the third use [27] The loss in activity was attributed by otherauthors to inactivation of enzyme due to continuous use [28]Although our carrier has shown better performance than thatof Novozyme 435 we think that the modified gel with epoxycould be further modified for future work

4 Conclusion

Novel biopolymer based on epoxy activated carrageenan wasprepared for immobilization of lactase as an example ofmedical enzyme The results were compared to those of ourprevious work using aldehyde activated carrageenan Theepoxy formula showed far better immobilization efficiencythat was triple that shown using the aldehyde one Thatcould be regarded to that the epoxy group is more activethan the aldehyde group The aldehyde group could onlybind to the enzymersquos free amino groups whereas the epoxygroup could bind to three groups ndashSH ndashNH

2 and ndashOH

The results showed in Figure 9 hydrolysis of lactose usingfree and immobilized lactase revealed that the immobilizedenzyme could attain its maximum efficiency and act withits highest velocity as fast as the free enzyme That wasregarded to the gel beads carrying the enzyme that reachedits maximum swelling and overcame its substrateproductdiffusion limitation and followed the same trend as thefree enzyme which is advantageous in industries The highactivity of the epoxy formulation is highly recommended tobe used for immobilization of other enzymesproteins andordrug delivery systems

Abbreviations

Carr CarrageenanCarr-Ch Carrageenan-chitosanCarr-Ch-Epo Carrageenan-chitosan-epoxyCarr-Ch-Epo-Ch Carrageenan-chitosan-epoxy-

chitosanCarr-Ch-Epo-Ch-Epo Carrageenan-chitosan-epoxy-

chitosan-epoxy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q Husain ldquo120573-Galactosidases and their potential applications areviewrdquo Critical Reviews in Biotechnology vol 30 no 1 pp 41ndash62 2010

[2] MM Elnashar G E Awad M E Hassan M S Mohy Eldin BM Haroun and A I El-Diwany ldquoOptimal immobilization of120573-galactosidase onto 120581-Carrageenan gel beads using responsesurface methodology and its applicationsrdquoThe Scientific WorldJournal vol 2014 Article ID 571682 7 pages 2014

[3] M M M Elnashar and M A Yassin ldquoLactose hydrolysisby 120573-galactosidase covalently immobilized to thermally stablebiopolymersrdquo Applied Biochemistry and Biotechnology vol 159no 2 pp 426ndash437 2009

BioMed Research International 9

[4] G F Bickerstaf ldquoImpact of genetic technology on enzymetechnologyrdquo The Genetic Engineer and Biotechnologist vol 15pp 13ndash30 1995

[5] M M Elnashar ldquoThe art of immobilization using biopoly-mers biomaterials and nanobiotechnologyrdquo in Biotechnology ofBiopolymers pp 1ndash30 Intech 2011

[6] G Roberts ldquoStructure of chitin and chitosanrdquo in Chitin Chem-istry G A F Roberts Ed pp 1ndash53 MacMillan HoundmillsUK 1992

[7] R Hejazi andM Amiji ldquoChitosan-based gastrointestinal deliv-ery systemsrdquo Journal of Controlled Release vol 89 no 2 pp 151ndash165 2003

[8] B Krajewska ldquoApplication of chitin- and chitosan-based mate-rials for enzyme immobilizations a reviewrdquo Enzyme andMicro-bial Technology vol 35 no 2-3 pp 126ndash139 2004

[9] M M Elnashar O A Ali and H A Ragaa ldquoImmobilizedpenicillin G acylase onto grafted k-carrageenan hypothesis onthe effect of pH on the gel-enzyme interactionrdquoArabian Journalof Chemistry In press

[10] K C Chao M M Haugen and G P Royer ldquoStabiliza-tion of kappa-carrageenan gel with polymeric amines useof immobilized cells as biocatalysts at elevated temperaturesrdquoBiotechnology and Bioengineering vol 28 no 9 pp 1289ndash12931986

[11] J S Chang C Chou and S Y Chen ldquoDecolorization of azo dyeswith immobilized Pseudomonas luteolardquo Process Biochemistryvol 36 no 8-9 pp 757ndash763 2001

[12] S H Moon and S J Parulekar ldquoCharacterization of 120581-carrageenan gels used for immobilization of Bacillus firmusrdquoBiotechnology Progress vol 7 no 6 pp 516ndash525 1991

[13] E N Danial M M M Elnashar and G E A Awad ldquoImmobi-lized inulinase on grafted alginate beads prepared by the one-step and the two-steps methodsrdquo Industrial and EngineeringChemistry Research vol 49 no 7 pp 3120ndash3125 2010

[14] D K Boadi and R J Neufeld ldquoEncapsulation of tannase for thehydrolysis of tea tanninsrdquo Enzyme and Microbial Technologyvol 28 no 7-8 pp 590ndash595 2001

[15] A M Eberhardt V Pedroni M Volpe and M L FerreiraldquoImmobilization of catalase from Aspergillus niger on inorganicand biopolymeric supports for H

2O2decompositionrdquo Applied

Catalysis B Environmental vol 47 no 3 pp 153ndash163 2004[16] M M Elnashar ldquoReview article immobilized molecules using

biomaterials and nanobiotechnologyrdquo Journal of Biomaterialsand Nanobiotechnology vol 1 pp 61ndash76 2010

[17] EMagnan I Catarino D Paolucci-Jeanjean L Preziosi-Belloyand M P Belleville ldquoImmobilization of lipase on a ceramicmembrane activity and stabilityrdquo Journal of Membrane Sciencevol 241 no 1 pp 161ndash166 2004

[18] S Rocchietti A S V Urrutia M Pregnolato et al ldquoInfluenceof the enzyme derivative preparation and substrate structureon the enantioselectivity of penicillin G acylaserdquo Enzyme andMicrobial Technology vol 31 no 1-2 pp 88ndash93 2002

[19] MMM Elnashar andM A Yassin ldquoCovalent immobilizationof 120573-galactosidase on carrageenan coated with chitosanrdquo Jour-nal of Applied Polymer Science vol 114 no 1 pp 17ndash24 2009

[20] A A El-Sanabary M M Elnashar A A Magda and B MBadran ldquoPreparation and evaluation of some new corrosioninhibitors in varnishesrdquo Anti-Corrosion Methods and Materialsvol 48 no 1 pp 47ndash58 2001

[21] C Tapia Z Escobar E Costa et al ldquoComparative studies onpolyelectrolyte complexes and mixtures of chitosan-alginate

and chitosan-carrageenan as prolonged diltiazem clorhydraterelease systemsrdquo European Journal of Pharmaceutics and Bio-pharmaceutics vol 57 no 1 pp 65ndash75 2004

[22] W J Sung and Y H Bae ldquoA glucose oxidase electrode basedonpolypyrrolewith polyanionPEGenzyme conjugate dopantrdquoBiosensors and Bioelectronics vol 18 no 10 pp 1231ndash1239 2003

