)JOEBXJ1VCMJTIJOH$PSQPSBUJPO #JPDIFNJTUSZ3FTFBSDI ...downloads.hindawi.com/journals/bri/2016/9781216.pdf · Biochemistry Research International Activity (U/g) 1250 1000 750 500 250

Research ArticleEffect of pH Temperature and Chemicals onthe Endoglucanases and 120573-Glucosidases from the ThermophilicFungus Myceliophthora heterothallica F214 Obtained bySolid-State and Submerged Cultivation

Vanessa de Caacutessia Teixeira da Silva1 Amanda Lais de Souza Coto1

Rafael de Carvalho Souza1 Marcello Bertoldi Sanchez Neves1 Eleni Gomes2

and Gustavo Orlando Bonilla-Rodriguez1

1Laboratorio de Bioquımica de Proteınas Departamento deQuımica eCienciasAmbientais Universidade Estadual Paulista (UNESP)Rua Cristovao Colombo 2265 15054-000 Sao Jose do Rio Preto SP Brazil2Laboratorio de Bioquımica e Microbiologia Aplicadas Departamento de Biologia Universidade Estadual Paulista (UNESP)Rua Cristovao Colombo 2265 15054-000 Sao Jose do Rio Preto SP Brazil

Correspondence should be addressed to Gustavo Orlando Bonilla-Rodriguez gustavobonillasjrpunespbr

Received 16 November 2015 Revised 3 March 2016 Accepted 12 April 2016

Academic Editor Gary A Lorigan

Copyright copy 2016 Vanessa de Cassia Teixeira da Silva et al 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

This work reports endoglucanase and beta-glucosidase production by the thermophilic fungus Myceliophthora heterothallica insolid-state (SSC) and submerged (SmC) cultivation Wheat bran and sugarcane bagasse were used for SSC and cardboard for SmCHighest endoglucanase production in SSC occurred after 192 hours 11706 plusmn 08Ug and in SmC after 168 hours 2642 plusmn 561UgThe endoglucanases and beta-glucosidases produced by both cultivation systems showed slight differences concerning their optimalpH and temperatureThe number of endoglucanases was also different six isoforms in SSC and ten in SmC Endoglucanase activityremained above 50 after incubation between pH 30 and 90 for 24 h for both cultivation systems The effect of several chemicalsdisplayed variation between SSC and SmC isoenzymes Manganese activated the enzymes from SmC but inhibited those fromSSC For 120573-glucosidases maximum production on SmC was 244 plusmn 48Ug after 168 hours using cardboard as carbon source InSSC maximum production reached 109 plusmn 03Ug after 240 h with 1 1 wheat bran and sugarcane bagasse Manganese exerted asignificant activation on SSC 120573-glucosidases and glucose inhibited the enzymes from both cultivation systems FeCl

3exerted the

strongest inhibition for endoglucanases and 120573-glucosidases

In memoriam of Vanessa de Cassia Teixeira da Silva

1 Introduction

Cellulose is the most abundant polysaccharide in the plantcell wall and consists in a linear chain composed of a varyingnumber of 120573-D-glucopyranose residues linked by 120573(1 rarr 4)glycosidic bonds Cellobiose is considered as the smallestrepetitive unit of cellulose and can be hydrolyzed into glucose

residues [1 2] Cellulose differs from other polysaccharidesby its insolubility and a stiff structure naturally resistant tobiological degradation suitable for its structural role in plants[3] Complete cellulose hydrolysis to glucose demands thesynergistic action of endoglucanases exoglucanases and 120573-glucosidases Endo-120573-14-glucanases (EC 3214) cleave theamorphous regions of the cellulose chain exoglucanases

Hindawi Publishing CorporationBiochemistry Research InternationalVolume 2016 Article ID 9781216 9 pageshttpdxdoiorg10115520169781216

2 Biochemistry Research International

or cellobiohydrolases (EC 32191) attack the nonreducingor reducing chain ends and 120573-14-glucosidases (EC 32121)hydrolyze cellobiose releasing glucose [4]

Fungal cellulolytic enzymes have biotechnological appli-cations in food animal feed wine agriculture biomassrefining and pulp and paper industries [5 6] The biggestchallenge for the enzymesrsquo biotechnological application istheir high cost of production The search for methodolo-gies that could reduce the cost of enzyme production andmicroorganismswith high growth rate and are able to developin cheap and easily accessible carbon sources is the currentapproach to change this paradigm [7]Thus various cellulosicsubstrates such as sugarcane bagasse corn stover wheatstraw and wheat bran have been tested by several authors forproduction of cellulolytic enzymes [8 9]

The composition of the medium can influence the profileof enzymatic expression by the microorganism Cellulasescan be obtained more economically by microorganismsthrough submerged or solid-state cultivation The low wateractivity in solid-state cultivation (SSC) shows a simple tech-nique of high productivity but it can affect the heat transfer tothe substrate and thus could affect the growth of themicroor-ganism [10] The use of thermophilic microorganisms can bean alternative to this technical limitation since they toleratehigher temperatures

The present study aimed to compare the production ofendoglucanases and 120573-glucosidases in solid-state and sub-merged cultivation media by thermophilic Myceliophthoraheterothallica and to characterize the crude extracts obtainedfrom both systems

2 Materials and Methods

21Microorganism The thermophilic fungusMyceliophthoraheterothallica F214 was isolated from sugar cane bagassecompost and maintained at the Laboratory of Applied Bio-chemistry and Microbiology UNESP

22 Inoculum Preparation The inoculum was prepared bygrowing the fungusM heterothallica F214 in 250mL Erlen-meyer flasks with 50mL medium composed of 10 sugar-cane bagasse (ground to 1-2mm) 014 (NH

4)2SO4 020

KH2PO4 003 CaCl

2 002 MgSO

2sdot7H2O 050 beef

peptone 020 yeast extract 003 urea 010 micronu-trients solution (5mgL FeSO

4sdot7H2O 16mgLMnSO

4sdotH2O

14mgL ZnSO4sdot7H2O and 20mgL CoCl

2) 02 glucose

and 20 agar pH 50 All the solutions and the culturemediawere sterilized by autoclaving at 121∘C for 20 minutes Thismedium was inoculated with suspension obtained scrappingthemycelium from culture grown for 3 days at 45∘C in 150mLof nutrient solution (gL) 35 (NH