[23] I Gancarz J Bryjak M Bryjak G Pozniak and W TylusldquoPlasma modified polymers as a support for enzyme immobi-lization 1 Allyl alcohol plasmardquo European Polymer Journal vol39 no 8 pp 1615ndash1622 2003

[24] MMM ElnasharM A Yassin and T Kahil ldquoNovel thermallyandmechanically stable hydrogel for enzyme immobilization ofpenicillin G acylase via covalent techniquerdquo Journal of AppliedPolymer Science vol 109 no 6 pp 4105ndash4111 2008

[25] A Tanriseven and S Dogan ldquoA novel method for the immobi-lization of 120573-galactosidaserdquo Process Biochemistry vol 38 no 1pp 27ndash30 2002

[26] Q Z K Zhou and X Dong Chen ldquoImmobilization of 120573-galactosidase on graphite surface by glutaraldehyderdquo Journal ofFood Engineering vol 48 no 1 pp 69ndash74 2001

[27] B Chen J Hu E M Miller W Xie M Cai and R AGross ldquoCandida antarctica Lipase B chemically immobilized onepoxy-activated micro- and nanobeads catalysts for polyestersynthesisrdquo Biomacromolecules vol 9 no 2 pp 463ndash471 2008

[28] K Nakane T Ogihara N Ogata and Y Kurokawa ldquoEntrap-immobilization of invertase on composite gel fiber of celluloseacetate and zirconium alkoxide by sol-gel processrdquo Journal ofApplied Polymer Science vol 81 no 9 pp 2084ndash2088 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

0

5

10

15

20

25238

182

(Ug

bea

ds)

CarrndashChndashEpo CarrndashChndashEpondashChndashEpo

Figure 4 Effect of epoxy chain length on 120573-galactosidase loadingcapacity

chain immobilized 238 Ug gel beds compared to 182 Uggel beads for the long chain These results are in accordancewith that of Gancarz et al 2003 [23] who observed that anincrease in surface epoxy groups led to an increase in quantityof immobilized enzyme but a decrease in retained enzymeactivity

To understand this phenomenon we calculated from thesupernatants the expected amounts of immobilized enzymeson the short and long chains formulas and they were foundto be 18 and 21Ug respectively These results were in favorof the long chain formula however the retained activity ofthe immobilized enzymes was in favor of the short chainwhere 238Ug were immobilized showing 133 retention ofactivity This increase in the enzyme activity after immobi-lization could be regarded to the hydrogen bond interactionsbetween the modified gel (polysaccharide) containing ndashOHndashOSO

3H ndashC=O ndashNH

2 and the lactose substrate containing

ndashOH ndashC=O groups These H-bonding interactions couldalso increase the lactose concentration surrounding the gelsurface more than the bulk solution and thus the activity ofthe immobilized enzyme increases till reaching saturation ofthe gel surface with lactase [24]

On the other hand the long chain formulation wasexpected to immobilize 21Ug and in practice it showedonly 182Ug which revealed 86 retention of activity ofthe immobilized enzyme This could be regarded to the longchain havingmultipoint attachment orandmultilayers of theimmobilized enzymesteric hindrance that resulted in lossof the enzymersquos 3D structure and consequently its activityAccordingly for further experiments the short chain wasused

322 Effect of 120573-Galactosidase Concentration 120573-Galacto-sidase was immobilized onto gel beads treated with shortchain epoxy activated carrageenan Carr-Ch-Epo as shownin Figure 5

Results revealed that by increasing the concentrationof 120573-galactosidase from 10U to 60U the ELC increasedgradually till it reached its maximum of 36Ug gel beadsusing 50U of free enzyme after which any more addedenzyme has almost no effect on the ELC This could beregarded to all free epoxy groups that have been engagedwith the enzymes [25] However we have chosen for furtheroptimization the ELC of 36Ug gel beads as it shows better

05

10152025303540

10 20 30 40 50 60

(Ug)

8958854327 1239676625 2492457653 3101527564 3602115799 362855251

(Ug

bea

ds)

Figure 5 pH profile of the free and immobilized 120573-galactosidase

0

20

40

60

80

100

120

20 30 40 50 60 70 80

Rela

tive a

ctiv

ity (

)

FreeImmo

Temperature (∘C)

Figure 6 Michaelis constants of free and immobilized 120573-galactosidase

enzyme loading efficiency of 49 which is more economicas it saves unloaded enzyme from being wasted

33 Evaluation of Catalytic Activity of Free and Immobilized120573-Galactosidase At this stage five experiments were studiedFirstly the optimum reaction temperature pH and substrateconcentrations were examined for both the free and immo-bilized enzyme Secondly the best results from the first stepwere used to obtain themaximum substrate hydrolysis as wellas the operational stability of the immobilized enzyme

331 Optimum Temperature for the Free and Immobilized 120573-Galactosidase The optimum temperature for the free andimmobilized enzyme was examined Results as shown inFigure 6 revealed that the optimum temperature for theimmobilized enzyme was found to be at a slightly highertemperature (37ndash40∘C) compared to the free enzyme (30ndash37∘C)

The shift of the optimum temperature towards highertemperatures when the biocatalyst is immobilized indicatesthat the enzyme structure is strengthened by the immobiliza-tion process and the formation of a molecular cage aroundthe protein molecule (enzyme) was found to enhance theenzyme thermal stability The increase of the immobilized

BioMed Research International 7

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10

Rela

tive a

ctiv

ity (

)

pH

ImmoFree

Figure 7 Lactose hydrolysis using free and immobilized 120573-galactosidase

enzyme temperature tolerance may also be due to diffusionaleffects where the reaction velocity is more likely to bediffusion limited so that improvements in thermal diffusionwould correspondingly result in proportionally higher reac-tion rates [3]

332 pH Profile Figure 7 illustrates the pH activity profile ofthe free and immobilized 120573-galactosidase The optimum pHvalues for free and immobilized enzyme were 45ndash5 and 4-6 respectively which showed that the immobilized enzymewas more stable at higher and wider range of pH [25] Theseproperties could be very useful for lactolysis in sweet wheypermeate which has a pH range of 55ndash6 Moreover at pH4 the immobilized enzyme retained more than 95 of itsrelative activity compared to only 56 for the free enzyme

The shift in the pH activity profile of the immobilized 120573-galactosidase and the better pH stability may be attributedto the partition effects that were arising from differentconcentrations of charged species in the microenvironmentof the immobilized enzyme and in the domain of the bulksolution [3]

333 Determination of Kinetic Parameters of Free and Immo-bilized 120573-Galactosidase The kinetic constants of free andimmobilized 120573-galactosidase as shown in Figure 8 weretabulated in Table 2

The apparent 119870119898

after immobilization 1314mM ishigher than that of the free enzyme 589mMwhich indicatesthat a higher concentration of substrate 2-fold is needed forthe immobilized enzyme compared to the free enzyme Nev-ertheless higher 119870

119898values for immobilized 120573-galactosidase

have been reported by other authors with increases from 12-fold up to 54-fold [26] These results are most likely dueto the fact that the immobilized enzyme surfaces are notaccessible to all the reacting species However no substrate orproduct inhibition by the increase of substrate concentrationup to 200mM could be observed during our experiment as

020406080

100120140160180

minus150 minus100 minus50 0 50 100 150 200 250[S] (mM)

ImmoFree

y = 0481x + 63219

R2 = 09332

y = 05566x + 32786

R2 = 09889

[S]V

Figure 8 Reusability of the immobilized 120573-galactosidase

Table 2 Michaelis-Menten constants and maximal reaction ratevalues for free and immobilized lactase

120573-Galactosidase form Kinetic constants119870119898(mM) 119881max (120583molsdotminminus1)

Free 589 327Immobilized 1314 632

shown by the straight line of the Hanes-Wolf representation(Figure 8)

On the other hand the maximum reaction velocity119881maxvalues for the immobilized enzyme were remarkable it wasfound to double that of the free enzyme that is it increasedfrom 327 to 632 120583molsdotminminus1This result is in agreement withthe speculation that the improvement in the immobilizedenzyme thermal stability as in Section 331 could result in ahigher reaction velocity It is worth noting that the increasein the reaction velocity is generally favored in industries

334 Lactose Hydrolysis Using Free and Immobilized 120573-Galactosidase This experiment has been carried out so thatthe immobilized enzyme could attain its maximum efficiencyand act with its highest velocity using almost double the 119870

119898

concentration of substrate and using the enzymersquos optimumconditions A high concentration of substrate 200mM atpH 45 and 37∘C was used in this study as this enzymewas supposed to be suitable for hydrolysis of higher lactoseconcentrations found in mammal milk (88ndash234mM lactose)and whey permeate (85 lactose) [25]

The results as shown in Figure 9 revealed that for the firsthour the rate of conversion of the free enzyme was higherthan that of the immobilized one This could be attributedto the fact that the gel needed longer time to reach itsmaximum swelling This swelling will allow more substratesto penetrate into the pores and consequently decrease thediffusion limitation However at 90min both enzyme formsfollowed the same trend and the same speed till they reachedmaximum relative conversion at 120min It is worth notingthat at 90min the gel beads carrying the enzyme reached its

8 BioMed Research International

0

20

40

60

80

100

120

0 50 100 150 200 250

Hyd

roly

sis (

)

Time (min)

ImmoFree

Figure 9 Effect of 120573-galactosidase concentration on the enzymersquosloading capacity

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Rela

tive a

ctiv

ity (

)

Cycle number

Figure 10 Temperature profile of free and immobilized enzyme

maximum swelling overcame its substrateproduct diffusionlimitation and followed the same trend as the free enzymewhich is advantageous in industries That means that thesmall gel beads used in this work could overcome the problemthe authors previously had when they used big gel disks asthe enzyme suffered fromdiffusion limitation and hydrolyzedonly 63 of the free enzyme [3]

335 Reusability of Immobilized Enzyme To evaluate thereusability of the immobilized enzyme the beadswere soakedin 200mM lactose for 120min till full conversion of lactoseto glucose and galactose The gel beads were removed fromthe product washed with buffer solution after use and thenresuspended in a fresh aliquot of a substrate to measure theenzymatic activity

This procedure was repeated until the enzyme lost itsactivity The turn over number of the enzyme catalyzed pro-cess was calculated As shown in Figure 10 the immobilizedenzyme retained 60 of its relative activity by the seconduse and 21 by the 3rd use Nevertheless these results werein agreement with those obtained by other authors usingthe commercial carrier Novozym 435 as the immobilizedactivity decreased to 23 after the second use and to 37 by

the third use [27] The loss in activity was attributed by otherauthors to inactivation of enzyme due to continuous use [28]Although our carrier has shown better performance than thatof Novozyme 435 we think that the modified gel with epoxycould be further modified for future work

4 Conclusion

Novel biopolymer based on epoxy activated carrageenan wasprepared for immobilization of lactase as an example ofmedical enzyme The results were compared to those of ourprevious work using aldehyde activated carrageenan Theepoxy formula showed far better immobilization efficiencythat was triple that shown using the aldehyde one Thatcould be regarded to that the epoxy group is more activethan the aldehyde group The aldehyde group could onlybind to the enzymersquos free amino groups whereas the epoxygroup could bind to three groups ndashSH ndashNH

2 and ndashOH

The results showed in Figure 9 hydrolysis of lactose usingfree and immobilized lactase revealed that the immobilizedenzyme could attain its maximum efficiency and act withits highest velocity as fast as the free enzyme That wasregarded to the gel beads carrying the enzyme that reachedits maximum swelling and overcame its substrateproductdiffusion limitation and followed the same trend as thefree enzyme which is advantageous in industries The highactivity of the epoxy formulation is highly recommended tobe used for immobilization of other enzymesproteins andordrug delivery systems

Abbreviations

Carr CarrageenanCarr-Ch Carrageenan-chitosanCarr-Ch-Epo Carrageenan-chitosan-epoxyCarr-Ch-Epo-Ch Carrageenan-chitosan-epoxy-

chitosanCarr-Ch-Epo-Ch-Epo Carrageenan-chitosan-epoxy-

chitosan-epoxy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q Husain ldquo120573-Galactosidases and their potential applications areviewrdquo Critical Reviews in Biotechnology vol 30 no 1 pp 41ndash62 2010

[2] MM Elnashar G E Awad M E Hassan M S Mohy Eldin BM Haroun and A I El-Diwany ldquoOptimal immobilization of120573-galactosidase onto 120581-Carrageenan gel beads using responsesurface methodology and its applicationsrdquoThe Scientific WorldJournal vol 2014 Article ID 571682 7 pages 2014

[3] M M M Elnashar and M A Yassin ldquoLactose hydrolysisby 120573-galactosidase covalently immobilized to thermally stablebiopolymersrdquo Applied Biochemistry and Biotechnology vol 159no 2 pp 426ndash437 2009

BioMed Research International 9

[4] G F Bickerstaf ldquoImpact of genetic technology on enzymetechnologyrdquo The Genetic Engineer and Biotechnologist vol 15pp 13ndash30 1995

[5] M M Elnashar ldquoThe art of immobilization using biopoly-mers biomaterials and nanobiotechnologyrdquo in Biotechnology ofBiopolymers pp 1ndash30 Intech 2011

[6] G Roberts ldquoStructure of chitin and chitosanrdquo in Chitin Chem-istry G A F Roberts Ed pp 1ndash53 MacMillan HoundmillsUK 1992

[7] R Hejazi andM Amiji ldquoChitosan-based gastrointestinal deliv-ery systemsrdquo Journal of Controlled Release vol 89 no 2 pp 151ndash165 2003

[8] B Krajewska ldquoApplication of chitin- and chitosan-based mate-rials for enzyme immobilizations a reviewrdquo Enzyme andMicro-bial Technology vol 35 no 2-3 pp 126ndash139 2004