4)2SO4 30 KH

2PO4 05

MgSO4sdot7H2O 05 CaCl

2 and 01 vv Tween 80 pH 50

23 Submerged Cultivation (SmC) Endoglucanase and 120573-glucosidase production in SmC was carried out in 250mLErlenmeyer flasks using cardboard as a carbon source previ-ously cut and ground in a blender The mixture contained 1cardboard and 20mLof the nutrient solution described above

was inoculated with 2mL of the microorganism inoculumas described The cultures were incubated on a rotary shaker(100 RPM) at 45∘C for 10 days and samples were taken every24 h filtered through vacuum usingWhatman paper number1 and centrifuged at 6000timesg for 20minutesThe supernatantwas used as a crude enzyme solution

In order to evaluate the use of agroindustrial residuessuch as wheat bran and sugar cane bagasse as the substratein SmC they were washed ground and oven-dried (60∘C for48 h) The sugarcane bagasse was sieved allowing selectingparticles ranging from 1 to 2mm Subsequently cultivationwas carried out in 250mL Erlenmeyer flasks containing 1(1 1 ww) of wheat bran and sugarcane bagasse 20mL nutri-ent solution and 2mL inoculum suspension The mediumwas previously autoclaved as described The cultures wereincubated on a rotary shaker (100 RPM) at 45∘C for 10 daysand sampling was performed as explained above

24 Solid-State Cultivation (SSC) The substrates used in theSSC were composed of used 5 grams of cardboard or amixture of wheat bran and sugar cane bagasse (1 1) Thesubstrates were inserted in polypropylene bags (12 times 20 cm)and sterilized at 121∘C for 40minThe fungus inoculation wascarried out by adding 20mL of themycelium suspensionThemoisture content of around 80 was reached with the addi-tion of sterile distilled water The fermentation was at 45∘Cfor 14 days Every 48 h one bag was taken and the fermentedmaterial was mixed with 20mL of distilled water per gramstirred for 30min filtered and centrifuged at 10000timesg at10∘CThe supernatant was used as a crude enzyme solution

25 Enzymatic Assay Endoglucanase activity was measuredat 60∘C in a reactional mixture composed of 09mL of100mMLminus1 sodium acetate buffer pH 50 containing 4of carboxymethyl cellulose (CMC) (Sigma-Aldrich USA)and 01mL of crude enzyme solution The reducing groupsexpressed as glucose released were assayed by using theDNS (35-dinitrosalicylic acid) method [11] One unit ofendoglucanase activity was defined as the amount of enzymeable to release 1 120583mol of reducing sugars per min under theassay conditions [12]120573-D-Glucosidase activity was measured in a mixture of

50mm sodium acetate buffer pH 50 500 120583L of 2mMLminus1p-nitrophenyl-120573-D-glucopyranoside (pNP-Glc) as substrateand 50120583L of the enzyme solution incubated at 60∘C for10min The reaction was stopped by addition of 2mL of2M Na

2CO3and the absorbance was read at 410 nm One

unit of 120573-glucosidase was defined as the amount of enzymethat releases 1 120583mole of nitrophenolmin in the reactionconditions

All the enzyme assays were performed in triplicate

26 Zymogram for Enzyme Activity The endoglucanase solu-tion was analyzed by electrophoresis on denaturing 10sodium dodecyl sulphate (SDS) polyacrylamide gel usingTris-Glycine buffer pH 83 After electrophoresis the enzymewas renatured with 1 triton X-100 for 30 minutes andsubsequently the gel was overlaid with a mixture of 1 (wv)

Biochemistry Research International 3

Activ

ity (U

g)

1250

1000

750

500

250

0

Time (h)24019214496480

(a)

Time (h)264240216192168144120967248240

(Um

L)

(Ug

)

08

06

04

02

00

80

60

40

20

0

(b)

Time (h)264240216192168144120967248240

(Ug

)

(Um

L)

3500

3000

2500

2000

1500

1000

500

0 0

5

10

15

20

25

30

35

(c)

Figure 1 (a) Endoglucanase production (expressed as Ug) byM heterothallica F214 in solid-state cultivation (◼) SSCWB (e) SSCP (998771)SSCLB Endoglucanase production in submerged cultivation expressed as Ug and UmL (b) (◻) SmCWB (c) (I) SmCP

CMC (Sigma-Aldrich) as substrate and 1 (wv) agarose andincubated for 30min at 50∘C For detection of endoglucanaseactivity the gel was immersed in a 01 (wv) Congo Redsolution for 15 minutes and then washed with 1MLminus1 NaCluntil visualization of the bright spots corresponding to theenzyme [13]

For 120573-glucosidase the gel was incubated with 02MLminus1acetate buffer pH 50 for 10 minutes at room temperatureunder gentle agitation After that the gel was treated witha solution of esculin 01 ferric chloride in 003 02MLminus1acetate buffer pH 50 until the appearance of bands [14]

27 Enzyme Characterization The effect of pH on theenzyme activity was evaluated as described above in thepH range from 20 to 110 using 01M buffers pH 2-3sodium citrate pH 3ndash55 sodium acetate pH60ndash75HEPESpH 80ndash95 glycine and pH 100ndash110 CAPS The effect oftemperature was assayed incubating the reactional mixturein temperature range from 35 to 85∘C in the optimal pH

The effect of pH on enzyme stability was studied byincubating the crude enzyme solution in various buffers withpH ranging from 25 to 110 during 24 h at 25∘C followed bythe determination of endoglucanase or 120573-glucosidase resid-ual activity Thermal stability was determined by incubatingthe crude enzyme from 40 to 70∘C for 60min and residualactivitywas assayed as described above For both experimentsthe data were compared to a control without preincubationdenoted as 100

28 Effect of Different Compounds on Endoglucanase120573-Glucosidase Activity The enzyme activity assays were donein the presence of metal ions and other compounds ethanolglucose triton and metal ions The metal ions (chloridesalts) were of analytical grade and were dissolved in ultrapurewater (UPW) The extract was dialyzed against UPW beforethe assays The results were compared by Studentrsquos 119905-test forindependent samples using BioEstat 53 [15]

3 Results and Discussion

31 Endoglucanase Production by SSC and SmC The cultiva-tion of Myceliophthora heterothallica by SSC and SmC usingdifferent lignocellulosic residues as substrates revealed thatthe extracellular enzyme production was highly dependenton the chemical composition of the tested culture media(wheat bran sugarcane bagasse and cardboard) When theratio of 9 1 sugarcane bagassewheat bran (SSCSB) (5 g oftotal carbon source) was used endoglucanase activity was3030 plusmn 17 Ug after 144 hours and the production in SSCusing cardboard (SSCP) achieved 1827 plusmn 16Ug Changingthe ratio of sugarcane bagassewheat bran to 1 1 (SSCWB)the production rosemore than three times to 11706plusmn 08Ugafter 192 h (Figure 1(a)) The values found for SSCSB agreewith those reported for Aspergillus terreus [16] Anotherfungus Fusarium oxysporum showed a similar production[17] High yields as those found by SSCP in this work werealso shown by the mutant Penicillium janthinellum EU2D21[18]