[9] M M Elnashar O A Ali and H A Ragaa ldquoImmobilizedpenicillin G acylase onto grafted k-carrageenan hypothesis onthe effect of pH on the gel-enzyme interactionrdquoArabian Journalof Chemistry In press

[10] K C Chao M M Haugen and G P Royer ldquoStabiliza-tion of kappa-carrageenan gel with polymeric amines useof immobilized cells as biocatalysts at elevated temperaturesrdquoBiotechnology and Bioengineering vol 28 no 9 pp 1289ndash12931986

[11] J S Chang C Chou and S Y Chen ldquoDecolorization of azo dyeswith immobilized Pseudomonas luteolardquo Process Biochemistryvol 36 no 8-9 pp 757ndash763 2001

[12] S H Moon and S J Parulekar ldquoCharacterization of 120581-carrageenan gels used for immobilization of Bacillus firmusrdquoBiotechnology Progress vol 7 no 6 pp 516ndash525 1991

[13] E N Danial M M M Elnashar and G E A Awad ldquoImmobi-lized inulinase on grafted alginate beads prepared by the one-step and the two-steps methodsrdquo Industrial and EngineeringChemistry Research vol 49 no 7 pp 3120ndash3125 2010

[14] D K Boadi and R J Neufeld ldquoEncapsulation of tannase for thehydrolysis of tea tanninsrdquo Enzyme and Microbial Technologyvol 28 no 7-8 pp 590ndash595 2001

[15] A M Eberhardt V Pedroni M Volpe and M L FerreiraldquoImmobilization of catalase from Aspergillus niger on inorganicand biopolymeric supports for H

2O2decompositionrdquo Applied

Catalysis B Environmental vol 47 no 3 pp 153ndash163 2004[16] M M Elnashar ldquoReview article immobilized molecules using

biomaterials and nanobiotechnologyrdquo Journal of Biomaterialsand Nanobiotechnology vol 1 pp 61ndash76 2010

[17] EMagnan I Catarino D Paolucci-Jeanjean L Preziosi-Belloyand M P Belleville ldquoImmobilization of lipase on a ceramicmembrane activity and stabilityrdquo Journal of Membrane Sciencevol 241 no 1 pp 161ndash166 2004

[18] S Rocchietti A S V Urrutia M Pregnolato et al ldquoInfluenceof the enzyme derivative preparation and substrate structureon the enantioselectivity of penicillin G acylaserdquo Enzyme andMicrobial Technology vol 31 no 1-2 pp 88ndash93 2002

[19] MMM Elnashar andM A Yassin ldquoCovalent immobilizationof 120573-galactosidase on carrageenan coated with chitosanrdquo Jour-nal of Applied Polymer Science vol 114 no 1 pp 17ndash24 2009

[20] A A El-Sanabary M M Elnashar A A Magda and B MBadran ldquoPreparation and evaluation of some new corrosioninhibitors in varnishesrdquo Anti-Corrosion Methods and Materialsvol 48 no 1 pp 47ndash58 2001

[21] C Tapia Z Escobar E Costa et al ldquoComparative studies onpolyelectrolyte complexes and mixtures of chitosan-alginate

and chitosan-carrageenan as prolonged diltiazem clorhydraterelease systemsrdquo European Journal of Pharmaceutics and Bio-pharmaceutics vol 57 no 1 pp 65ndash75 2004

[22] W J Sung and Y H Bae ldquoA glucose oxidase electrode basedonpolypyrrolewith polyanionPEGenzyme conjugate dopantrdquoBiosensors and Bioelectronics vol 18 no 10 pp 1231ndash1239 2003

[23] I Gancarz J Bryjak M Bryjak G Pozniak and W TylusldquoPlasma modified polymers as a support for enzyme immobi-lization 1 Allyl alcohol plasmardquo European Polymer Journal vol39 no 8 pp 1615ndash1622 2003

[24] MMM ElnasharM A Yassin and T Kahil ldquoNovel thermallyandmechanically stable hydrogel for enzyme immobilization ofpenicillin G acylase via covalent techniquerdquo Journal of AppliedPolymer Science vol 109 no 6 pp 4105ndash4111 2008

[25] A Tanriseven and S Dogan ldquoA novel method for the immobi-lization of 120573-galactosidaserdquo Process Biochemistry vol 38 no 1pp 27ndash30 2002

[26] Q Z K Zhou and X Dong Chen ldquoImmobilization of 120573-galactosidase on graphite surface by glutaraldehyderdquo Journal ofFood Engineering vol 48 no 1 pp 69ndash74 2001

[27] B Chen J Hu E M Miller W Xie M Cai and R AGross ldquoCandida antarctica Lipase B chemically immobilized onepoxy-activated micro- and nanobeads catalysts for polyestersynthesisrdquo Biomacromolecules vol 9 no 2 pp 463ndash471 2008

[28] K Nakane T Ogihara N Ogata and Y Kurokawa ldquoEntrap-immobilization of invertase on composite gel fiber of celluloseacetate and zirconium alkoxide by sol-gel processrdquo Journal ofApplied Polymer Science vol 81 no 9 pp 2084ndash2088 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

0

20

40

60

80

100

120

2 3 4 5 6 7 8 9 10

Rela

tive a

ctiv

ity (

)

pH

ImmoFree

Figure 7 Lactose hydrolysis using free and immobilized 120573-galactosidase

enzyme temperature tolerance may also be due to diffusionaleffects where the reaction velocity is more likely to bediffusion limited so that improvements in thermal diffusionwould correspondingly result in proportionally higher reac-tion rates [3]

332 pH Profile Figure 7 illustrates the pH activity profile ofthe free and immobilized 120573-galactosidase The optimum pHvalues for free and immobilized enzyme were 45ndash5 and 4-6 respectively which showed that the immobilized enzymewas more stable at higher and wider range of pH [25] Theseproperties could be very useful for lactolysis in sweet wheypermeate which has a pH range of 55ndash6 Moreover at pH4 the immobilized enzyme retained more than 95 of itsrelative activity compared to only 56 for the free enzyme

The shift in the pH activity profile of the immobilized 120573-galactosidase and the better pH stability may be attributedto the partition effects that were arising from differentconcentrations of charged species in the microenvironmentof the immobilized enzyme and in the domain of the bulksolution [3]

333 Determination of Kinetic Parameters of Free and Immo-bilized 120573-Galactosidase The kinetic constants of free andimmobilized 120573-galactosidase as shown in Figure 8 weretabulated in Table 2

The apparent 119870119898

after immobilization 1314mM ishigher than that of the free enzyme 589mMwhich indicatesthat a higher concentration of substrate 2-fold is needed forthe immobilized enzyme compared to the free enzyme Nev-ertheless higher 119870

119898values for immobilized 120573-galactosidase

have been reported by other authors with increases from 12-fold up to 54-fold [26] These results are most likely dueto the fact that the immobilized enzyme surfaces are notaccessible to all the reacting species However no substrate orproduct inhibition by the increase of substrate concentrationup to 200mM could be observed during our experiment as

020406080

100120140160180

minus150 minus100 minus50 0 50 100 150 200 250[S] (mM)

ImmoFree

y = 0481x + 63219

R2 = 09332

y = 05566x + 32786

R2 = 09889

[S]V

Figure 8 Reusability of the immobilized 120573-galactosidase

Table 2 Michaelis-Menten constants and maximal reaction ratevalues for free and immobilized lactase

120573-Galactosidase form Kinetic constants119870119898(mM) 119881max (120583molsdotminminus1)

Free 589 327Immobilized 1314 632

shown by the straight line of the Hanes-Wolf representation(Figure 8)

On the other hand the maximum reaction velocity119881maxvalues for the immobilized enzyme were remarkable it wasfound to double that of the free enzyme that is it increasedfrom 327 to 632 120583molsdotminminus1This result is in agreement withthe speculation that the improvement in the immobilizedenzyme thermal stability as in Section 331 could result in ahigher reaction velocity It is worth noting that the increasein the reaction velocity is generally favored in industries

334 Lactose Hydrolysis Using Free and Immobilized 120573-Galactosidase This experiment has been carried out so thatthe immobilized enzyme could attain its maximum efficiencyand act with its highest velocity using almost double the 119870

119898

concentration of substrate and using the enzymersquos optimumconditions A high concentration of substrate 200mM atpH 45 and 37∘C was used in this study as this enzymewas supposed to be suitable for hydrolysis of higher lactoseconcentrations found in mammal milk (88ndash234mM lactose)and whey permeate (85 lactose) [25]

The results as shown in Figure 9 revealed that for the firsthour the rate of conversion of the free enzyme was higherthan that of the immobilized one This could be attributedto the fact that the gel needed longer time to reach itsmaximum swelling This swelling will allow more substratesto penetrate into the pores and consequently decrease thediffusion limitation However at 90min both enzyme formsfollowed the same trend and the same speed till they reachedmaximum relative conversion at 120min It is worth notingthat at 90min the gel beads carrying the enzyme reached its

8 BioMed Research International

0

20

40

60

80

100

120

0 50 100 150 200 250

Hyd

roly

sis (

)

Time (min)

ImmoFree

Figure 9 Effect of 120573-galactosidase concentration on the enzymersquosloading capacity

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Rela

tive a

ctiv

ity (

)

Cycle number

Figure 10 Temperature profile of free and immobilized enzyme

maximum swelling overcame its substrateproduct diffusionlimitation and followed the same trend as the free enzymewhich is advantageous in industries That means that thesmall gel beads used in this work could overcome the problemthe authors previously had when they used big gel disks asthe enzyme suffered fromdiffusion limitation and hydrolyzedonly 63 of the free enzyme [3]

335 Reusability of Immobilized Enzyme To evaluate thereusability of the immobilized enzyme the beadswere soakedin 200mM lactose for 120min till full conversion of lactoseto glucose and galactose The gel beads were removed fromthe product washed with buffer solution after use and thenresuspended in a fresh aliquot of a substrate to measure theenzymatic activity

This procedure was repeated until the enzyme lost itsactivity The turn over number of the enzyme catalyzed pro-cess was calculated As shown in Figure 10 the immobilizedenzyme retained 60 of its relative activity by the seconduse and 21 by the 3rd use Nevertheless these results werein agreement with those obtained by other authors usingthe commercial carrier Novozym 435 as the immobilizedactivity decreased to 23 after the second use and to 37 by

the third use [27] The loss in activity was attributed by otherauthors to inactivation of enzyme due to continuous use [28]Although our carrier has shown better performance than thatof Novozyme 435 we think that the modified gel with epoxycould be further modified for future work

4 Conclusion

Novel biopolymer based on epoxy activated carrageenan wasprepared for immobilization of lactase as an example ofmedical enzyme The results were compared to those of ourprevious work using aldehyde activated carrageenan Theepoxy formula showed far better immobilization efficiencythat was triple that shown using the aldehyde one Thatcould be regarded to that the epoxy group is more activethan the aldehyde group The aldehyde group could onlybind to the enzymersquos free amino groups whereas the epoxygroup could bind to three groups ndashSH ndashNH

2 and ndashOH

The results showed in Figure 9 hydrolysis of lactose usingfree and immobilized lactase revealed that the immobilizedenzyme could attain its maximum efficiency and act withits highest velocity as fast as the free enzyme That wasregarded to the gel beads carrying the enzyme that reachedits maximum swelling and overcame its substrateproductdiffusion limitation and followed the same trend as thefree enzyme which is advantageous in industries The highactivity of the epoxy formulation is highly recommended tobe used for immobilization of other enzymesproteins andordrug delivery systems

Abbreviations

Carr CarrageenanCarr-Ch Carrageenan-chitosanCarr-Ch-Epo Carrageenan-chitosan-epoxyCarr-Ch-Epo-Ch Carrageenan-chitosan-epoxy-

chitosanCarr-Ch-Epo-Ch-Epo Carrageenan-chitosan-epoxy-

chitosan-epoxy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q Husain ldquo120573-Galactosidases and their potential applications areviewrdquo Critical Reviews in Biotechnology vol 30 no 1 pp 41ndash62 2010

[2] MM Elnashar G E Awad M E Hassan M S Mohy Eldin BM Haroun and A I El-Diwany ldquoOptimal immobilization of120573-galactosidase onto 120581-Carrageenan gel beads using responsesurface methodology and its applicationsrdquoThe Scientific WorldJournal vol 2014 Article ID 571682 7 pages 2014

[3] M M M Elnashar and M A Yassin ldquoLactose hydrolysisby 120573-galactosidase covalently immobilized to thermally stablebiopolymersrdquo Applied Biochemistry and Biotechnology vol 159no 2 pp 426ndash437 2009

BioMed Research International 9

[4] G F Bickerstaf ldquoImpact of genetic technology on enzymetechnologyrdquo The Genetic Engineer and Biotechnologist vol 15pp 13ndash30 1995

[5] M M Elnashar ldquoThe art of immobilization using biopoly-mers biomaterials and nanobiotechnologyrdquo in Biotechnology ofBiopolymers pp 1ndash30 Intech 2011

[6] G Roberts ldquoStructure of chitin and chitosanrdquo in Chitin Chem-istry G A F Roberts Ed pp 1ndash53 MacMillan HoundmillsUK 1992

[7] R Hejazi andM Amiji ldquoChitosan-based gastrointestinal deliv-ery systemsrdquo Journal of Controlled Release vol 89 no 2 pp 151ndash165 2003

[8] B Krajewska ldquoApplication of chitin- and chitosan-based mate-rials for enzyme immobilizations a reviewrdquo Enzyme andMicro-bial Technology vol 35 no 2-3 pp 126ndash139 2004

[9] M M Elnashar O A Ali and H A Ragaa ldquoImmobilizedpenicillin G acylase onto grafted k-carrageenan hypothesis onthe effect of pH on the gel-enzyme interactionrdquoArabian Journalof Chemistry In press