(Ug

)

70

60

50

40

30

20

10

0

Time (h)33628824019214496480

(a)

Time (h)264240216192168144120967248240

0

30

60

90

120

150

180

(Ug

)

00

03

06

09

12

15

18

(Um

L)

(b)

Time (h)

0

50

100

150

200

250

300

(Ug

)00

05

10

15

20

25

30

(Um

L)

264240216192168144120967248240

(c)

Figure 2 (a) 120573-Glucosidase production (expressed as Ug) byM heterothallica F214 in solid-state cultivation (◼) SSCWB (e) SSCP (998771)SSCLB 120573-Glucosidase production in submerged cultivation expressed as Ug and UmL (b) (◻) SmCWB (c) (I) SmCP

For SmC (Figure 1(b)) cardboard or wheat bran inmixture with sugarcane bagasse at 1 1 in concentration of1wv (04 g of total carbon source) was used In mediumwith wheat bran the lowest production was obtained 635 plusmn41 Ug after 192 h andusing cardboard (SmCP) the endoglu-canase production was increased to 2642 plusmn 561Ug in 168 h(Figure 1(c))

Wheat bran is the most common substrate for solidcultivation since it is rich in proteins carbohydrates andminerals that promote supplying macro- and micronutrientsfor microbial growth [19 20] The sugarcane bagasse iscomposed of cellulose (44) hemicellulose (25ndash27) lignin(20ndash22) ashes and minerals [21] The addition of wheatbran to sugarcane bagasse can supply nutritional conditionfor growth and enzyme production in SSC On the otherhand cardboard is the substrate with the highest percentageof cellulose 63 plusmn 16 and also 14 hemicellulose and lessthan 5 of lignin [22] This lower lignin content makes thecellulose more accessible to the microorganism and couldimprove the liberation of cellulase inducers

The analysis of enzyme by zymography showed differentprofiles for the endoglucanases according to the system andcarbon source used in the cultivation (Figure 3(a)) For theratio (1 1) of sugarcane bagasse and wheat bran in SSCand SmC six isoenzymes were observed whose molecularmasses follow around 116 66 and 30 kDa The middle rangeisoenzymes have faded bands This profile was modifiedwhen using cardboard as the carbon source the same sixisoenzymes previously mentioned were also expressed inSSC but the 66 kDa bands displayed higher activity and four

other isoforms with intermediary molecular weight appearedclearly in submerged cultivation suggesting that the higheraccessibility of cellulose from cardboard in SmC may haveinfluenced the production of more isoenzymes to facilitatedigestion The profile for the SSC using cardboard did notdisplay a defined profile but also suggests the expression ofintermediate molecular weight isoforms

32 120573-Glucosidase Production by SSC and SmC The peak ofenzyme production by SSC was obtained after 192 hours ofcultivationWhen the 9 1 ratio of sugarcane bagasse (SSCSB)(5 g of carbon source in total) was used120573-glucosidase activitywas 109 plusmn 03Ug after 240 h and replacing the proportion ofsugarcane bagasse with 1 1 (SSCWB) production increasedto 673 plusmn 08Ug after 288 h (Figure 2(a)) When using card-board in SSC the production decreased to 362 plusmn 001UgThe fungusFomitopsis sp RCK2010 produced 5368Ug usingonly wheat bran and Thermoascus aurantiacus produced48Ug with the same substrate [8 23] On the other handusing SmC (Figure 2(b)) only two types of substrates werecompared (1 of total carbon source 04 grams) cardboardand wheat bran with sugarcane bagasse (1 1) (SmCWB)Under these experimental conditions the lowest production129plusmn 11 Ugwas obtained after 192 hours but using cardboard(SmCP) production almost doubled to 244 plusmn 48Ug after 168hours of cultivation (Figure 2(c))

The zymogram of beta-glucosidases (Figure 3(b)) showeddifferent expression levels according to the cultivation systemand the carbon source used In all cultivation systems only


(a) (b)

Figure 3 Expression profile analyzed by zymography (a) endoglu-canase isoforms produced byM heterothallica F214 in submerged(Sm) and solid-state (SS) cultivation Line 1 SSCWB Line 2SmCWB Line 3 SSCP and Line 4 SmCP (b) 120573-glucosidasesproduced by submerged and solid-state cultivation Line 1 SSCWBLine 2 SmCWB Line 3 SSCP and Line 4 SmCP

two isozymes around 146 and 66 kDa were produced Appar-ently the use of the ratio (1 1) isoenzyme lower molecularweight had increased expression

33 Comparison of the Results Obtained by Both CultivationSystems Although by submerged cultivation (SmC) highervalues of productivity were obtained in terms of Ug com-pared to cultivation in the solid state two aspects must beconsidered first the volume of liquid obtained in the processIn SmC the enzyme was diluted 100 times while in SSC it wasdiluted only 20 times Since the aim of this project was theproduction of enzymes for use in their crude form the initialconcentration of the solution is important because it avoidsa step to concentrate the enzyme reducing the process costsand showing a frequent decreased yield of the enzyme due tononspecific adsorption to membranes and concentrators forexample an advantage of SSC

On the other hand previous data from our group(unpublished) showed no linearity between the increase inconcentration of the substrate (C source) from 1 to 25 andenzyme production a fact also described by Kumar et al[24] working with Aspergillus niger who in fact verified adecrease in cellulase andpectinase production above a certainconcentration of the carbon source

In addition the cultivation system also influences enzymeproduction since SSC has absence of water among theparticles of the solid substrate which have been reported as animportant factor that interferes in the extracellular enzymessecretion [25] The endoglucanase production in SSC has thebest benefit-cost ratio since it allows using agroindustrialresidues resembles the environmental conditions found bythe fungus has high productivity and demands a small areafor production [10]

34 Crude Enzyme Characterization

341 Endoglucanases A biochemical characterization wascarried out for the highest SSC and SmCproductions namelySSCWB and SmCP respectively The effect of pH on theendoglucanase activity was studied using CMC (4) assubstrate and the endoglucanases produced by both systemsshowed maximum activity at pH 55 (Figure 4(a))

Reported values have been found for optimum pH in thecrude extract in the range from pH 35 to 60 independentof the cultivation system [26 27] The crude endoglucanasesolution obtained from Pycnoporus sanguineus in SmCshowed optimum pH around 35ndash40 [27] Similarly [28 29]obtained the maximum activity at pH 40 for endoglucanasesfrom Trichoderma atroviride 676 and Penicillium sp CR-316respectively cultivated in SmC while others found a higherrange from 45 to pH 60 for the endoglucanases from thefungusAcremonium sp produced in SSC [26] and those fromM thermophila M77 exhibited an optimum pH of 50 [30]The endoglucanases produced by A niger in SSC exhibitedhigher activity at pH 40 [31 32]