[10] K C Chao M M Haugen and G P Royer ldquoStabiliza-tion of kappa-carrageenan gel with polymeric amines useof immobilized cells as biocatalysts at elevated temperaturesrdquoBiotechnology and Bioengineering vol 28 no 9 pp 1289ndash12931986

[11] J S Chang C Chou and S Y Chen ldquoDecolorization of azo dyeswith immobilized Pseudomonas luteolardquo Process Biochemistryvol 36 no 8-9 pp 757ndash763 2001

[12] S H Moon and S J Parulekar ldquoCharacterization of 120581-carrageenan gels used for immobilization of Bacillus firmusrdquoBiotechnology Progress vol 7 no 6 pp 516ndash525 1991

[13] E N Danial M M M Elnashar and G E A Awad ldquoImmobi-lized inulinase on grafted alginate beads prepared by the one-step and the two-steps methodsrdquo Industrial and EngineeringChemistry Research vol 49 no 7 pp 3120ndash3125 2010

[14] D K Boadi and R J Neufeld ldquoEncapsulation of tannase for thehydrolysis of tea tanninsrdquo Enzyme and Microbial Technologyvol 28 no 7-8 pp 590ndash595 2001

[15] A M Eberhardt V Pedroni M Volpe and M L FerreiraldquoImmobilization of catalase from Aspergillus niger on inorganicand biopolymeric supports for H

2O2decompositionrdquo Applied

Catalysis B Environmental vol 47 no 3 pp 153ndash163 2004[16] M M Elnashar ldquoReview article immobilized molecules using

biomaterials and nanobiotechnologyrdquo Journal of Biomaterialsand Nanobiotechnology vol 1 pp 61ndash76 2010

[17] EMagnan I Catarino D Paolucci-Jeanjean L Preziosi-Belloyand M P Belleville ldquoImmobilization of lipase on a ceramicmembrane activity and stabilityrdquo Journal of Membrane Sciencevol 241 no 1 pp 161ndash166 2004

[18] S Rocchietti A S V Urrutia M Pregnolato et al ldquoInfluenceof the enzyme derivative preparation and substrate structureon the enantioselectivity of penicillin G acylaserdquo Enzyme andMicrobial Technology vol 31 no 1-2 pp 88ndash93 2002

[19] MMM Elnashar andM A Yassin ldquoCovalent immobilizationof 120573-galactosidase on carrageenan coated with chitosanrdquo Jour-nal of Applied Polymer Science vol 114 no 1 pp 17ndash24 2009

[20] A A El-Sanabary M M Elnashar A A Magda and B MBadran ldquoPreparation and evaluation of some new corrosioninhibitors in varnishesrdquo Anti-Corrosion Methods and Materialsvol 48 no 1 pp 47ndash58 2001

[21] C Tapia Z Escobar E Costa et al ldquoComparative studies onpolyelectrolyte complexes and mixtures of chitosan-alginate

and chitosan-carrageenan as prolonged diltiazem clorhydraterelease systemsrdquo European Journal of Pharmaceutics and Bio-pharmaceutics vol 57 no 1 pp 65ndash75 2004

[22] W J Sung and Y H Bae ldquoA glucose oxidase electrode basedonpolypyrrolewith polyanionPEGenzyme conjugate dopantrdquoBiosensors and Bioelectronics vol 18 no 10 pp 1231ndash1239 2003

[23] I Gancarz J Bryjak M Bryjak G Pozniak and W TylusldquoPlasma modified polymers as a support for enzyme immobi-lization 1 Allyl alcohol plasmardquo European Polymer Journal vol39 no 8 pp 1615ndash1622 2003

[24] MMM ElnasharM A Yassin and T Kahil ldquoNovel thermallyandmechanically stable hydrogel for enzyme immobilization ofpenicillin G acylase via covalent techniquerdquo Journal of AppliedPolymer Science vol 109 no 6 pp 4105ndash4111 2008

[25] A Tanriseven and S Dogan ldquoA novel method for the immobi-lization of 120573-galactosidaserdquo Process Biochemistry vol 38 no 1pp 27ndash30 2002

[26] Q Z K Zhou and X Dong Chen ldquoImmobilization of 120573-galactosidase on graphite surface by glutaraldehyderdquo Journal ofFood Engineering vol 48 no 1 pp 69ndash74 2001

[27] B Chen J Hu E M Miller W Xie M Cai and R AGross ldquoCandida antarctica Lipase B chemically immobilized onepoxy-activated micro- and nanobeads catalysts for polyestersynthesisrdquo Biomacromolecules vol 9 no 2 pp 463ndash471 2008

[28] K Nakane T Ogihara N Ogata and Y Kurokawa ldquoEntrap-immobilization of invertase on composite gel fiber of celluloseacetate and zirconium alkoxide by sol-gel processrdquo Journal ofApplied Polymer Science vol 81 no 9 pp 2084ndash2088 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

0

20

40

60

80

100

120

0 50 100 150 200 250

Hyd

roly

sis (

)

Time (min)

ImmoFree

Figure 9 Effect of 120573-galactosidase concentration on the enzymersquosloading capacity

0

20

40

60

80

100

120

0 1 2 3 4 5 6

Rela

tive a

ctiv

ity (

)

Cycle number

Figure 10 Temperature profile of free and immobilized enzyme

maximum swelling overcame its substrateproduct diffusionlimitation and followed the same trend as the free enzymewhich is advantageous in industries That means that thesmall gel beads used in this work could overcome the problemthe authors previously had when they used big gel disks asthe enzyme suffered fromdiffusion limitation and hydrolyzedonly 63 of the free enzyme [3]

335 Reusability of Immobilized Enzyme To evaluate thereusability of the immobilized enzyme the beadswere soakedin 200mM lactose for 120min till full conversion of lactoseto glucose and galactose The gel beads were removed fromthe product washed with buffer solution after use and thenresuspended in a fresh aliquot of a substrate to measure theenzymatic activity

This procedure was repeated until the enzyme lost itsactivity The turn over number of the enzyme catalyzed pro-cess was calculated As shown in Figure 10 the immobilizedenzyme retained 60 of its relative activity by the seconduse and 21 by the 3rd use Nevertheless these results werein agreement with those obtained by other authors usingthe commercial carrier Novozym 435 as the immobilizedactivity decreased to 23 after the second use and to 37 by

the third use [27] The loss in activity was attributed by otherauthors to inactivation of enzyme due to continuous use [28]Although our carrier has shown better performance than thatof Novozyme 435 we think that the modified gel with epoxycould be further modified for future work

4 Conclusion

Novel biopolymer based on epoxy activated carrageenan wasprepared for immobilization of lactase as an example ofmedical enzyme The results were compared to those of ourprevious work using aldehyde activated carrageenan Theepoxy formula showed far better immobilization efficiencythat was triple that shown using the aldehyde one Thatcould be regarded to that the epoxy group is more activethan the aldehyde group The aldehyde group could onlybind to the enzymersquos free amino groups whereas the epoxygroup could bind to three groups ndashSH ndashNH

2 and ndashOH

The results showed in Figure 9 hydrolysis of lactose usingfree and immobilized lactase revealed that the immobilizedenzyme could attain its maximum efficiency and act withits highest velocity as fast as the free enzyme That wasregarded to the gel beads carrying the enzyme that reachedits maximum swelling and overcame its substrateproductdiffusion limitation and followed the same trend as thefree enzyme which is advantageous in industries The highactivity of the epoxy formulation is highly recommended tobe used for immobilization of other enzymesproteins andordrug delivery systems

Abbreviations

Carr CarrageenanCarr-Ch Carrageenan-chitosanCarr-Ch-Epo Carrageenan-chitosan-epoxyCarr-Ch-Epo-Ch Carrageenan-chitosan-epoxy-

chitosanCarr-Ch-Epo-Ch-Epo Carrageenan-chitosan-epoxy-

chitosan-epoxy

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] Q Husain ldquo120573-Galactosidases and their potential applications areviewrdquo Critical Reviews in Biotechnology vol 30 no 1 pp 41ndash62 2010

[2] MM Elnashar G E Awad M E Hassan M S Mohy Eldin BM Haroun and A I El-Diwany ldquoOptimal immobilization of120573-galactosidase onto 120581-Carrageenan gel beads using responsesurface methodology and its applicationsrdquoThe Scientific WorldJournal vol 2014 Article ID 571682 7 pages 2014

[3] M M M Elnashar and M A Yassin ldquoLactose hydrolysisby 120573-galactosidase covalently immobilized to thermally stablebiopolymersrdquo Applied Biochemistry and Biotechnology vol 159no 2 pp 426ndash437 2009

BioMed Research International 9

[4] G F Bickerstaf ldquoImpact of genetic technology on enzymetechnologyrdquo The Genetic Engineer and Biotechnologist vol 15pp 13ndash30 1995

[5] M M Elnashar ldquoThe art of immobilization using biopoly-mers biomaterials and nanobiotechnologyrdquo in Biotechnology ofBiopolymers pp 1ndash30 Intech 2011

[6] G Roberts ldquoStructure of chitin and chitosanrdquo in Chitin Chem-istry G A F Roberts Ed pp 1ndash53 MacMillan HoundmillsUK 1992

[7] R Hejazi andM Amiji ldquoChitosan-based gastrointestinal deliv-ery systemsrdquo Journal of Controlled Release vol 89 no 2 pp 151ndash165 2003

[8] B Krajewska ldquoApplication of chitin- and chitosan-based mate-rials for enzyme immobilizations a reviewrdquo Enzyme andMicro-bial Technology vol 35 no 2-3 pp 126ndash139 2004

[9] M M Elnashar O A Ali and H A Ragaa ldquoImmobilizedpenicillin G acylase onto grafted k-carrageenan hypothesis onthe effect of pH on the gel-enzyme interactionrdquoArabian Journalof Chemistry In press

[10] K C Chao M M Haugen and G P Royer ldquoStabiliza-tion of kappa-carrageenan gel with polymeric amines useof immobilized cells as biocatalysts at elevated temperaturesrdquoBiotechnology and Bioengineering vol 28 no 9 pp 1289ndash12931986

[11] J S Chang C Chou and S Y Chen ldquoDecolorization of azo dyeswith immobilized Pseudomonas luteolardquo Process Biochemistryvol 36 no 8-9 pp 757ndash763 2001

[12] S H Moon and S J Parulekar ldquoCharacterization of 120581-carrageenan gels used for immobilization of Bacillus firmusrdquoBiotechnology Progress vol 7 no 6 pp 516ndash525 1991

[13] E N Danial M M M Elnashar and G E A Awad ldquoImmobi-lized inulinase on grafted alginate beads prepared by the one-step and the two-steps methodsrdquo Industrial and EngineeringChemistry Research vol 49 no 7 pp 3120ndash3125 2010

[14] D K Boadi and R J Neufeld ldquoEncapsulation of tannase for thehydrolysis of tea tanninsrdquo Enzyme and Microbial Technologyvol 28 no 7-8 pp 590ndash595 2001

[15] A M Eberhardt V Pedroni M Volpe and M L FerreiraldquoImmobilization of catalase from Aspergillus niger on inorganicand biopolymeric supports for H

2O2decompositionrdquo Applied

Catalysis B Environmental vol 47 no 3 pp 153ndash163 2004[16] M M Elnashar ldquoReview article immobilized molecules using

biomaterials and nanobiotechnologyrdquo Journal of Biomaterialsand Nanobiotechnology vol 1 pp 61ndash76 2010

[17] EMagnan I Catarino D Paolucci-Jeanjean L Preziosi-Belloyand M P Belleville ldquoImmobilization of lipase on a ceramicmembrane activity and stabilityrdquo Journal of Membrane Sciencevol 241 no 1 pp 161ndash166 2004

[18] S Rocchietti A S V Urrutia M Pregnolato et al ldquoInfluenceof the enzyme derivative preparation and substrate structureon the enantioselectivity of penicillin G acylaserdquo Enzyme andMicrobial Technology vol 31 no 1-2 pp 88ndash93 2002

[19] MMM Elnashar andM A Yassin ldquoCovalent immobilizationof 120573-galactosidase on carrageenan coated with chitosanrdquo Jour-nal of Applied Polymer Science vol 114 no 1 pp 17ndash24 2009

[20] A A El-Sanabary M M Elnashar A A Magda and B MBadran ldquoPreparation and evaluation of some new corrosioninhibitors in varnishesrdquo Anti-Corrosion Methods and Materialsvol 48 no 1 pp 47ndash58 2001

[21] C Tapia Z Escobar E Costa et al ldquoComparative studies onpolyelectrolyte complexes and mixtures of chitosan-alginate

and chitosan-carrageenan as prolonged diltiazem clorhydraterelease systemsrdquo European Journal of Pharmaceutics and Bio-pharmaceutics vol 57 no 1 pp 65ndash75 2004

[22] W J Sung and Y H Bae ldquoA glucose oxidase electrode basedonpolypyrrolewith polyanionPEGenzyme conjugate dopantrdquoBiosensors and Bioelectronics vol 18 no 10 pp 1231ndash1239 2003

[23] I Gancarz J Bryjak M Bryjak G Pozniak and W TylusldquoPlasma modified polymers as a support for enzyme immobi-lization 1 Allyl alcohol plasmardquo European Polymer Journal vol39 no 8 pp 1615ndash1622 2003

[24] MMM ElnasharM A Yassin and T Kahil ldquoNovel thermallyandmechanically stable hydrogel for enzyme immobilization ofpenicillin G acylase via covalent techniquerdquo Journal of AppliedPolymer Science vol 109 no 6 pp 4105ndash4111 2008

[25] A Tanriseven and S Dogan ldquoA novel method for the immobi-lization of 120573-galactosidaserdquo Process Biochemistry vol 38 no 1pp 27ndash30 2002