The effect of the pH on the stability (Figure 4(b)) wasanalyzed in the pH range from 25 to 11 after incubation for 24hours at room temperature (25∘C) Endoglucanases producedby SSC and SmC exhibited high activity above pH 35ndash40 upto pH 90 The residual activity falls sharply at more acid oralkaline pH values

The effect of temperature on endoglucanase activity (Fig-ure 5(a)) was analyzed at pH 55 for the extracts obtained inboth cultivation systems The profiles are uneven suggestingthat the isoforms have different values for the temperatureoptima For enzyme from SSC the maximum activity wasreached at 60∘C which decreased to 40 at 80∘C On theother hand for the enzymes obtained in SmC the optimumtemperature was 65∘C the same value found for endoglu-canases obtained in SSC from A niger [32] EndoglucanasesfromA niger obtained by SSC have an optimum temperatureof 60∘C [31] and those obtained by SSC fromM thermophilaM77 exhibited maximum activity at 70∘C [30]

On the other hand analyzing the enzymes obtained bySmC those of P sanguineus displayed an optimum temper-ature of 60∘C [27] and values between 60 and 70∘C for theendoglucanases fromT atroviride 676 [28] and similar valueswere reported for the enzymes from the fungusPenicillium spCR-316 [29]

Concerning the thermal stability of endoglucanases (Fig-ure 5(b)) the residual activity was measured after incubationof the extract at different temperatures between 40 and 70∘Cfor 1 hour The endoglucanases produced by SmC displayedactivity above 80 between 40 and 60∘C On the other handthe endoglucanases produced by SSC exceed 100 of residualactivity at 50∘C and only decay after 60∘C Endoglucanasesproduced by SSC and SmC lost around 80 of their activityduring 1 hour at 70∘CThese endoglucanasesweremore stablethan those produced by SSC fromM thermophilaM77 [30]

The effect of several compounds on endoglucanase activ-ity was analyzed using the crude extract produced by SSCWBand SmCP (Table 1) The effects showed some discrepanciesbetween the enzymes produced in both conditions showing


pH12111098765432

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

(a)

pH

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

12108642

(b)

Figure 4 Effect of pH on the activity of the endoglucanases from the crude extract from M heterothallica F214 (a) (◼) SSCWB and (I)SmCP respectively Effect of the pH on the stability of the endoglucanases (b) (◼) SSCWB and (I) SmCP respectively

Temperature (∘C)

Rela

tive a

ctiv

ity (

)

85807570656055504540

100

80

60

40

20

0

(a)

Temperature (∘C)

Resid

ual a

ctiv

ity (

)

100

80

60

40

20

0

757065605550454035

(b)

Figure 5 Effect of temperature on the activity of endoglucanase of the crude extract fromM heterothallica F214 at pH 55 (a) (◼) SSCWBand (I) SmCP respectively Temperature effect on the stability of the endoglucanases (b) (◼) SSCWB and (I) SmCP respectively

opposite effects or different degrees of activationinhibitionThe most remarkable difference occurred for MnCl

2 which

caused a strong inhibition for the endoglucanases obtainedby SSC while increasing the activity of the enzymes fromSmC suggesting that the ldquonewrdquo isoenzymes expressed in thiscultivation system anddescribed abovewould be significantlyactivated by that cation Since EDTA inhibited the endoglu-canases produced by both cultivation systems it was expectedthat a divalent cation would increase the enzyme activityIron inhibition could be attributed to the effect of Fe3+ at thereducing ends of cellulose [33]

342 120573-Glucosidases The 120573-glucosidase activity producedby both cultivation systems showed maximum activity at pH50 (Figure 6(a)) Reported values for optimum pH of fungal120573-glucosidases fall into a wide range 2ndash25 [34] 45 [23 27]48 [35] and 60 [36] but most of these enzymes display

higher activity in the pH range 4-5 [37] The effect of thepH on the stability (Figure 6(b)) was analyzed on the pHrange from 25 to 11 after incubation for 24 hours at roomtemperature (25∘C)The120573-glucosidases produced by SSC andSmC exhibited high activity above pH 35 up to 100 Theresidual activity falls sharply at more acid or alkaline pHvalues

The effect of temperature on 120573-glucosidase activity isshown in Figure 7(a) analyzed at pH 50 for the extractsobtained in both cultivation systems The profile for SmCsuggests that the isoforms have different values for thetemperature optima For SSC the maximum value was 65∘Cand for SmC 70∘C Falkoski et al [27] found an optimumtemperature of 55∘C and for Iembo et al [34] it was 65∘C

The thermal stability (Figure 7(b)) was determined bymeasuring residual activity after incubation at 40ndash70∘Cduring 1 hour Both crude extracts were stable up to 60∘C


Table 1 Effect of different compounds on endoglucanase and beta-glucosidase relative activity of the dialyzed crude extract expressed asmean values plusmn SD NT = not tested ND = not detected The asterisks represent significant differences against the control with 119901 lt 005according to Studentrsquos 119905-test for means

Compound Conc (mM) Endoglucanases 120573-GlucosidasesRel act SSC () Rel act SmC () Rel act SSC () Rel act SmC ()

PVA 10 1209 plusmn 28lowast

1102 plusmn 104 NT NTIsopropanol 10 1167 plusmn 07

lowast741 plusmn 16

lowast1064 plusmn 41 929 plusmn 50

NaCl 10 1133 plusmn 07lowast

903 plusmn 39lowast

959 plusmn 39 715 plusmn 25lowast

DTT 10 1125 plusmn 09lowast

1214 plusmn 49lowast

864 plusmn 26lowast

929 plusmn 36

PMSF 1 1102 plusmn 03lowast


1012 plusmn 64

Glucose 10 1095 plusmn 09lowast


690 plusmn 11lowast

DMSO 10 1093 plusmn 23lowast

1089 plusmn 188 NT NTAcetone 10 1010 plusmn 12 959 plusmn 84 927 plusmn 34 1061 plusmn 54

Triton X-100 10 1012 plusmn 13 1143 plusmn 167 NT NTEthanol 10 1010 plusmn 29 714 plusmn 37


MgCl2

10 897 plusmn 21lowast

351 plusmn 34lowast


PEG 8000 3 855 plusmn 04lowast

937 plusmn 53 NT NTPEG 3350 10 1128 plusmn 17

lowast971 plusmn 76 NT NT

CaCl2


314 plusmn 25lowast

795 plusmn 76lowast

1007 plusmn 20

SDS 10 570 plusmn 08lowast

821 plusmn 45lowast

799 plusmn 16lowast

808 plusmn 29lowast

EDTA 10 845 plusmn 27lowast


892 plusmn 89 940 plusmn 25

AlCl3


17 plusmn 07lowast


212 plusmn 07lowast

MnCl2




1050 plusmn 17

FeCl3

10 110 plusmn 04lowast NDlowast 213 plusmn 141

lowast NDlowast

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

pH12111098765432

(a)

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

2 4 6 8 10 12

pH

(b)

Figure 6 Effect of pH on the activity of the 120573-glucosidases from the crude extract from M heterothallica F214 (a) (◼) SSCWB and (I)SmCP respectively Effect of the pH on the stability of the 120573-glucosidases (b) (◼) SSCWB and (I) SmCP respectively

The effect of several compounds on120573-glucosidase activitywas analyzed using the crude extract produced by SSCWBand SmCP (Table 1) The effects showed some discrepanciesbetween the enzymes produced in both conditions showingopposite effects or different degrees of activationinhibitionThe most remarkable difference occurred for MnCl

2 which

caused a strong activation for 120573-glucosidase obtained bySSC while increasing the activity of those enzymes fromSmC

4 Conclusions

The thermophilic fungus M heterothallica F214 proved tobe a great producer of endoglucanases using both sugarcanebagasse and wheat bran by solid-state cultivation or usingcardboard in submerged cultivation Cardboard in SmCwould be a residue of easy access and would be able toinduce the synthesis of more isoforms of endoglucanaseswhich when characterized in the crude extract was shown


Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0

Temperature (∘C)90858075706560555045403530

(a)

Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0

Temperature (∘C)70656055504540

(b)

Figure 7 Effect of temperature on the activity of 120573-glucosidase of the crude extract fromM heterothallica F214 at pH 50 (a) (◼) SSCWBand (I) SmCP Temperature effect on the stability of the 120573-glucosidases (b) (◼) SSCWB and (I) SmCP

to be thermostable and prompted us to perform further stud-ies of biotechnological applications The beta-glucosidasesobtained by SmC showed higher stability

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors thank FAPESP and CNPq (Brazil) for financialsupport and Dr Marcia Maria de Souza Moretti for herhelpful discussion of the results

References

[1] M Dashtban H Schraft andW Qin ldquoFungal bioconversion oflignocellulosic residues opportunities amp perspectivesrdquo Interna-tional Journal of Biological Sciences vol 5 no 6 pp 578ndash5952009

[2] R Kumar S Singh and O V Singh ldquoBioconversion of lig-nocellulosic biomass biochemical and molecular perspectivesrdquoJournal of Industrial Microbiology amp Biotechnology vol 35 no5 pp 377ndash391 2008

[3] M Linder and T T Teeri ldquoThe roles and function of cellulose-binding domainsrdquo Journal of Biotechnology vol 57 no 1ndash3 pp15ndash28 1997

[4] J Zhou Y-H Wang J Chu Y-P Zhuang S-L Zhang and PYin ldquoIdentification and purification of the main components ofcellulases from a mutant strain of Trichoderma viride T 100-14rdquoBioresource Technology vol 99 no 15 pp 6826ndash6833 2008

[5] O Kirk T V Borchert and C C Fuglsang ldquoIndustrial enzymeapplicationsrdquo Current Opinion in Biotechnology vol 13 no 4pp 345ndash351 2002

[6] R C Kuhad R Gupta and A Singh ldquoMicrobial cellulases andtheir industrial applicationsrdquoEnzymeResearch vol 2011 ArticleID 280696 10 pages 2011

[7] C V Nascimento F H M Souza D C Masui et al ldquoPurifi-cation and biochemical properties of a glucose-stimulated 120573-D-glucosidase produced by Humicola grisea var thermoideagrown on sugarcane bagasserdquo Journal of Microbiology vol 48no 1 pp 53ndash62 2010

[8] D Deswal Y P Khasa and R C Kuhad ldquoOptimization of cellu-lase production by a brown rot fungus Fomitopsis sp RCK2010under solid state fermentationrdquoBioresource Technology vol 102no 10 pp 6065ndash6072 2011

[9] M Narra G Dixit J Divecha K Kumar D Madamwar andA R Shah ldquoProduction purification and characterization ofa novel GH 12 family endoglucanase from Aspergillus terreusand its application in enzymatic degradation of delignified ricestrawrdquo International Biodeterioration amp Biodegradation vol 88pp 150ndash161 2014

[10] A Pandey C R Soccol and DMitchell ldquoNew developments insolid state fermentation I-bioprocesses and productsrdquo ProcessBiochemistry vol 35 no 10 pp 1153ndash1169 2000

[11] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[12] NC-IUB ldquoUnits of enzyme activityrdquo European Journal of Bio-chemistry vol 97 no 2 pp 319ndash320 1979

[13] A C Alfenas Eletroforese de Isoenzimas a Proteinas AfinsFundamentos e Aplicacoes em Plantas e Microrganismos Uni-versidade Federal de Vicosa Vicosa Brazil 1998

[14] K-S Kwon J Lee H G Kang and Y C Hah ldquoDetection of120573-glucosidase activity in polyacrylamide gels with esculin assubstraterdquoApplied and Environmental Microbiology vol 60 no12 pp 4584ndash4586 1994

[15] M Ayres J R M Ayres D L Ayres and A S Santos BioEstat50mdashAplicacoes Estatısticas nas Areas das Ciencias Biologicas eMedicas Sociedade CivilMamiraua CNPq Belem Brazil 2007

[16] J Gao H Weng Y Xi D Zhu and S Han ldquoPurificationand characterization of a novel endo-120573-14-glucanase from thethermoacidophilic Aspergillus terreusrdquo Biotechnology Lettersvol 30 no 2 pp 323ndash327 2008

[17] G Panagiotou D Kekos B J Macris and P Christakopou-los ldquoProduction of cellulolytic and xylanolytic enzymes by


Fusarium oxysporum grown on corn stover in solid statefermentationrdquo Industrial Crops and Products vol 18 no 1 pp37ndash45 2003

[18] M S Singhvi M G Adsul and D V Gokhale ldquoComparativeproduction of cellulases by mutants of Penicillium janthinellumNCIM 1171 and its application in hydrolysis of Avicel andcelluloserdquo Bioresource Technology vol 102 no 11 pp 6569ndash6572 2011

[19] M A Haque M Shams-Ud-Din and A Haque ldquoThe effect ofaqueous extracted wheat bran on the baking quality of biscuitrdquoInternational Journal of Food Science and Technology vol 37 no4 pp 453ndash462 2002

[20] R R Singhania R K Sukumaran K P Rajasree A MathewL Gottumukkala and A Pandey ldquoProperties of a major 120573-glucosidase-BGL1 from Aspergillus niger NII-08121 expresseddifferentially in response to carbon sourcesrdquo Process Biochem-istry vol 46 no 7 pp 1521ndash1524 2011

[21] GEPLACEA-ICIDCA MANUAL de los Derivados de la Canade Azucar Grupo de Paises Latinoamericanos y del CaribeExportadores de azucar-Instituto Cubano de Investigaciones delos Derivados de Cana de Azucar Mexico 1990

[22] T Kinnarinen and A Hakkinen ldquoInfluence of enzyme loadingon enzymatic hydrolysis of cardboard waste and size distribu-tion of the resulting fiber residuerdquo Bioresource Technology vol159 pp 136ndash142 2014

[23] EKalogeris P Christakopoulos P Katapodis et al ldquoProductionand characterization of cellulolytic enzymes from the ther-mophilic fungus Thermoascus aurantiacus under solid statecultivation of agricultural wastesrdquo Process Biochemistry vol 38no 7 pp 1099ndash1104 2003

[24] S Kumar H K Sharma and B C Sarkar ldquoEffect of substrateand fermentation conditions on pectinase and cellulase produc-tion by Aspergillus niger NCIM 548 in submerged (SmF) andsolid state fermentation (SSF)rdquo Food Science and Biotechnologyvol 20 no 5 pp 1289ndash1298 2011

[25] E B N Graminha A Z L Goncalves R D P B Pirota M AA Balsalobre R Da Silva and E Gomes ldquoEnzyme productionby solid-state fermentation application to animal nutritionrdquoAnimal Feed Science and Technology vol 144 no 1-2 pp 1ndash222008

[26] M N de Almeida V M Guimaraes K M Bischoff et alldquoCellulases and hemicellulases from endophytic Acremoniumspecies and its application on sugarcane bagasse hydrolysisrdquoApplied Biochemistry and Biotechnology vol 165 no 2 pp 594ndash610 2011

[27] D L Falkoski V M Guimaraes M N de Almeida A CAlfenas J L Colodette and S T de Rezende ldquoCharacterizationof cellulolytic extract from Pycnoporus sanguineus PF-2 and itsapplication in biomass saccharificationrdquo Applied Biochemistryand Biotechnology vol 166 no 6 pp 1586ndash1603 2012

[28] A L Grigorevski-Lima M M Q De Oliveira R P DoNascimento E P Da Silva Bon and R R R Coelho ldquoPro-duction and partial characterization of cellulases and xylanasesfrom Trichoderma atroviride 676 using lignocellulosic residualbiomassrdquo Applied Biochemistry and Biotechnology vol 169 no4 pp 1373ndash1385 2013

[29] P Picart P Diaz and F I Pastor ldquoCellulases from two Penicil-lium sp strains isolated from subtropical forest soil productionand characterizationrdquo Letters in Applied Microbiology vol 45no 1 pp 108ndash113 2007

[30] M M S Moretti D A Bocchini-Martins R Da Silva ARodrigues LD Sette and EGomes ldquoSelection of thermophilic

and thermotolerant fungi for the production of cellulases andxylanases under solid-state fermentationrdquo Brazilian Journal ofMicrobiology vol 43 no 3 pp 1062ndash1071 2012

[31] N Bansal R Tewari R Soni and S K Soni ldquoProduction ofcellulases fromAspergillus nigerNS-2 in solid state fermentationon agricultural and kitchenwaste residuesrdquoWasteManagementvol 32 no 7 pp 1341ndash1346 2012

[32] TNcube R LHoward EKAbotsi E L J vanRensburg and INcube ldquoJatropha curcas seed cake as substrate for production ofxylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentationrdquo Industrial Crops and Products vol 37 no 1pp 118ndash123 2012

[33] A Tejirian and F Xu ldquoInhibition of cellulase-catalyzed lig-nocellulosic hydrolysis by iron and oxidative metal ions andcomplexesrdquo Applied and Environmental Microbiology vol 76no 23 pp 7673ndash7682 2010

[34] T Iembo R Da-Silva F C Pagnocca and E Gomes ldquoProduc-tion characterization and properties of beta-glucosidase andbeta-xylosidase from a strain ofAureobasidium sprdquoPrikladnaiabiokhimiia i mikrobiologiia vol 38 no 6 pp 639ndash643 2002

[35] D Pericin and M Jarak ldquoProduction and some characteristicsof beta-glucosidase in Diaporthe (Phomopsis) helianthirdquo ActaMicrobiologica et ImmunologicaHungarica vol 42 no 1 pp 29ndash37 1995

[36] J Kaur B S Chadha B A Kumar G S Kaur and HS Saini ldquoPurification and characterization of 120573-glucosidasefrom Melanocarpus sp MTCC 3922rdquo Electronic Journal ofBiotechnology vol 10 no 2 pp 260ndash270 2007

[37] A Soslashrensen M Lubeck P S Lubeck and B K AhringldquoFungal beta-glucosidases a bottleneck in industrial use oflignocellulosic materialsrdquo Biomolecules vol 3 no 3 pp 612ndash631 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


or cellobiohydrolases (EC 32191) attack the nonreducingor reducing chain ends and 120573-14-glucosidases (EC 32121)hydrolyze cellobiose releasing glucose [4]

Fungal cellulolytic enzymes have biotechnological appli-cations in food animal feed wine agriculture biomassrefining and pulp and paper industries [5 6] The biggestchallenge for the enzymesrsquo biotechnological application istheir high cost of production The search for methodolo-gies that could reduce the cost of enzyme production andmicroorganismswith high growth rate and are able to developin cheap and easily accessible carbon sources is the currentapproach to change this paradigm [7]Thus various cellulosicsubstrates such as sugarcane bagasse corn stover wheatstraw and wheat bran have been tested by several authors forproduction of cellulolytic enzymes [8 9]

The composition of the medium can influence the profileof enzymatic expression by the microorganism Cellulasescan be obtained more economically by microorganismsthrough submerged or solid-state cultivation The low wateractivity in solid-state cultivation (SSC) shows a simple tech-nique of high productivity but it can affect the heat transfer tothe substrate and thus could affect the growth of themicroor-ganism [10] The use of thermophilic microorganisms can bean alternative to this technical limitation since they toleratehigher temperatures

The present study aimed to compare the production ofendoglucanases and 120573-glucosidases in solid-state and sub-merged cultivation media by thermophilic Myceliophthoraheterothallica and to characterize the crude extracts obtainedfrom both systems

2 Materials and Methods

21Microorganism The thermophilic fungusMyceliophthoraheterothallica F214 was isolated from sugar cane bagassecompost and maintained at the Laboratory of Applied Bio-chemistry and Microbiology UNESP

22 Inoculum Preparation The inoculum was prepared bygrowing the fungusM heterothallica F214 in 250mL Erlen-meyer flasks with 50mL medium composed of 10 sugar-cane bagasse (ground to 1-2mm) 014 (NH

4)2SO4 020

KH2PO4 003 CaCl

2 002 MgSO

2sdot7H2O 050 beef

peptone 020 yeast extract 003 urea 010 micronu-trients solution (5mgL FeSO

4sdot7H2O 16mgLMnSO

4sdotH2O

14mgL ZnSO4sdot7H2O and 20mgL CoCl

2) 02 glucose

and 20 agar pH 50 All the solutions and the culturemediawere sterilized by autoclaving at 121∘C for 20 minutes Thismedium was inoculated with suspension obtained scrappingthemycelium from culture grown for 3 days at 45∘C in 150mLof nutrient solution (gL) 35 (NH

4)2SO4 30 KH

2PO4 05

MgSO4sdot7H2O 05 CaCl

2 and 01 vv Tween 80 pH 50

23 Submerged Cultivation (SmC) Endoglucanase and 120573-glucosidase production in SmC was carried out in 250mLErlenmeyer flasks using cardboard as a carbon source previ-ously cut and ground in a blender The mixture contained 1cardboard and 20mLof the nutrient solution described above

was inoculated with 2mL of the microorganism inoculumas described The cultures were incubated on a rotary shaker(100 RPM) at 45∘C for 10 days and samples were taken every24 h filtered through vacuum usingWhatman paper number1 and centrifuged at 6000timesg for 20minutesThe supernatantwas used as a crude enzyme solution

In order to evaluate the use of agroindustrial residuessuch as wheat bran and sugar cane bagasse as the substratein SmC they were washed ground and oven-dried (60∘C for48 h) The sugarcane bagasse was sieved allowing selectingparticles ranging from 1 to 2mm Subsequently cultivationwas carried out in 250mL Erlenmeyer flasks containing 1(1 1 ww) of wheat bran and sugarcane bagasse 20mL nutri-ent solution and 2mL inoculum suspension The mediumwas previously autoclaved as described The cultures wereincubated on a rotary shaker (100 RPM) at 45∘C for 10 daysand sampling was performed as explained above

24 Solid-State Cultivation (SSC) The substrates used in theSSC were composed of used 5 grams of cardboard or amixture of wheat bran and sugar cane bagasse (1 1) Thesubstrates were inserted in polypropylene bags (12 times 20 cm)and sterilized at 121∘C for 40minThe fungus inoculation wascarried out by adding 20mL of themycelium suspensionThemoisture content of around 80 was reached with the addi-tion of sterile distilled water The fermentation was at 45∘Cfor 14 days Every 48 h one bag was taken and the fermentedmaterial was mixed with 20mL of distilled water per gramstirred for 30min filtered and centrifuged at 10000timesg at10∘CThe supernatant was used as a crude enzyme solution

25 Enzymatic Assay Endoglucanase activity was measuredat 60∘C in a reactional mixture composed of 09mL of100mMLminus1 sodium acetate buffer pH 50 containing 4of carboxymethyl cellulose (CMC) (Sigma-Aldrich USA)and 01mL of crude enzyme solution The reducing groupsexpressed as glucose released were assayed by using theDNS (35-dinitrosalicylic acid) method [11] One unit ofendoglucanase activity was defined as the amount of enzymeable to release 1 120583mol of reducing sugars per min under theassay conditions [12]120573-D-Glucosidase activity was measured in a mixture of

50mm sodium acetate buffer pH 50 500 120583L of 2mMLminus1p-nitrophenyl-120573-D-glucopyranoside (pNP-Glc) as substrateand 50120583L of the enzyme solution incubated at 60∘C for10min The reaction was stopped by addition of 2mL of2M Na

2CO3and the absorbance was read at 410 nm One

unit of 120573-glucosidase was defined as the amount of enzymethat releases 1 120583mole of nitrophenolmin in the reactionconditions

All the enzyme assays were performed in triplicate

26 Zymogram for Enzyme Activity The endoglucanase solu-tion was analyzed by electrophoresis on denaturing 10sodium dodecyl sulphate (SDS) polyacrylamide gel usingTris-Glycine buffer pH 83 After electrophoresis the enzymewas renatured with 1 triton X-100 for 30 minutes andsubsequently the gel was overlaid with a mixture of 1 (wv)


Activ

ity (U

g)

1250

1000

750

500

250

0

Time (h)24019214496480

(a)

Time (h)264240216192168144120967248240

(Um

L)

(Ug

)

08

06

04

02

00

80

60

40

20

0

(b)

Time (h)264240216192168144120967248240

(Ug

)

(Um

L)

3500

3000

2500

2000

1500

1000

500

0 0

5

10

15

20

25

30

35

(c)










(Ug

)

70

60

50

40

30

20

10

0

Time (h)33628824019214496480

(a)

Time (h)264240216192168144120967248240

0

30

60

90

120

150

180

(Ug

)

00

03

06

09

12

15

18

(Um

L)

(b)

Time (h)

0

50

100

150

200

250

300

(Ug

)00

05

10

15

20

25

30

(Um

L)

264240216192168144120967248240

(c)









(a) (b)















pH12111098765432

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

(a)

pH

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

12108642

(b)


Temperature (∘C)

Rela

tive a

ctiv

ity (

)

85807570656055504540

100

80

60

40

20

0

(a)

Temperature (∘C)

Resid

ual a

ctiv

ity (

)

100

80

60

40

20

0

757065605550454035

(b)



2 which











lowast741 plusmn 16



903 plusmn 39lowast




864 plusmn 26lowast

929 plusmn 36



1012 plusmn 64



690 plusmn 11lowast





MgCl2


351 plusmn 34lowast





CaCl2


314 plusmn 25lowast

795 plusmn 76lowast

1007 plusmn 20


821 plusmn 45lowast

799 plusmn 16lowast

808 plusmn 29lowast




AlCl3


17 plusmn 07lowast


212 plusmn 07lowast

MnCl2




1050 plusmn 17

FeCl3


lowast NDlowast

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

pH12111098765432

(a)

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

2 4 6 8 10 12

pH

(b)



2 which


4 Conclusions



Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0

Temperature (∘C)90858075706560555045403530

(a)

Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0


(b)



Competing Interests


Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Activ

ity (U

g)

1250

1000

750

500

250

0

Time (h)24019214496480

(a)

Time (h)264240216192168144120967248240

(Um

L)

(Ug

)

08

06

04

02

00

80

60

40

20

0

(b)

Time (h)264240216192168144120967248240

(Ug

)

(Um

L)

3500

3000

2500

2000

1500

1000

500

0 0

5

10

15

20

25

30

35

(c)










(Ug

)

70

60

50

40

30

20

10

0

Time (h)33628824019214496480

(a)

Time (h)264240216192168144120967248240

0

30

60

90

120

150

180

(Ug

)

00

03

06

09

12

15

18

(Um

L)

(b)

Time (h)

0

50

100

150

200

250

300

(Ug

)00

05

10

15

20

25

30

(Um

L)

264240216192168144120967248240

(c)









(a) (b)















pH12111098765432

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

(a)

pH

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

12108642

(b)


Temperature (∘C)

Rela

tive a

ctiv

ity (

)

85807570656055504540

100

80

60

40

20

0

(a)

Temperature (∘C)

Resid

ual a

ctiv

ity (

)

100

80

60

40

20

0

757065605550454035

(b)



2 which











lowast741 plusmn 16



903 plusmn 39lowast




864 plusmn 26lowast

929 plusmn 36



1012 plusmn 64



690 plusmn 11lowast





MgCl2


351 plusmn 34lowast





CaCl2


314 plusmn 25lowast

795 plusmn 76lowast

1007 plusmn 20


821 plusmn 45lowast

799 plusmn 16lowast

808 plusmn 29lowast




AlCl3


17 plusmn 07lowast


212 plusmn 07lowast

MnCl2




1050 plusmn 17

FeCl3


lowast NDlowast

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

pH12111098765432

(a)

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

2 4 6 8 10 12

pH

(b)



2 which


4 Conclusions



Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0

Temperature (∘C)90858075706560555045403530

(a)

Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0


(b)



Competing Interests


Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(Ug

)

70

60

50

40

30

20

10

0

Time (h)33628824019214496480

(a)

Time (h)264240216192168144120967248240

0

30

60

90

120

150

180

(Ug

)

00

03

06

09

12

15

18

(Um

L)

(b)

Time (h)

0

50

100

150

200

250

300

(Ug

)00

05

10

15

20

25

30

(Um

L)

264240216192168144120967248240

(c)









(a) (b)















pH12111098765432

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

(a)

pH

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

12108642

(b)


Temperature (∘C)

Rela

tive a

ctiv

ity (

)

85807570656055504540

100

80

60

40

20

0

(a)

Temperature (∘C)

Resid

ual a

ctiv

ity (

)

100

80

60

40

20

0

757065605550454035

(b)



2 which











lowast741 plusmn 16



903 plusmn 39lowast




864 plusmn 26lowast

929 plusmn 36



1012 plusmn 64



690 plusmn 11lowast





MgCl2


351 plusmn 34lowast





CaCl2


314 plusmn 25lowast

795 plusmn 76lowast

1007 plusmn 20


821 plusmn 45lowast

799 plusmn 16lowast

808 plusmn 29lowast




AlCl3


17 plusmn 07lowast


212 plusmn 07lowast

MnCl2




1050 plusmn 17

FeCl3


lowast NDlowast

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

pH12111098765432

(a)

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

2 4 6 8 10 12

pH

(b)



2 which


4 Conclusions



Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0

Temperature (∘C)90858075706560555045403530

(a)

Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0


(b)



Competing Interests


Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)















pH12111098765432

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

(a)

pH

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

12108642

(b)


Temperature (∘C)

Rela

tive a

ctiv

ity (

)

85807570656055504540

100

80

60

40

20

0

(a)

Temperature (∘C)

Resid

ual a

ctiv

ity (

)

100

80

60

40

20

0

757065605550454035

(b)



2 which











lowast741 plusmn 16



903 plusmn 39lowast




864 plusmn 26lowast

929 plusmn 36



1012 plusmn 64



690 plusmn 11lowast





MgCl2


351 plusmn 34lowast





CaCl2


314 plusmn 25lowast

795 plusmn 76lowast

1007 plusmn 20


821 plusmn 45lowast

799 plusmn 16lowast

808 plusmn 29lowast




AlCl3


17 plusmn 07lowast


212 plusmn 07lowast

MnCl2




1050 plusmn 17

FeCl3


lowast NDlowast

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

pH12111098765432

(a)

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

2 4 6 8 10 12

pH

(b)



2 which


4 Conclusions



Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0

Temperature (∘C)90858075706560555045403530

(a)

Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0


(b)



Competing Interests


Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


pH12111098765432

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

(a)

pH

20

40

60

80

100

0

Rela

tive a

ctiv

ity (

)

12108642

(b)


Temperature (∘C)

Rela

tive a

ctiv

ity (

)

85807570656055504540

100

80

60

40

20

0

(a)

Temperature (∘C)

Resid

ual a

ctiv

ity (

)

100

80

60

40

20

0

757065605550454035

(b)



2 which











lowast741 plusmn 16



903 plusmn 39lowast




864 plusmn 26lowast

929 plusmn 36



1012 plusmn 64



690 plusmn 11lowast





MgCl2


351 plusmn 34lowast





CaCl2


314 plusmn 25lowast

795 plusmn 76lowast

1007 plusmn 20


821 plusmn 45lowast

799 plusmn 16lowast

808 plusmn 29lowast




AlCl3


17 plusmn 07lowast


212 plusmn 07lowast

MnCl2




1050 plusmn 17

FeCl3


lowast NDlowast

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

pH12111098765432

(a)

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

2 4 6 8 10 12

pH

(b)



2 which


4 Conclusions



Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0

Temperature (∘C)90858075706560555045403530

(a)

Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0


(b)



Competing Interests


Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






lowast741 plusmn 16



903 plusmn 39lowast




864 plusmn 26lowast

929 plusmn 36



1012 plusmn 64



690 plusmn 11lowast





MgCl2


351 plusmn 34lowast





CaCl2


314 plusmn 25lowast

795 plusmn 76lowast

1007 plusmn 20


821 plusmn 45lowast

799 plusmn 16lowast

808 plusmn 29lowast




AlCl3


17 plusmn 07lowast


212 plusmn 07lowast

MnCl2




1050 plusmn 17

FeCl3


lowast NDlowast

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

pH12111098765432

(a)

100

80

60

40

20

0

Rela

tive a

ctiv

ity (

)

2 4 6 8 10 12

pH

(b)



2 which


4 Conclusions



Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0

Temperature (∘C)90858075706560555045403530

(a)

Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0


(b)



Competing Interests


Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0

Temperature (∘C)90858075706560555045403530

(a)

Rela

tive a

ctiv

ity (

)

20

40

60

80

100

0


(b)



Competing Interests


Acknowledgments


References
















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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