[26] Q Z K Zhou and X Dong Chen ldquoImmobilization of 120573-galactosidase on graphite surface by glutaraldehyderdquo Journal ofFood Engineering vol 48 no 1 pp 69ndash74 2001

[27] B Chen J Hu E M Miller W Xie M Cai and R AGross ldquoCandida antarctica Lipase B chemically immobilized onepoxy-activated micro- and nanobeads catalysts for polyestersynthesisrdquo Biomacromolecules vol 9 no 2 pp 463ndash471 2008

[28] K Nakane T Ogihara N Ogata and Y Kurokawa ldquoEntrap-immobilization of invertase on composite gel fiber of celluloseacetate and zirconium alkoxide by sol-gel processrdquo Journal ofApplied Polymer Science vol 81 no 9 pp 2084ndash2088 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 9

[4] G F Bickerstaf ldquoImpact of genetic technology on enzymetechnologyrdquo The Genetic Engineer and Biotechnologist vol 15pp 13ndash30 1995

[5] M M Elnashar ldquoThe art of immobilization using biopoly-mers biomaterials and nanobiotechnologyrdquo in Biotechnology ofBiopolymers pp 1ndash30 Intech 2011

[6] G Roberts ldquoStructure of chitin and chitosanrdquo in Chitin Chem-istry G A F Roberts Ed pp 1ndash53 MacMillan HoundmillsUK 1992

[7] R Hejazi andM Amiji ldquoChitosan-based gastrointestinal deliv-ery systemsrdquo Journal of Controlled Release vol 89 no 2 pp 151ndash165 2003

[8] B Krajewska ldquoApplication of chitin- and chitosan-based mate-rials for enzyme immobilizations a reviewrdquo Enzyme andMicro-bial Technology vol 35 no 2-3 pp 126ndash139 2004

[9] M M Elnashar O A Ali and H A Ragaa ldquoImmobilizedpenicillin G acylase onto grafted k-carrageenan hypothesis onthe effect of pH on the gel-enzyme interactionrdquoArabian Journalof Chemistry In press

[10] K C Chao M M Haugen and G P Royer ldquoStabiliza-tion of kappa-carrageenan gel with polymeric amines useof immobilized cells as biocatalysts at elevated temperaturesrdquoBiotechnology and Bioengineering vol 28 no 9 pp 1289ndash12931986

[11] J S Chang C Chou and S Y Chen ldquoDecolorization of azo dyeswith immobilized Pseudomonas luteolardquo Process Biochemistryvol 36 no 8-9 pp 757ndash763 2001

[12] S H Moon and S J Parulekar ldquoCharacterization of 120581-carrageenan gels used for immobilization of Bacillus firmusrdquoBiotechnology Progress vol 7 no 6 pp 516ndash525 1991

[13] E N Danial M M M Elnashar and G E A Awad ldquoImmobi-lized inulinase on grafted alginate beads prepared by the one-step and the two-steps methodsrdquo Industrial and EngineeringChemistry Research vol 49 no 7 pp 3120ndash3125 2010

[14] D K Boadi and R J Neufeld ldquoEncapsulation of tannase for thehydrolysis of tea tanninsrdquo Enzyme and Microbial Technologyvol 28 no 7-8 pp 590ndash595 2001

[15] A M Eberhardt V Pedroni M Volpe and M L FerreiraldquoImmobilization of catalase from Aspergillus niger on inorganicand biopolymeric supports for H

2O2decompositionrdquo Applied

Catalysis B Environmental vol 47 no 3 pp 153ndash163 2004[16] M M Elnashar ldquoReview article immobilized molecules using

biomaterials and nanobiotechnologyrdquo Journal of Biomaterialsand Nanobiotechnology vol 1 pp 61ndash76 2010

[17] EMagnan I Catarino D Paolucci-Jeanjean L Preziosi-Belloyand M P Belleville ldquoImmobilization of lipase on a ceramicmembrane activity and stabilityrdquo Journal of Membrane Sciencevol 241 no 1 pp 161ndash166 2004

[18] S Rocchietti A S V Urrutia M Pregnolato et al ldquoInfluenceof the enzyme derivative preparation and substrate structureon the enantioselectivity of penicillin G acylaserdquo Enzyme andMicrobial Technology vol 31 no 1-2 pp 88ndash93 2002

[19] MMM Elnashar andM A Yassin ldquoCovalent immobilizationof 120573-galactosidase on carrageenan coated with chitosanrdquo Jour-nal of Applied Polymer Science vol 114 no 1 pp 17ndash24 2009

[20] A A El-Sanabary M M Elnashar A A Magda and B MBadran ldquoPreparation and evaluation of some new corrosioninhibitors in varnishesrdquo Anti-Corrosion Methods and Materialsvol 48 no 1 pp 47ndash58 2001

[21] C Tapia Z Escobar E Costa et al ldquoComparative studies onpolyelectrolyte complexes and mixtures of chitosan-alginate

and chitosan-carrageenan as prolonged diltiazem clorhydraterelease systemsrdquo European Journal of Pharmaceutics and Bio-pharmaceutics vol 57 no 1 pp 65ndash75 2004

[22] W J Sung and Y H Bae ldquoA glucose oxidase electrode basedonpolypyrrolewith polyanionPEGenzyme conjugate dopantrdquoBiosensors and Bioelectronics vol 18 no 10 pp 1231ndash1239 2003

[23] I Gancarz J Bryjak M Bryjak G Pozniak and W TylusldquoPlasma modified polymers as a support for enzyme immobi-lization 1 Allyl alcohol plasmardquo European Polymer Journal vol39 no 8 pp 1615ndash1622 2003

[24] MMM ElnasharM A Yassin and T Kahil ldquoNovel thermallyandmechanically stable hydrogel for enzyme immobilization ofpenicillin G acylase via covalent techniquerdquo Journal of AppliedPolymer Science vol 109 no 6 pp 4105ndash4111 2008

[25] A Tanriseven and S Dogan ldquoA novel method for the immobi-lization of 120573-galactosidaserdquo Process Biochemistry vol 38 no 1pp 27ndash30 2002

[26] Q Z K Zhou and X Dong Chen ldquoImmobilization of 120573-galactosidase on graphite surface by glutaraldehyderdquo Journal ofFood Engineering vol 48 no 1 pp 69ndash74 2001

[27] B Chen J Hu E M Miller W Xie M Cai and R AGross ldquoCandida antarctica Lipase B chemically immobilized onepoxy-activated micro- and nanobeads catalysts for polyestersynthesisrdquo Biomacromolecules vol 9 no 2 pp 463ndash471 2008

[28] K Nakane T Ogihara N Ogata and Y Kurokawa ldquoEntrap-immobilization of invertase on composite gel fiber of celluloseacetate and zirconium alkoxide by sol-gel processrdquo Journal ofApplied Polymer Science vol 81 no 9 pp 2084ndash2088 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology