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CHAPTER 4IMMOBILIZATION OF DIASTASEa-AMYLASE
4.1 INTRODUCTION ................................................................................................... ..69
4.2 IMMOBILIZATION OF a-AMYLASE ON EMERALDINE SALT ................ ..70
4.2.1 COUPLING OF a-AMYLAsE A.\1D IMMOBILIZATION EFFICIENCY ............................. ..70
4.2.2 EFFECT OF PH ON ENZYME ACTIVITY ................................................................... ..75
4.2.3 EFFECT OF TEMPERATURE O.\J TIIE ACTIYITY ........................................................ .. 77
4.2.4 THERMAL STABILITY OF THE FREE AND IMMOBILIZED ENZYMES .......................... ..78
4.2.5 DETERMINATION OF MICHAELIS CoNsTANT ........................................................ ..80
4.2.6 STORAGE STABILITY OF THE IMMOBILIZED 0t—AMYLASE ...................................... .. 82
4.2.7 REUSAEILITY ....................................................................................................... ..83
4.3 KMMOBILIZATION OF a»AMYLASE ON EMERALDINE BASE ................ ..84
4.3.1 OPTIMIZATION OF IMMOBILIZATION CONDITIONS ................................................ ..84
4.3 .2 EFFECT OF PH ON THE ENZYME ACTIVITY ........................................................... .. 89
4.3.3 EFFECT OF TEMPERATURE O.\J ACTIVITY ............................................................... ..90
4.3.4 EFFECT OF TEMPERATURE ON STABILITY ............................................................. ..90
4.3.5 KINETIC C0NsTANTs ............................................................................................ ..92
4.3.6 STORAGE STABILITY ........................................................................................... ..94
4.3.7 ACTIVITY AFTER VARIOUS CYCLES ...................................................................... ..95
4.4 IMMOBILIZATION OF (Z-AMYLASE ON POLY(O-TOLUIDINE) SALT.....96
4.4.1 ENZYME IMMOBIL-IZATlO.\I PARAMETERS ............................................................. .. 96
4.4.2 EFFECT OF PH ON TIIE ACTIVITY OF FREE AND IMMOBILIZED OL—A]\/IYLASE ......... .. 100
4.4.3 EFFECT OF TEMPERATURE ON ACTIYITY ............................................................. .. 101
4.4.4 THERMAL STABILITY ......................................................................................... .. 102
4.4.5 KINETIC PARAMETF.Rs OF TIIE AMYLASE IMMOBILIZED ON TS ........................... ..103
4.4.6 STORACE STABILITY .......................................................................................... .. 105
4.4.7 REUsE OF IMMOBILIZEI) F.\‘ZYMF.S ..................................................................... .. 106
4.5 IMMOBILIZATION OF a-AMYLASE ON POLY(O-TOLUIDINE) BASE...107
4.5.1 OPTIMAL PARAMETERS FOR (1-AMYL-ASE IMMOBILIZATION ............................... .. 107
4.5.2 EFFECT OF PH ow ACTIVITY ............................................................................... ..1 I 1
4.5.3 EFFECT OF TEMPERATURE ON THE ACTIVITY ...................................................... .. 1 1 1
4.5.4 TI-IERMAL STABILITY ......................................................................................... .. 1 12
4.5.5 KINETIC PARAMETERS ....................................................................................... .. 1 14
4.5.6 STORAGE STABILITY .......................................................................................... .. 116
4.5.7 REUSABILITY ..................................................................................................... .. l 16
4.6 CONCLUSION ...................................................................................................... ..1l7
REFERENCE-S ............................................................................................................ ..ll9
§hapter 4 g Immobilization of Diastase a-amylasel
4.1 Introduction
Diastase (from Greek time-totazg, "separation") is any member of a class of enzymes
occuring in the seed of grains and malt which catalyses the breakdown of starch into
smaller carbohydrate molecules such as maltose. lt was the first type of enzymes
discovered, in 1833, by Anselme Payen [1], who found it in malt solution. Today, diastase
means any 0:-, /3-, or )1-amylase (all of them hydrolases which differ in the way they attack
the bonds of the starch molecules) that can break down carbohydrates.
a-amylase (EC 3.2.1.1; l, 4 a-d-glucan glucanohydrolase) is a widespread enzyme
occurring in mammals as salivary and pancreatic 0:-amylase; in the seeds of plants, it is
produced by large numbers of microorganisms. The a-amylases are calcium
metalloenzymes, completely unable to fimction in the absence of calcium. This enzyme
catalyses the hydrolysis of a-l, 4-glucosidic linkages in amylose, amylopectin and
glycogen in an endo-fashion [2, 3]. lt does not hydrolyse the a-1, 6 linkages or any other
branch points and so produces maltose and limit dcxtrins; the precise action pattem
depends on the source of the 0:-amylase [4]. Amylases, which are starch hydrolyzing
enzymes, are the most important industrial enzymes and are used in different processes in
food, textile, pharmaceutical industries, beverage industries etc. [2]. Bacillus subri1i.s',
Bacilizts amylo/iquefacines, Bacillus iicheniformis and Aspergillus oriyzae are very
important 0:-amylase sources [5-8] because their enzymes are highly thcrmostable. 0:
Amylase is known to attack both insoluble starch and starch granules held in aqueous
suspension [9].
To improve their economic feasibility in food, pharmaceutical, medical, industrial
and technological processes, soluble enzymes are usually immobilized onto a solid support.
ln order to achieve the desired catalytic activity, support material can have a critical role in
the process of immobilization. Several attempts have been carried out in the past to
immobilize the enzyme a-amylase on different supports [7, 8, 10-33]. Some examples
involve zirconium dynamic membrane [7], glass beads and glutaraldehyde fixation [10].
69
A Study on the Immobilization of Enzymes on Polymeric Supports
covalent cross-linking onto silica gel [1 l], coconut fiber [12], and adsorption on zirconia
[13]. Many of the immobilization processes were done by using polymer based supports
[14-19]. T iimtiirk et al. [14] covalently immobilized a-amylase on po1y(2-hydroxyethyl
methacrylate) and poly(styrene-2-hydroxyethyl methacrylate) micro spheres, which were
activated using epichlorohydrin. Varlan et al. had reported [15] paramagnetic polyacrolein
beads as a support for immobilization. Bajpai et al. used [17] semi-interpenetrating
polymer network of poly(ethylene glycol), poly(vinyl alcohol) and polyacrylamide as
support for immobilization of a-amylase.
This chapter describes the immobilization of diastase a-amylase under very mild
conditions on polyaniline and poly(0-toluidine), both were prepared as acidic and basic
forms. Immobilization efficiency and enzyme activity of a-amylase were examined at
various pH and temperature and discussed in detail.
4.2 Immobilization of a-amylase on Emeraldine Salt
4.2.1 Coupling of a-amylase and immobilization efficiency
The uptake of enzyme varied significantly according to the support, method chosen,
immobilization medium, enzyme concentration and contact time of enzyme to support.
Therefore these parameters were tested to optimize enzyme immobilization on emeraldine
salt (ES), glutaraldehyde activated emeraldine salt (ESI), and ascorbic acid activated
emeraldine salt (ES2). The effect of solution pH dLu'ing the immobilization step on enzyme
activity is shown in the fig. 4.2.1. The best results were obtained in the pH range from 4.5
to 5.5, with maximum activity retention at pH 5.0. The optimum pH is that which favors the
bonding between support and enzyme, and the pH which favors the enzyme to retain its
activity. The pH stability of the enzyme also has to be considered. Selecting an optimum
solution pH for enzyme immobilization is therefore important. Even if the enzyme loading
is achieved maximum at any other pH, it is not worth if the enzyme shows less activity.
Hence taking into consideration of all the above facts, we have measured the remaining
70
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Chapter 4 Immobilization of Diastase G-amllase
On the addition of protein to the support, immobilization amount increases and
reaches a saturation point. Further addition did not make any remarkable increase in protein
loading. The saturation point depends upon the properties of the support and the methods of
immobilization. Immobilization via adsorption depends mainly on the surface area
available for immobilization. It incorporated almost 2 mg protein per 1 g ES at the plateau.
For ESI it was above 2.3 mg and for ES2 it was nearly 1.5 mg when initial enzyme taken
was 3. lmg.
However, the protein content is not the only parameter that characterizes an
immobilized enzyme. The coupling procedure may have an adverse effect on enzymatic
activity and so activity yield should also have to be determined.
ty EU)
i
ctvO’)
’>_/ /
... — ‘ml_ —I— ES-' -0- ES1
-4- ES21 ' 5 ' 5 ' 1 I-, 'Initial amonut of protein (mg)
Fig. 4.2.4 Eflect of initial protein concentration onimmobilized a-amylase activity.
It can be noted from the fig. 4.2.4 that the maximum activity showed by ESI and
ES2 are when the ratio of protein added to them is up to 3 mg protein g" polymer support,
and for ES it is 2 mg protein g" ES. During the addition of protein to PANI, the amount of
protein immobilized is increased gradually and finally get saturated.
73
fig#&ll‘ ‘If “ H ‘ " ‘$6 Q3 fig §_ _d Nwm5%tag Q3 ; S 2 amfigNaQM if fix; W; __N mm>_\>< “M:A__\;5_$_U_|=M_><3°‘‘ J I ‘ lq ‘ ‘ l ‘ _| I‘ |__ ‘ _ >_ Lj W \>_;_2E__m ___w :5M 93:3 QE%N=QA___2_H__;__w “EV ac: _58 & Qx; =3; ___Q_9___ ___°§____o_~wNm_%_______ (_V_@_> §§< §__3oEE_ {_£€Ua_fl_=___ =O€N___£oE=__ _§___€EE_ _§=_ §i_o__£2; %%33_a:$_\U M kg‘ Q%3_é\__~3_a kc _5_:sN_$©_:__:~__\U >_U:Q_~_Vm__Qm Nfi N35“
_Chapter 4 _ g Imm0bil*iz_atiogn of Diastase a-amylase _
Beyond this saturation level there was a slow increase in protein loading but the
activity was found to be decreasing. Covalcntly coupled enzyme showed maximum activity
at this saturation level, but adsorbed enzymes followed a different trend. Here in the ease of
ES the enzyme adsorbed at the saturation level is 2.1 mg g“l ES, but the activity was very
low, and showed maximum activity when the protein loaded was 1.5 mg g'1 ES. The
decrease in activity of enzyme (fig. 4.2.4) before (for adsorbed) or beyond (for covalently
bound) this saturation level may be attributed to multilayer adsorption on the single layered
adsorbed or covalently bonded enzyme, so that a considerable amount of enzymes are less
accessible for starch. Similar conclusions were put forward for the decrease in enzyme
activity when glueoamylase was covalently immobilized onto surface grafted polyaniline
[35].
Immobilization yields, activity yields, and the efficieney of immobilization are
shown in table 4.1. Maximum activity yield obtained was 37.4 % which represents the
amount of actively bound enzyme. Immobilization effieiency is defined as the ratio of
activity yield to immobilization yield. In all cases immobilization yield is higher than
activity yield, and hence efficiency of immobilization is low. Tanyolac et ul. [36]
immobilized 0:-amylase onto nitrocellulose membrane and the maximum enzyme yield was
around 21 % and Sun er al. reported [37] about 33 % recovery of activity when thermal
composite hydrogcl membranes were used as support. This fact is usually explained with
difficult accessibility of the substrate to the immobilized enzyme due to steric restrictions
or unfavorable orientation of the immobilized enzyme molecules [12]. As a result of these
effects interaction between the substrate and the active site of the immobilized enzyme is
blocked.
4.2.2 Effect of pH on enzyme activity
Enzymes are very fragile molecules when removed from their natural medium.
They are amphoterie molecules containing a large number of acidic a11d basic groups,
situated mainly on the surface. The charges on these groups will vary with the pH of their
environment. This will affect the total net charge of the enzyme and the distribution of
charge on its exterior surface, in addition to the reactivity of the eatalytieally active groups.
75
__ A Study on the Immobilization of Enzymes on Polymeric Supports
Hence a change in pH or ionic media may cause denaturation of the protein structure and
complete loss of the activity of the enzyme. So the pH value has considerable influence on
the cfficiency and stability of all enzymes.
The pH effect on the activity of the free and immobilized forms of or-amylase has
been studied in buffer solution at different pH values (pH 4-8) and the results are presented
in fig. 4.2.5. Activity under optimum conditions was assigned a value of 100 %. The pH
values for optimum activity of the free amylase are 5 and 5.5. The present result is
comparable to earlier reports in the literature [8, 12].
¥ 3% Q :
0/0/ //.// /,/
501 \—O— Free °\ES \"\:Or F I4 5 6 7
pH
——4— E81 A—v— ES2 1
o.,.i
Fig. 4.2.5 Eflecl of pH on actiw'I_v of free and immobilized(1-amylase.
Upon immobilization there was a slight change in the nature of pH profile
depending upon the method of immobilization, and spacer used. The adsorbed enzyme
showed its maximum activity at pH 5 and for covalently bound enzymes, the value was at
5.5. The shift of optimum pH towards acidic region was reported when a-amylase
covalently immobilized to functionalized glass beads [26] and zirconium dynamic
membrane [7]. The slight positive charge on the surface of ES has the effect that more ions
of opposite charges are drawn in to the vicinity of the bound enzyme, so enzyme will
76
Chapter 4 g Immobiligzatjon (if Diastase a-amylase__§
encounter this pH value at its micro environment but the bulk of this solution has higher H
ions. Hence the optimum for the ES shifted towards the lower value. In activated ES1,
glutaraldchyde spacer enabled the attached molecule to act like free enzyme but in ES2 the
spacer not long so the property of the surface somewhat affected the action of attached
enzyme. Hence the pH-dependent activity profile for the immobilized a-amylase on ES and
ES2 is considerably narrowed to acidic region. But for ESl it is broadened and showing
less susceptibility to pH change. Similar observations, with resistance to pH change were
reported by other researchers [29, 30]. Thus, the immobilized amylase on ES] displays
significantly improved stability to pH changes over the free form and other immobilized
forms.
4.2.3 Effect of temperature on the activity
The effect of temperature on the activity of free and immobilized a-amylase was
studied by subjecting them to various temperatures ranging from 30-60 °C and is given in
fig. 4.2.6.
t ve act v ty (%)U1
Rea
V ., O1-\
2
FOO
p— —Free— -ES,— —ES1— — ES20 T-7:;
O<1)
co 140-1
hict
0"o
0"»oi
Temperature (°C)
Fig. 4.2.6 Eflécr ofremperarure on relative activity offree and in-mzobi/izcd a-am_vlase.
77
+
A Study on the Immobilization of Enzymes on Polymeric Supportsii __ — _ _ _ __ 7 __ _ * _The optimum temperature of native enzyme was 50 °C which decreased to 35-40 °C
when it immobilized. Handa el al. reported [8] a 5 “C decrease in optimum temperature
when or-amylase was immobilized on to polyacrylonitrile. The decrease in the optimum
temperature might arise from changing the conformational integrity of the enzyme structure
by immobilization [5, 38] which favored amylase activity below 50 “C. Otherwise the
attachment on the support might have affected the three dimensional protein structure
which either caused the denaturation of the protein structure or affected the conformation of
protein which resulted in an alteration of enzyme substrate affinity [l2] and hence showed a
decrease in the immobilized enzyme catalytic activity at higher temperature.
4.2.4 Thermal stability of the free and immobilized enzymes
Thermal stability experiments were carried out with free and immobilized enzymes,
which were incubated for l hour in the absence of substrate at various temperatures. F ig.
4.2.7 shows the thermal inactivation curves between 30 and 60 °C for the free and
immobilized enzymes.
1,
100-J
-i:... ‘ O‘v .._ 507‘
A— ~—Free.. _ -53 .— —-ES1 V-— —ES2 y— t— T ‘ - e T -F r A30 40 50 60
Re at ve actvty (%)
4 > 0 O
O
Temperature (°C)
Fig. 4.2. 7 Effect Qflemperature on stability Qfflee andinimoliilized a-aniylase.
78
ghapter 4?? g U f _lmmobi_l,izationof Diastase a-amylase
There was no change in the activity of free and immobilized enzyme for 1 hour at
30 °C. As the temperature rises, the stability dropped significantly for both free and
immobilized amylase. At 40 °C, the immobilized and free enzymes retained their activity
about to a level of 80-90 °/0. At higher temperature the immobilized amylase wasinactivated at a much slower rate than that of the free form. The free and immobilized
enzyme lost 60-80 % their activity at 60 “C after I hour heat treatment. Fig. 4.2.8 shows the
effect of incubation time at optimum temperature on the activity of each immobilized
enzyme. Almost 90 % of the immobilized enzymes were active even after 120 minutes of
incubation at their respective optimum temperatures. These results suggest that the thermal
stability of a-amylase increased considerably as a result of immobilization onto emeraldine
salts, and is suitable for long term process. Improved thermal stability of the immobilized
amylase was reported by Bqjak [39] when using acrylic carriers as the support.
100 -i 0 Q i_i § __‘3[ 4v \. Evmv-Zhfig 31 3at
@U'IC)c 0 oQQO
so-A4"-M ‘
ve act v'ty (%
._ \_\_—0—- Free \*0: kg.—lr-- E51__G_ \.at O C1*‘ i_' I" T ' I T ' IO 20 40 60 80 100 120
Re at
40
Time (minutes)
Fig. 4.2.8 E_f/ec! Qfpre-incubation time on smbih'ryQ/free and immobilized a'—amy!asc.
The immobilized enzyme, especially in the activated ES bound system, was more
resistant than that of the soluble form against heat. The strong interaction with the support
material is supposed to preserve the tertiary structure of the enzyme which enables the
enzyme being protected from conformational changes imposed by heat [26]. lt has been
79
A Study on the Immobilization of“Enzymes on PolymerieSupports
reported [40] that the multipoint attachment to a complementary surface of a relatively rigid
support drastically increased the thermal stability of the enzyme.
4.2.5 Determination of Michaelis constant
lt seems interesting to analyze the enzymatic hydrolysis with immobilized a
amylase in the framework of the Michaelis-Menten mechanism. The kinetic parameters, the
Michaelis constant Km and maximum velocity Vmax for free and immobilized a-amylase,
were determined by varying the concentration of starch in the reaction medium.
Lineweaver-Burk plots and Hancs-Woolf plots for free and immobilized a-amylase are
shown in fig. 4.2.9. The kinetic parameters of free and immobilized enzymes are
summarized in table 4.2. The apparent Km values for the immobilized a-amylase were
higher than those of free 0:-amylase. ln general Km of an immobilized enzyme is different
from that of free enzyme due to the alteration in enzyme conformation, microenvironmental effects of the carrier, and bulk and diffusional effects [44]_ The decrease in
affinity towards substrate is also caused by the structural changes introduced in the enzyme
by immobilization procedure and hence resulted in the lower the accessibility of the
substrate to the active sites of the immobilized enzyme [42]. On the other hand, the Vnm
values followed the opposite trend, suggesting the residual activity of the immobilized cz
amylasc decreased. Such observations have been generally reported [43-45] during
immobilization of enzymes.
Table 4.2 Kinetic parameters Q/'r/ze_/Tee and imniobilized a-an7_vluse.
Free enzyme ES i ESI T ES2Jim. 9 .9 ._‘_ . - .Km(mg mu‘) 0.40 d: 0.09 y 2.15 i: 0.11 i 0.83 3% 0.08 A 2.89 i 0.03. . fly . _ __l it ;(EU pg" protein) 7.95 i 0.05 2.71 1*. 0.07 4.73 r 0.04 3.09 i 0.13
80
4 ‘ 'i
Chapter 4 _ j : lmm0lfil[§§_tion of Diastase a-zjlqylase
00 I 2'§ 1 5- 2‘ I24 . |_J/'; '15 M 5 4 7 5 1: -c-’s c.'-on 0'5 ‘ IL 11us_] [so]
..,J ‘O; I bE 5- gs08/ l1ll
C15-I
20
1s
10$2' I 5:10" ‘ ann-0
5..
|‘!,_,_ /I _/ I1:‘. -1.0 o=- 0.0 05 10 I -(ls. GT0 ufs 10 1» zr1:|s‘| |s 1“ d.4%..20- , I
R aoQ2: LiP \
\
I5...la
~l§.1 15.1I ll
\o.a 1 s -2 -1 o 1 2 3 ,S145.1 ' -'
F ig. 4.2.9 Linewe:aver'-Burl: plots (I) and Hanes- W00/fplols (H) Qf_'fi'ee a—um_v1a.s'e (a) andimnmbi/ized on ES (b) on ES] (C) and on ES2 (H).
81
A Study on the Immobilization of Enzymes on Polymeric Supports
4.2.6 Storage stability of the immobilized a-amylase
One of the most important parameters to be considered in enzyme immobilization is
storage stability. The stability for long term storage of the immobilized a-amylase on
emeraldine salt powders was examined by storing them at 4 °C in semidry condition for a
period of time. Free a-amylase in buffer solution was also prepared and stored under the
same conditions. The free enzyme in buffer solution lost all of its activity within 2 days.
Under the storage conditions, less reduction in activity for the immobilized enzymes was
I ESV//l. ES1w E82
observed.
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Month
Fig. 4.2.10 Eflect of storage time on the relative activityof immobilized a-amylase stored for 6 months.
As shown in fig. 4.2.10, the adsorbed a-amylase retained more than 50 % of their
original activity over a period of four months, and the activity of the covalently bonded
enzyme on ESI was more than 80 % of its original activity afier 6 months while that on
BS2 retained only 40 % of its original activity over the same period of time. This result
readily suggests that the immobilized amylase exhibits improved storage stability over the
free enzyme. Kahraman et al. reported [26] 80 % activity retention after 25 days of storage
for a-amylase immobilized on glass beads and Lim et al. reported [18] a 10 % loss of
82
Chapter 4 Immobilization of Diastase a-amylase
activity in every 12 days of storage for amylase on silanized silica particles. The stability of
the immobilized a-amylase can be attributed to its improved resistance to heat and
denaturing agents [46], as a result of adsorption and covalent fixation on the surface of the
emeraldine salt powders.
4.2.7 Reusability
In order to improve enzymatic process economically, the enzyme could be used in
continuous process over a long period of time to exploit it completely. Inactivation is the
most prominent problem when exposed to inadequate ambient conditions, such as extreme
pH or temperature [47], organic solvent and surfactant [48]. On this accotmt, improvement
in the reusability of immobilized enzymes is of great importance. The excellent reusability
will prolong the half-life of enzymes. The easy separation of catalyst from the product
mixture followed by reuse is one of the most important advantages of heterogeneous
catalysis.
I ES100- V////4 ES1% ES2
. :4.O
W (%)
\\\\\\
\
Q
Q
'- vs
Re at ve act v
O
Q
:11 50- ..
3;;
:-:~ O I
2::
to'. P0‘.191'~ O O
3 5 e a 9 10Number of cycles
-L
N)
b
\|
Fig 4.2.11 Reusability of immobilized a-amylase.
In our study activity for 10 cycles of use for the immobilized enzymes was
monitored and the results are shown in the fig. 4.2.11. It was examined in a batch wise
manner by using the same condition repeatedly 10 times within 8 hours. lt was observed
83
A Study on the Immobilization of Enzymes on Polymeric Supports
that the immobilized enzyme activity decreased when recycling number was increased.
Both of the covalently bonded enzymes retained 80 % activity after 4 runs and about 50-70
% activity even after 10 cycles of use and further washing. But the adsorbed enzyme was
leached out during repeated use and could retain only 30 % activity after l0 rtms. This
excellent reusability could be explained by improved resistance to denaturation and
conformational changes caused by the surrounding conditions such as buffer solution, as a
result of covalent immobilization on the support [26]. This agreed well with the reported
results, the reuse capabilities of a-amylase was in the range of 74-88 % when immobilized
on UV curable support [16] and in another study, reuse capabilities of a-amylase on
poly(dimer acid-co-alkyl polyamine) particles [49] demonstrated more than 99 % activity
after 20 runs and 91.3 % activity after 40 runs.
4.3 Immobilization of 0:-Amylase on Emeraldine Base
4.3.1 Optimization of immobilization conditions
The effects of enzyme coupling time and pH on the enzyme activity were studied
with glutaraldehyde activated EBI, ascorbic acid activated EB2 and non-activated EB at
room temperature and at different reaction coupling pH and time. The effect of
immobilization pH on the activity of immobilized amylase is shown in fig. 4. 3.1. The best
activity showed after immobilization was found in the pH region of 5-5.5. The possible
reasons are lower loading and enzyme conformational change due to unfavorable charge
distribution of amino acid residues by the medium which may reduce the activity of the
enzyme [13]. Since the free a-amylase used in this experiment was stable at narrow pH
range (4.5-5.5) the possible denaturation of enzyme in alkaline solution was also expected.
The activity yield of the immobilized a-amylase coupled at different coupling
reaction time is presented in fig. 4.3.2. An increase in the coupling duration time up to 30
45 minutes led to an increase in the activity of the immobilized enzyme on EBI and EB2.
The maximum enzyme activity of adsorbed enzyme on EB powders was observed after 75
84
8ShmQaCS3tS_mDf0n_mt3_mMOmmI4retahCGS3€rC€d3dGW0_hSBEdetaW_tCaHOHdH3dm3V_nC3mOBPMm.1_Ig_mMuOCnO_nC3CrfOSCtumm12BBBEEE- Mm”H_H_u_“_“_H_n_“lH______ ~§___v_‘_____%______________________ ____'___l___I______________________ __'_'_.___._Q_' ‘ ‘ _ ' ' _ _ I _ _ _ _ _ _ __ ________________ '_'_O_‘__‘>______'___~___'__I___‘___‘__1_‘_‘_______‘_.___I_'_U____q___I_'_‘__._________________ "'__““________I."I:__A'I___"'____I__fi__'_____‘__l_‘_____v_._A____‘.__>__________________ __‘____'_'_§___'________._________'_‘__P_‘_____v___‘___.______‘_§_‘_._'_"_____‘_‘_‘___'_’____‘_‘__ '_,_‘______________ _§'__'_‘_‘_§____‘I.'_I'_'_>_§___'__‘_ ‘I... ->‘.-'->.,..‘-‘-4-‘.'lf v _ _‘_ .__ :_9__‘_II‘__I__' ‘u 6- _ U‘ _ I ’ V _ _-IQ‘_.-l'§l‘-I‘.._I'III.-“l"Q.U.--_-.-.'~‘.."“‘-.-.-.-“'l_ Q-~.O"--IQ""‘_‘."‘.".-'_""iI“"".._-"-"~'-G-‘II‘.-..’.‘-‘-‘I“._"‘-‘.‘_‘~""h.b‘.‘-_~"AI.’.‘-.I‘-l..4'.U.-‘-1-.‘I _ _ _ _ _ _ ‘ ___::__:____:___::_:_________:___'1___’____::_:::____I I O . ‘ _ _ _ 7 Q:____:_::_________:__;:____:____'___:_:I_________::Q~_ _ _ _ _ _ ‘ ~ ___:_________::_' ‘ __:___‘_ ___ :.__:____:_: :;:_'______: . ______t_I______‘____C:___:__‘____.____:__::_:___‘_________::D‘m_m_u.U_m.H_m_M_M_U_U.m_m_W_m_Tu‘m.U_~_u_Tn_u_u'__u_m_'-MA“_m_Ts"_"‘_'_'_._‘__‘_‘_l___I_:‘_“'_ _ _ _ _ _ _ _ _ _ __ I ‘ _ I ~ 0 ‘ i ' _ _ _ _ _‘_ ‘ _$_ _ _ _ ‘ "-.I.‘..-I_I.-..'-_"‘§.'Q.l'."-II_ _ _ . _ _ _ _ ’ Q D__’_____::' __'I__:_____:‘:_' :::__ _ _ _ _ _ _ _ _ _ _‘:_:_:__:‘_‘_1‘:::_:_____:‘_::I‘1g a_>__g m_>___w_°”_rm€m8__htHOmuH8Hm“mamwUOccWuIQM4SmmWmIamdbfMM[WM12BBBEEEV.mO_O_O|O‘O‘. O O O_O_Q‘O‘O_.'O‘._O_O‘O_O_Ov_QOOOQOOQOO.‘DOQ.OOOOOOOOQOOOOOOOOOOOOOOO’“O"O"O4.w."O“OJ/W _O O ‘QQO Q O . . . Q‘O‘O‘OvO'O O O‘ O_O‘O_Q_OyO__- IOO OOOO0.00000000000.000000_Q. OOOO.QOOOO..‘QOQOOQOOO:9’ _ _ ' 8*WOWOMOMOWOWO?£0".£O£&OvgOKh_%A“_§.OOOOOOOOO‘UGO...O.OOOOO.OCO..OO.QOO.OOO.OOOOOOOO..O.O.OOO.OOOQO'OOOOOOO.“GOOOOOOOOOOOOOOOOOQOO.OQ.QO'OOOOOOO.OOOO.00.00OQ.OOOQOQOOOOOOOOOOOOOOOOO‘__’O_’,O;_O_O_O_”O‘O_O_O_’O_O_”’O'O_O_O_O_’O_’O‘O"O_”O__Q.OOOOOOOOOOO.OOOQO.OOO.OOOOO.OOO..OOOOOOOO.OOO.OOO§OO.OOOOOOOQOOOOO.:OOO~OO.’OOOOOQOOOO.COCO.OOOQOOOO.OOOOOOOO.OOOOOOOOOOOOOO‘OOOO.OOOQOOOOOO‘OOOOOOOOOOOQ.O’OOOOOOQOOOO0.0.QQOOOO..OOOOOQOO‘OOOOOOOOv“O“'“'H'“““O"O"O"O”O”O“r“t”O"Out"OxO“O"O"O“ONt"ONONO“O“O“O“'“'“é“'".“O“Q“.OOOOOOOOOOOOOOOOOQOOOOO0.000000.000000QO_O‘.OOOOvOO_OOv0.000QOO“_OOOOOOOQOOOOOOOOOOOOOOOQ.OOOOOOOQOOOOO’_ _W H w % mo M wA5 b_>_§ m>__~_°~_57O6540351WWM‘MTn0n_0Ua_Zm____D€0%mlmmW mugm_m WH OC___DG‘WedMMHmJ34byFIdHOWCw
A Study on the Immobilization of lflgnzymesgon Polymeric Supports _
On the other hand, as time prolonged a large amount of amylase was immobilized to
EB, on the basis of protein determinations, but the specific activity of these preparations
was low. The decrease in the immobilized enzyme activity can be due to the multipoint
attachment of the enzyme molecules to the activated EB1 and EB2 or overcrowding of the
immobilized amylase on non-activated EB, as a result of which substrate diffusion
limitations occur [50]. In view of these observations, coupling time of 30, 45 and 75
minutes were used in the rest of the experiment for EB1, EB2, and EB respectively.2.1 ; /7;/II . ./‘/
ed mgProte n oad
O
1?—--EB iy -—0—— EB11 -4- EB2 ;. [— -- -— | ~ I2 4
Initial protein (mg)
Fig 4.3.3 Influence Qfinirial protein concentrationon the protein loading.
Fig. 4.3.3 shows the effect of protein loading on the immobilization yields of the
supports. EB incorporated higher amount of 3.5 mg protein g" EB, which was 66.9 % of
the enzyme offered for immobilization. EB2 coupled almost 50 % of the offered enzyme.
On EB1 a saturation level reached when the loading was 2 mg protein g'1 EB1. Further
addition only made a negligible change in the protein loading. This nonlinear relationship
after a particular interval of protein loading was reported by F erncmdes er al. [34] and Chen
er al. [35].
86
Chapter 4 Immobilization of Diastase a-amylase
Fig. 4.3.4 shows the recovery of the enzyme activity upon the addition of enzyme to
support. The maximum activity is shown by enzyme immobilized on EBlwith activity of
more than 6 EU.
6 _ '\: K: ;.2_
-0- EB1—A— EB27 i I l l i1 2 3 4 5
Initial protein (mg)
ty (EU)
LEnzyme act'v'
Fig. 4.3.4 Effect of initial protein amount on the activityof immobilized enzynie.
Table 4.3 also shows this immobilization yield, activity yield and immobilization
efficiency for the enzymes and it can be observed that high immobilization effieieney was
obtained for EB1 (42.4%). These values show that the covalent coupling of amylase to
glutaraldehyde linked EB1 through their free amino groups, provided immobilized
derivatives with good enzymatic activities. But in all eases activity yield less than one,
which was expected as usual after enzyme immobilization as a result of conformational
changes, shear and non productive binding [1 1].
87
_ \ I’ _|| 11fig Q: $6 Q3iv M: _@ gmWfiw 3 #6 _3& WEQ4 2Em‘Wm QM: 2“ Q3“; GNU2 ME _>_;< H E >< >_:__o_E__m M DEV _?Agv 2_;N=Q __§_~_o_,_: % A__\_;$_@_> _§__:_§_____ 53 gt;__2__E_E°EE_ rM>_3< _ob_>_B< H_£_éufl__E____ =o=mN=3°EE_¢_2E_; __wME: ___3°_____;N____H_°=_E_\ IJ 3+ l |__ : ‘ é IA [Y _ ‘ 1A95 (=§o________£____ :___%_o___mv_S\ _w__%3;$:__w’_\U M bi mM__q\_:__a|B _:~\___fi_:$____v“_§v =c_:3N_c_:~_::E\ “Q _u3_§
Chapter 4 gg 7 Immobilizationgof Diastase a-argnylgase
4.3.2 Effect of pH on enzyme activity
As a result of immobilization the pH optimum and the activity curve for enzyme
may change depend upon the ionic conditions of its immediate environment. Hence, the pH
of the assay medium was altered from 4 to 8 and the resulting effect on the enzyme
activities were measured (fig.4.3.5).
v 7?/li§§\ v1: i 2/ \\;
Re at ve act v ty (%)
.%/
‘ —0—- Free—O—~ EB—A-— E81"-*V— EB2-r —|—' | "1 -1 - is I4 5 6 7 B
pH
Fig. 4.3.5 Effect of pH on {he activity Qffree and1'mm0bz'/ized a-amylase.
Immobilized enzymes showed its optimum activities at pH 5.5. Within the range of
pH 4.0-7.5, the pH profile for both free and immobilized on EB2 a-amylase were very
similar. Compared to them amylase immobilized on EB and EB] exhibited higher activity
above pH 6.0 and lower activity below pH 5.0. This indicated that the immobilization
procedure improved the stability of a-amylase appreciably particularly in the alkaline
region. A possible explanation of the results obtained is that immobilization on these
supports may be created some secondary interactions like hydrogen bonding, ionic and
polar interactions between enzyme and polymeric matrix which cause conformational
changes in the enzyme molecules resulting in changes of protein’s charge [17, 41].
89
__ A Qtudy on the Immobilization of Enzy_mes on Polymeric Supports V _
4.3.3 Effect of temperature on activity
Enzymes are sensitive to temperature changes. The effect of temperature on relative
enzyme activity is illustrated in fig. 4.3.6. Upon immobilization on EB the optimum
temperature for the activity of amylase is decreased from 50 "C to 40 °C. This indicates that
less energy of activation required for immobilized enzyme, or higher temperatures favor the
formation of some multiple linkages between support and enzyme leading to a decrease in
the activity of the enzyme [51].
100- IO
Re at've act v ty (%
- <1 > o 0 \
-f-'-— —Free \’i — —EB o— —EB1 f— —EB29 if t30 40 50 BO
O .7/\%,/
* ‘—i——~ | ' - -e | 'Temperature (°C)
Fig. 4. 3.6 Effect Qflemperature on the activity of freeand immobilized a-amylase.
4.3.4 Effect of temperature on stability
The effect of temperature on the stability of the free and immobilized 01-amylase is
shown in fig. 4.3. 7. The stability of the a-amylase is strongly dependent on temperature and
the activity of the or-amylase shows a more critical temperature dependence at temperatures
above the optimum temperature. The free and immobilized amylases retain only less than
30 % of their optimum activities at temperature above 60 °C. The temperature stability of
90
Qhapter 4 _ Immobfilizationpf Dlastase a amylase___
both free and immobilized enzyme with respect to the preineubation t1me at the1r opnmum
temperature is shown in the/ig.4. 3.8.
100
Y (°/=)eactvtRe atvact v'ty (%)at veReU1Q
Fig. 4.3. 7 Efléc-I Qflenrperanwe on (he stab?/i/J’ Qffree
U5O
Q
7
J
DDOO
O
O
—Free ‘— EB 0- EB1- EB21 * F_' 7 I30 40 50 60
Temperature (°C)
and inmzobilizcd a—am_1-*la.ve.
1DOJ A\POI
_.EB1 at5O°C- -EB2 \
G
% i—_:Q=.-_.;—-2
o%0__._.-o%O_;ai;1>0 °C
O\.— Free \ ,-IEB .““-~.- 1 - r - 1 - rso so 90 120
Time (minutes)
F ig. 4.3.8 Ejfccl Qfpreincubation linze on {he acn'wT!_1-' Qflee and immobilized a-umy/a.s"e at oprimum zenzpemnu'e*
9|
A Study on the Immobilization of Enzymes om Polymeric gSupp0rtslg__
The results suggested that the immobilized amylase holds much more excellent heat
resistance than that of the free enzyme. Almost 90 % of immobilized enzymes retained
their activity after 120 minutes of heat treatment, while free enzyme could retains only 30
% of its initial activity.
An explanation for the difference in temperature activity profiles between the free
and immobilized amylase is that the latter is less susceptible to temperature-induced
conformational changes after immobilization on EB powders. The decrease of relative
activity of the free enzyme was probably due to its thermal denaturation, while the relative
activity of the immobilized a/-amylase decreased slowly because of some protections of the
carrier for the immobilized enzyme [26]. lt was also suggested that [11] immobilization can
help to distribute the thermal energy imposed to the protein at higher temperatures.
4.3.5 Kinetic constants
Kinetic parameters, the Miehaelis constant Km and the I/max for free and
immobilized a-amylases were determined using soluble starch as substrate and are
summarized in table 4.4. In the free and immobilized enzyme preparations, Michaelis
Menten kinetic behavior was observed. The Lineweaver-Burk plots and Hanes-Woolf plots
for the immobilized 0:-amylases are presented in fig. 4.3.9. For the free enzyme, Km was
found to be 0.46 mg ml.” whereas V,,,,,, was calculated as 7.95 EU ,ug'l proteins. Kinetic
constants of the immobilized amylase were also detemiined in the batch system. The Km
values were found to be as 4.58, 1.99, 1.03 mg ml." for adsorbed, glutaraldehyde coupled
and ascorbic acid coupled enzymes respectively. The V,m,, values of immobilized enzyme
for adsorbed, glutaraldehydc and ascorbic acid activated preparations were estimated from
the data as 2.23, 3.8 and 2.48 EU pg” protein of immobilized enzyme onto the emeraldine
base, respectively. ln this study, as expected, the Km and I/',,m values were significantly
affected after immobilization of amylase onto emeraldine base. These effects were more
pronounced when enzyme physically adsorbed on the support. The change in the affinity of
the enzyme to its substrate is probably caused by structural changes in the enzyme
introduced by the immobilization procedure and by lower accessibility of the large
92
Chapter 4 L Immobilization of Djastase a-amylase
substrate to the active site of the immobilized enzyme. Since immobilization process does
not control proper orientation of the enzyme on the support, this may change the property
of the active site, which might hinder the active site for binding the substrates [52].1 1 - a4- I2\1 . .§2_H - r_
-0.5 /
10
5° 5'
A
Aco us / 3 ' ‘ti '1 3 4:
3-F '
syvo-h
+1, / I I ‘1 2 2 1 D 1 2
~- 5.Eu 11 0.5-H
2.)- 1 ' it IU 4 8 10 U5 00 O5 101 {[80] [501‘ 1 IIFig. 4.3.9 Lineweavar-Bzzrk plots (I ) and Hcmes- W00{fpl0!s (II)_fbr a-amylase immobilized on EB
(a), EB] (b) and EBZ (0).
93
A Study on the Immobilization of Enzymes on Polymeric Supports
Table 4.4 Kinetic constantsfor the a-amylase immobilized on emeraldine base.
FY99 EB EBI EB2enzyme
K... (mg mu‘) 0.46 1 0.09 4.58 i 0.05 1.99 :1: 0.04 1.03 :t 0.06
V,,,,, (EU pg‘ protein) 7.95 ¢ 0.05 2.23 :t 0.03 3.8 i 0.05 2.48 :t 0.09
4.3.6 Storage stability
Stability for long term storage is one of the important parameters to be considered in
enzyme immobilization. The immobilization of enzyme to a support often limits its
freedom to undergo drastic conformational changes, and hence results in increased stability
towards denaturation. In general the enzymes solutions are not stable during storage.
Storage stabilities of free and immobilized enzymes were studied at 4 °C is depicted in fig.
4.3.10.
Z EB1°°— W//% EB1E EB2
Re atve actvty %O ' 8°S E V
I.--I 0 III!- :::: 2 I22:' '"' "' 2 222!IIII§§§ :::: : ::::"- —I|-21 :22:'— III|IIIIII| III‘I." IIII—_ I‘-; III!III! .-M01 IIIo..., III III|-n, u out..-| I IIIIIIIII
II-IIIIIII U‘ IIIIII II1222! 12!Month
Fig. 4.3.10 Storage stabilities of bound a-amylasestored at 4 “C.
94
Chapter 4 Immobilization of Diastase a-amylase
Free amylase solution stored at 4 °C lost its original activity within 2 days, while
immobilized enzymes stored in semi-dry condition at 4 °C retained above 60 % of its
activity after a period of 3 months. The covalently coupled enzymes stored at 4 °C lost
nearly 50 % of their activity, but the adsorbed enzyme stored at 4 °C lost about 80 % of its
activity afier 6 months storage. Bayramoglu et al. reported [29] the enhanced stability of a
amylase when immobilized on reactive polymer membranes. Hence it is clear that
immobilization definitely held the enzyme in a stable position in comparison to the free
counterpart.
4.3.7 Activity after various cycles
In order to check the reusability of the immobilized enzymes, the system was
submitted to ten consecutive reaction cycles. The results (fig. 4.3.11) indicate that the
catalytic activity of the immobilized enzyme is durable under repeated use. Thus, the
immobilized enzyme is able to maintain good starch hydrolysis above 60 % even after five
runs.
I EBW///A EB1B21 -" I:|
Re at ve actvty(%)
..IInIII: an- -.. 3:2 ggg"' 9" IIIill III Ill..| ‘I, ‘M IIIIII III gm -.| IIII II‘ III In-— Ill III gm -.| ,Ill III --| II ,an In ‘I, It ,III III -I, on |III an -. In , 'an III -, II I 1In -. III I II ,Hi " ::: ' :1 'III -:: III : lo :III ..‘ III I III |'1 not . III ‘ III . I.‘III 0 III III I ,III 0 'l| I IIIII I 1 ..|'3 ::' II , In 1 umI ll7 "' ' Iv ::: 0:: l ::lI50 1 :: .. ::: : ::. III In ..I I |-II III -.| ..' | |- I ::: ::: --' - : 'am |:9: III III I gm | |::: "' In ¢ '5' : '--I -‘I ll -U ‘ um I 101 IV ..| III 1' I‘ III I4 ' am I |I III in II "' ' III ' | .nu an --, .,, III III -‘I III , ,ll nun on In "ll an -‘I III ,—- '5' III gm um :‘ an\ on no .., II ' II' III Ill -I, II ' I' In not , nu ' |' III III " ' II ' I |I" ' II‘ III "' III ' I |Ilt l III lo: "' III ' u 0I" III III "' an ' I nIlv nun in In --, I um ,I" Ill an In "' In IlI' In III gm "' um |‘ III III not pm gm "' In |III III III III um "' an |Ill III --| 1 I-I Ill I‘, |"' "' in ' III "l III |an III I, 1 III ‘Nan III ‘I 1 In -.,III III ., 1 um '.|an III um ._,In on Inan III | anIII an aO ii? iii iii ii ::: ,::: :
10
Number of cycles
Fig. 4.3.11 Relative activity of the immobilizeda-amylase after repeated use.
95
A Stud on the Immobilization of En mes on Pol meric Su ortsY Z3’ Y PPThere was no drastic decrease in percent hydrolysis by covalently coupled enzymes,
especially coupled with glutaraldehyde even after ten uses, due to reduction of enzyme
leakage from the support. This result suggests that the covalently immobilized amylase is
more stable on the polyaniline surface. Similar to this, Sadhukhan et al. [53] reported that
the activity loss of immobilized a-amylase was 40 % after the sixth cycle, whereas
Bayramoglu er al. reported [54] that 0:-amylase film strip hardened with chromium sulphate
was able to retain 41.2 % of activity after 20 I'l1I'lS. It should be noted that this high
operational stability could significantly reduce the operation cost in practical applications.
A big advantage gained with this immobilization is the washing, reusing possibility and
long term storage in semi-dry condition without much activity loss.
4.4 Immobilization of a-amylase on Poly(o-toluidine) Salt
4.4.1 Enzyme immobilization parameters
The effect of pH of immobilization medium on the enzyme activity is shown in fig.
4.4.1. The best pH for immobilization was observed in the range of 5-5.5, since most of
free amylase taken for immobilization is less stable above and below this pH range.
_100 -—“'1': ' / | S1r.;.;.;. A.. ...... ..-:~:-:-. r:-:-:-:-:-:-:~:-:- I S2Q Q Q .O Q QI'Z'Z~I '-,',~,*_ ‘Q'Q'Q'<- ~ ~ ~ ‘Q'Q'Q'<
Re at ve ac: my %3222;§;5;i;&;i;§;E; §
. . . . .'Q'Q‘Q‘- ‘"5",-,-,~. .;.;.;.'Q'Q'-‘- ¢,*,',*Q°Q'-‘- 0 ' ' 4- - - .;.;.;.I-1'1-1. . .
rI~I'I~Zp§ -1.3.0 4 n I ' . V ‘I Q Q ,~,~,'. I Q . , , , O Q Q_- I Q . -3,‘,. . Q Q Q_ , ‘ , ,i O:O:1:1 .:.:Q:Q_ .O'0‘I‘OIO1 --QQ ,3’),I I ._ _. I . . , , ,‘Q'Q'Q Q'Q‘Q'- ' ' ' 'I‘D.I.\ ‘I‘0.0' 3,‘),Q Q Q ~ Q Q_Q_ ,',',',, -:-:-:~I.I.I.| .I‘I.i. ,¢,¢,¢,I.O~I.\ .Q.I‘I‘ ,:,:,:,I I I . Q Q -I , , , ,I1 Q'Q‘Q‘- ‘Q‘Q'Q 3,0,",Q'Q'Q‘. ‘Q‘Q'-‘ 3,0,4Q Q Q Q Q Q . , , ,I - . . - Q I ._ . . . . . . - Q I,I,',¢, ‘pp, ‘I.O'l' Q_Q_Q_<Q'Q'Q‘. 'Q‘Q'Q' 0 °,' I ~ Q ~~ ~ ~ 2 - - ' 'Q'Q Q‘ *Q'Q‘Q'.:-:-:»: -:-:-:.0.0-O‘ 0.0.0.1 _-.5... 4.I.I.l‘‘,_,_,_ ,_,-,_, Q.-.5. -.5.-.l‘,,, ,,,, --Q. .QQQQQ-- .QQQ '34,‘, '0"Q Q I Q Q Q < ,~ » v I ~III1 QQQ ‘,,¢,', 1";'1' '10- QQQ u'Q'Q<0 ' ' ' ~ I ' Q Q I . Q I O Q . Q Q O 4an I:O:8:| -,-.-,- -,-.5-_ ,:,:.:. :.:,:.:. . . . I Q I - Q Q . > , _Q Q Q_ 9,0,‘, °,°,~,' »'Q'Q'Q‘ >I.I'I 1‘I.t.6 5 ' ' . 9 5 ‘ ‘I"‘l. I...I‘¢l.Q-O.I 0.3-; ‘A,-,1, I.O.4'I 'Q‘I O., I I I - O Q . , , — :-:-:-. :-:-:-.QQQ 4" Orv‘ QQQ I‘I:O:II l Q . I I Q - - Q I _‘.'.-.2 .;.;.;- ;-3-;- 2-2~I-. -.-.'.- ¢ Q Q ~ - Q I I I Q IQ Q Q 4 ~_-.-.- 'l.Q.Q. Q . Q - Q Q Q,;.;.;. ~.*.-.- .-.-.-. .;.;.;. .;.;.;.;\:I:I:I‘ :¢:I:~: ‘r:~:v:< \:I.I:O. ‘Q:Q:Q:..,.‘ III QQQ< ~,' 'Q Q Q - Q’0.0.4 -.355 Q'Q'Q 4 .°,',','‘.;.;.;. ._._._. ._._._._ i.-.-.-. .~.-,-,'Q Q Q ~ ~,~,¢_- ,~,~,~, O.i'O‘l 'Q‘Q'Q‘‘Q'Q'Q'. Q_Q Q - V Q Q Q ',',',-, ,5‘,-g' ' ‘. Q Q‘Q'. ‘Q'<'Q' ~ I ' ¢ ~ I '.'Q'Q QQQ QQQ- ¢<~~ ~00._. . . ._._._. _Q_-,Q_ .;.~.;. .-.;.-.- _._._._QQ‘Q‘. QQQ~ -QQQ ~Q‘Q~ ' " QQQI Q Q Q Q Q I . , 1 ~ '_ .. ac. :_:_:_. .7_:_:_;‘ .;.;.;. ;.;.;._ *.*.'. - Q QQQ' QQQ- C000 QQQ.... .Q. .II.Q QQQ..;.;.;. -.'.-.- _.;._.; _.;.;.;.Q Q Q Q_¢_Q_.- Q Q .. Q O t I Q Q , , , ,,:,:,:, :,:,:,~ --O'0.0- O.l~I.I' » 0 O I I I Q 4 l:,:,:,: ’:,:,:, 3.5%. \'I.I.O. / ‘
pH
Fig. 4.4.1 Influence of immobilization pH on the activity of a-amylase.
96
Chapter 4 Immobilization of Diastase a-amylase
The time necessary for irmnobilization of a-amylase is shown in fig. 4.4.2. The
figure shows significant differences in the immobilized enzyme activity up to 30-60
minutes. After this point, a decrease in activity retention was observed. Apparently, the
immobilization reaction occurred in the first 30-60 minutes. Contact with the free enzyme
in the solution and those iimnobilized after a particular time may produce layers of
adsorbed enzyme that presents lower activity than those covalently immobilized in the first
minutes. Another possibility was pointed out that increasing the contact of enzyme and
support might promote multiple bonds and result in enzyme deformations, compromising
the active site integrity [55].
ITS100- TS1TS2.;.;.:.;'0-O.I.o"3"" '¢'>'a‘'¢‘-’-H - - n ~._..._., .._._._., , . , . . . ., , ,_, ._._._._;.:.;._. ._._._._I . . . . ..;.;.;._ 4.1.1.; _~_-_-,‘¢'-‘Q5 'I'o'0'- -5'.‘-'o I ~ . O.I‘I.O. ,-,',~,~_ _ _ , . . . . .
Re at've act v ty (%)
8
- . . .-....'.'.'~' -‘.'.'.‘ I-I-I-§~Z~I'I~I *2-Z~Z~I 4.3.1.;'39.‘. ‘Q’-'.'. -.5-.5'¢'»'¢'- 'o'0'n'- ._¢'-‘-‘01 '<’¢'¢'¢ '.'.'.‘~ Q v ~ ~3-_»_¢_ Q‘-3.5 ,~,~,',''~'-‘-‘- '-'-‘R. Q‘-'3.‘. -.1 ~_¢ -.~ O O ,¢,~_¢,'Z - -' ' '¢'.‘ 0 o o o¢ -'u'< ‘-'| - - -,I.~,',‘¢'¢'.'l 'C.I’Q-Q 0.-.3-_. . - . . . . . i _o‘»'¢'-‘ o'¢'u'|‘ ‘I 3-‘- - n - 1 n . . -.I,¢,v_. ~ . . . . . . _ _ _ ,-" -:-:-:-: -:-:-:-1 . . .IOII III! "5' 10'0l.-.- QOOI .... “., .¢n- ¢¢-- "" 000:-,~:-I-I .:.'.°.' .‘-'.'.‘ I-1-I-§ .'.'-°-‘1 ' * ' I a'~'-' c'n‘-'-' ~ ~ I ~ Jo‘-'.',',',',' ‘u.-.-3 3...-; 1,50,», .0.-3.1,:,:,:,: _~_..~_- -5.‘... :.:.:,-, '-_-.-_., , ...- .... __,-, ....1 3,", , o.-.-‘q. -0- ¢ -I , , , _ - 0 1 OQ... _.._ .51 --¢. ,I¢l*,~,:,v,- 0..-.5. :-.-3} ~,~,',~, :3‘:,:,.,:,: .5-.-3 3...... :,:,:,-, '55-.~ - - ~ .'.'.'.' -'.'.'.' - ¢ -'- -‘.'.'.''5'" 0.1. o-~- ~--I 00!:Z :,:,_,:. -J.-.5 -3'...‘ .:_:_:‘- 5..-_¢_,~,~,-,- '.'.'.'. '.‘.'.'< -,-,-_~j '-'.'-'.,-_~_-,- '.'|'-'- ‘-‘J-'< -,-,5‘, ‘~'-'¢'_~,-,-,- '.'.'.'. '.'.'.'- ,-,-,-,~, °.'-'.'0 * I - ‘-"5': '-‘o'o'- 1 n v ~ '¢'-‘-'--~» ...- .-.- ~--~ -~..q- _~_¢,-,~ .0.o.o.4 .~‘¢_¢_¢ '53,‘, 3.5..‘I""'*' »‘»'|'¢‘ Q‘-'~'¢‘ "'3-3 J.‘-‘J,:,:‘:,: ‘-3.... ‘-3.5, :,:,:_:, .-.--0;;-;-:-;- -I-2-2-2 -2-I-2'2 .~.;.;.; -2-:-I-I_,‘,_,_, 5..-,._ -3....‘ .:._,'._ ..._._._,',¢,v_¢ ‘-'¢'¢'- ‘-'¢'.'> 50,03, ‘.'.'o'»,',',' ' 'n'-'-'- ‘¢'-'¢‘. v,v,*,~ ‘-3'1‘., ,5 0000 0000 _~ nu.,_,:,_,_ _.'._.'. _._._._. :,:,_,:, _._._-‘_ , , , _._-A. _-_»_-_- , , , , _-_...‘."" 0001 -¢|~< "" 00::‘II-_ coca Inga --¢- llll,:,:,:,_ .5-_-_. v...._._. :,:,:,:, _»_-‘._,_,.._,_ .¢_..._. ‘....._. _,_,_,', .._.‘._.,...,_,_ _-_-‘-_- _.'-.t_- _,_,_,_, _._-.._.,_,.,___ _._._._. ».._._._t .,_,_,_, _._._-_.,,,, 0004 .._t_._. ,,,_ OQQ4"0' '-°-'¢'< lov< "'*2-I-2+ ‘-t-?-?- -2'.-2».15 so 45 so 15Time (minutes)
I~Z~I'Z‘. _.,-_
Fig 4.4.2 Influence of coupling time on the activityof immobilized a-amylase.
The best protein loading was obtained (l.7mg g'1 of support) with activating and
cross-linking by glutaraldehyde (TSI), as shown in fig. 4.4.3. This immobilized enzyme has
an activity of 4.9 EU, which in terms of relative activity, was only 14.9 % (table 4.5). The
relative activity is the effective activity of the immobilized enzyme compared with the
activity in solution. This low relative activity reflects that much of the enzyme was
immobilized in an inactive form owing to the instability towards immobilization process.
97
A Study on the Immobilization of Enzymes on Polymeric Supports
"9 W9)
&lI
01
oadProte n
In the case of ascorbic acid activated po1y(0-toluidinc) the relative activity of
immobilized a-amylase was increased by increasing the amount of protein in the solution
‘J 1If-i*I/I-—-—'-"""'_"'
—I-— TS-—o— TS1—A— T82* *—| --1 I I ‘I1 2 3 4 5
Initial protein (mg)
Fig. 4.4.3 Variation ofprolein loading relative tothe inilial amount of protein taken.
up to 4 mg, as shown infig. 4.4.4.
Y (EU)Enzyme act v t
—I—TS-—-— TS1 ‘-A— TS2 4I -fi I lily I j I ':‘— I 7‘ it ll1 2 3 4 5
Initial amount of protein(mg)
Fig. 4.4.4 Influence ofiniliai protein concemralionon the acrivilj-' of immobilized enzjvme.
98
/rtI {4', __r_ V I:_ __.JI I_ _n_ 5T;Tjmh__i __\_____.__ \_‘__\\ \II \_\\_ ll] _Q2 M; N*HMwfiB__m 2LA IZ“NZ?Q? Q: Q?fig0:“ h__T3“Gr?mm Q2 2“MWOM“ Q3g>_;< H m__ ><c._2_H___m__w :5 _€>__g0E_hN_;A__\__V523% A5SH:E:__2E__mAg 7% “EVE0; ___2°_aQ5___3°_:_=o:~N__B°_______ u_~_>_:_>:U< _gN___H_oEE_ bm>3U_W_a=____ *_ __°:flN__5oEE_ _E___€=_E_ _“E___\ _ I | \ I___w___%_oLMg so _a_E__$=B___Q_\U ____2_%_§% ~:~_:3N__\_:~c:==\ mg gig
_ “Study on the immobilization of Enzymes on Polymeric Supports
When more enzymes were added, the amount of protein bound on the TS2 surface
increased, but the activity per mg of protein did not increase any further. This suggests that
a higher coverage of protein could either hide or damage some active sites of the
immobilized enzymies and thus lead to a decline in total activity. The adsorbed enzyme on
TS could incorporate only a small amount of enzyme around 0.3 mg in the saturation level.
The maximum activity yield was 15.9, which has highest value for immobilization
efficiency of 53 % indicated that most of the immobilized enzymes retained their original
activity.
In the literature there are many different loading values available for different
supports with various levels of activity retention for 0:-amylase immobilization. For
example coupling capacities were reported as 3 and 29 mg" g respectively, on polystyrene
and silica based supports [21], 25-90 mg g"l for celluloic supports [56] and 94 mg g'l for
UV curable polymeric materials [57].
4.4.2 Effect of pH on the activity of free and immobilized a-amylase
The effect of pH on the activity of free and immobilized 0:-amylase in starch
hydrolysis was determined in the pH range 4.0-7.0 and the results are presented in fig.
4.4.5. The optimum activity for free enzyme was observed for both at pH 5 and 5.5. Similar
to our previous results of immobilization on PAN], the optimum pH value of a-amylase
narrowed to a single value of 5 and 5.5, after immobilization via adsorption and covalent
coupling on the poly(o-toluidine), respectively. lt is said that polyionic matrices cause the
partitioning of protons between the bulk phase and the enzyme mieroenvironment causing a
shift in the optimum pH value, and shift depends on the method of immobilization as well
as on the structure and charge of the matrix [29, 41, 58, 59]. Usually the shift of pH
optimum to the acidic side is explained with increase of positive charge. As a result of that
the concentration of H+ ions in the mieroenvironment of the immobilized enzyme decreases
which means that the pH of the immobilized enzyme region is more alkaline than that of
the external solution. Since the effect of pH on the enzyme activity is expressed based on
100
Chapter} immobilization ofDiastase q—amvlase
the pH of the extemal solution, which in this case decreases, the pH optimum shifts to the
acidic side [7].
100-’
. . I-—n
Re at ve act v ty (%)
l1\\\
ll —0—Free °—o— TS *0—~ -A— TS1
—v— TS24 I " " “—| ' I“ W ' “‘4 5 6 I
7
pH
Fig. 4.4.5 E_[]ecrt Qfpl-I on the acliirily Qffice andinmiobi/ized a-am_1=1a,s'e.
4.4.3 Effect of temperature on activity
Optimum activity for free and both immobilized preparations were observed at 50
“C and 40 "C, respectively (fig. 4.4.6). Such significant change of temperature optimum
after immobilization of amylase has also been reported by other authors [26, 30, 41].
Compared with the free a-amylase immobilized one exhibited lower activity at temperature
above 40 “C. Probably upon immobilization conformational changes in the enzyme
molecules occurred that favored amylase activity at temperature 40 “C. On the other hand,
these changes caused decreasing of the immobilized enzyme catalytic activity in the
interval 45-60 “C compared to the free enzyme. In general, the effect of changes in
temperature on the rates of enzyme-catalyzed reactions does not provide much information
on the mechanism of biocatalysts. However, these effects can be important in indicating
structural changes in enzyme.
l0l
__ d i A Study on the Immobilization of Enzymes on Polymeric Stlpports
've act v ty (%)Re at
Fig. 4. 4. 6 Effect of temperature on Ihe activity offree
4.4.4 Thermal stability
Fig. 4.4.7 represents the stability of free and immobilized enzymes at elevated
temperatures, which is recorded by measuring enzyme activity after 111Cl.lb8Il0l'l of enzyme
U1QI;
ta ;/ V
X \/44/
‘ —0— Free—O—TS—A—TS1—v— TS2t *1 1 t30 40 50 60
Temperature (°C)
and immobilized 0:-amylase.
in buffer for l h at different temperatures.
100
a4'\~
\in-Q
_
Re at've act'v ty %
F ig. 4.4. 7 Effect oftcmperarure on {he .s'tabi/:'!_1> Qfrhe
50
" —%A0
%
. -0- Free vO—TS‘ A—TS1--V'—TS20. 1 V ~ ,e J- ~ 1 - 1 - — eso 40 so 60
Temperature (°C)
_/Pee and immobilized a-amy/asc.
102
Chapter 4 pg g _ Immobilization 0f_Diastase qgamylase
l- - mQ5 %ix‘_%__‘—_'_‘mA--—'-'-"“1_
v 8i—v"-*‘°'Rv~»*v—" 4-990 "' I \ O*% 0&0 3! 40 OCO LOLO. I \': I.\.
(°/vie act v tyO‘)O#1
O__
Re at‘v
. —O---- Free \—A-— TS1 X_V_ T52 at§0 “q30 -P - ~ - 5;! - n *- ii0 30 eo 90 120
Time (minutes)
Fig. 4.4.8 Variation ofactivity Qfenzymes relative topreincubation time at their optimum temperature.
At 40 "C immobilized enzymes showed good stability hence good activity than free
enzyme, after that their stability decreased. From the fig. 4.4.8 it is clear that immobilized
enzymes are retaining more than 90 % activity at their optimum temperature even after 120
minutes of incubation. As it is evident from the fig. 4.4.7 and fig. 4.4.8 the immobilized
enzyme possessed a better he-at-resistance than free enzyme. Probably this can also be
explained by the fact that the immobilization procedure could protect the enzyme active
conformation from distortion or damage by heat exchange [60].
4.4.5 Kinetic parameters of the amylase immobilized on TS
The values of Km and I/max were calculated from Linewcaver-Burk and Planes
Woolf plots (fig. 4.4.9). As seen from the table 4.6, the calculated Km for the immobilized
enzymes were 6.35, 1.57 and 2.95 mg mL" on TS, TSl and TS2 respectively, which are
higher than that of free enzyme (0.46 mg mljl); whereas the I/ma,‘ of immobilized enzyme
on the toluidine were 4.2, 3.08 and 2.68 EU pg" protein on TS, TSl and TS2 which are
smaller than that of the free enzyme (7.95 EU ,ug'l protein). These data indicated that
lO3
A Study on the Immobilization of Enzymes on Polymeric Supports
affinity between immobilized enzyme and substrate decreased in comparison with free
enzyme.
ll‘ II10- I2° 52. an
n /5.
| ' I. . ' 4I 9 v02 O0 02 O4 4 O1I[S'] [SJ20 » b2' 51 '10' ‘-10
/I w I ,/o 1 2 o148.1 18.1 2
4 (3
Q‘.
5
I 2E 204 00 .. . 04 oa 12 a 2 1 ' 6 ' 1 2ms; [s,1I ll
Fig. 4.4.9 Lineweaver-Burk plots (I) and Hanes- Woolf plots (II) for a-amylase immobilized on TS(a) TSI (b) and TS2 (c).
This might be related to diffusion barriers and steric effects, conformational
changes, or the new microenvironment effects [42, 61]. On the other hand, the change in
104
Chapter 4 Immobilization of Diastase a-amylase
affinity could be also owing to the structural changes caused by enzyme immobilization
onto the supports, and hence resulted in the lower accessibility of the substrate to the active
sites of the immobilized enzyme [62].
Table 4.6 Kinetic constants for free and immobilized a-amylase.
Free TS TSl TS2enzyme
Km (mg mL") 0.46 :1: 0.01 6.35 i 0.05 1.57 i 0.04 2.95 :t 0.09
V,,,,, (EU pg‘ protein) 7.95 i 0.05 4.2 1 0.03 3.08 1 0.08 2.68 :1: 0.08
4.4.6 Storage stability
The stability of an immobilized enzyme is dictated by many factors such as the
number of bonds formed between the enzyme and support, the nature of the bonds, the
degree of confinement of enzyme molecules in the matrix, and the immobilization
100 _ : T3TS1conditions.
Re at've actvty(%8
w<» &
..... .......... 22!" .... ..-...t ..... ""::.. .....lm ::::: :::::: :::::;£3255. iiiiit 1"" 255550 =====1.-..-. :::::' 3: 5,..... 232:: ::::: ::::: -....::::: :::::, :::::‘,_ :::::t ::::: ::::: =====::::: ::::: :::::: ::::: 11115 ;g;;;§..... ____ _, ,-- ::::: ::::: :::::: ::::: 5555; :;;¢;;
7
%/%%/%%:::::: 33;;%%%- e%%/é%
----6 "6... .. . . ..... "6... _ _v~---- an-1 ,,:,:t ---n "'0 11,1,6-6-6 ...... ...... ..... »-~- ........... ..... -.... ..... -~-~" -N... 6-....... -..-. ..... -..-- I0lhI- ----. --“
""nun9!!"
-;-3 ..... ..... 33:; ..... ......:.:.. ::::: ::::: ::::: ..... ::::: 31113121‘ Z1222 ""' 221'!‘ 5:335 ""' 22222— 22122 Z2222 2222!‘ Z2212‘ Z1322 222211 35;;.:... ..... ...... ..... ----- ;::::" -....I -.. ..--- -----V nu. "I" - . -".6.... ..... ...... ..... ..... _____, .....:-.1. ..... --um ---.. :31; ...... ""-' .::.: :::::‘ :::::: :::z: :::::: :::::22212 Z1211 $1222‘ 5:!!! Z1212 --.... :13;..... ...... :::::: .......... ...... ..... --- - . ......33: ...... ...... ..... 33:5; Z1222, .........- 22222 2111:‘ 22:72 ---n ;;;;;1 -.6-~~::::: ::::: :::::- ::::: ;;;;; ::::::..... ..... . . ..... ..... .....t..... ....- 1.221 ,3... -6-.» ggggg ----......., 6.... --... . ... ..... mu .....-----6 ..... ...... .....\ -6." "--.,--6" u 6 “.1-t "~ ""'. -----uw .23.! 12:2: ...... 6--u :53; .......... IIIOI ...... ..... ..... _ __ ..-..3... ..... .....t ..... ..... ...... ......... _.... ...... ...... ..... _ .....'--H nu: 6---.. nun "'“ :33. -nu..... .... ....., ..... ----- _____, .....00000 ..... .... .. ...... .6--.22121 I222: ---w Z121!11212 22222‘ -----V 22222onus: II Qt anon: """ nnnn It.-..‘ - ' llnbm .... .‘0 ,::::: E5553‘ :2 1313151 :::::‘Month
Fig. 4.4.10 Storage stability of immobilized a-amylase.
105
A Stud on the Immobilization of En mes on Pol meric Su ortsY Z)’ Y PPUnder the semi dry storage conditions, the starch hydrolyzing activity of the a
amylase immobilized via covalent coupling decreased at a slower rate than that of the
adsorbed enzyme (fig. 4.4.10). Upon 5 months of storage, the adsorbed enzyme preserved
only 32 % of its original activity, while the covalently bound enzyme had about 60 %
retention in the activity.
4.4.7 Reuse of immobilized enzymes
The recovery and recycling of enzymes could lower the overall cost of the enzyme
immobilization process. However, the applications of immobilization are often challenged
by difficulties that arise with regard to enzyme recovery and recycling. In this study, the
batch reaction repetitions, the activity of the immobilized enzyme remained stable
throughout the experiment, and above 70 % activity were retained for covalently bound
enzyme after consecutively repeating the reactions 10 times. This value is higher than that
reported by Laska et al. [63] for urease immobilized on polyaniline, which exhibited an 80
% loss in activity after seven batch reactions.
- T5::EI ::::I\l| ,,,.@ T52.... -.-. ,
Re at ve act'v ty (%)8 8 5‘. 8
on
1551 11:‘ "'1 :::;5:: 555‘ E251 “"E555 ii? 555 iii? :::..... ..-. .... "-- .-..:::, .... "__ :::; ..-.:::; ::: :::: :::; -....... 53:1 ;;;- u 2222 Z221---l ,- ' I ' on 4:::: .... 3; :::, ,,,. :22."-- 222' ;;;- --- .... 12:: ::::53:1 2.. ,,_, ::: up .... "Z-.-- ::: ;--i :::: :::: :::::::. .. . ::::I-I ;;-- |0|- '"' --- .... .... ,,,_" --.- ‘ZZZ I" nu nu. "-..., out Z... -*- .... --.. ..¢.iii? :::: ;;; :::; :::: /::::1 3:3 .... 1221 3;; -.-. ---t /....;;;- :.: :::: ::: /:::::::: ;;;; :::: /::::;;; v--~ .... '"' ---~ ---- .-... .... .... ,,,_ ';'; ---i ---- ".:::: :::: I-it ::::.-.. -... .... :::; :::; -... ... %.,,,3;; :::::1: :::; Q... .... ___, .... --- .--.. ::: :::: :::' mi :::. ;;;; ;;;, ---11: :::; um Z!!! :::: :::; --- ".:::' :::: »--' 3111 :::: ---.... .... .... -;-; ...- .... :::: :::‘ ~:::3;; :3. __ :.:. :::: ::::1 :::: :::; §::: ::: =31 :::: :::‘ :::: :::“ 31¢~--- .... .... :::; --;- ---i . 1 ;;;;-~-i ---~ .... -... .... ::_; ---- .... .22. ....:::; :::; :::~ -.-- .... -..- :::‘ --~ n-- ....;;;; :::; ---- ---- Z22; 2:21 ;g;, 313‘ iii‘ 12!;--;; .... ZZZ! Z221 :::: :::; ::: 1212 Iii: ::::51-» :::; :::: :::: :::, ---- --.1 :::: :::: /""---» .... ...- .... .... :::; ---- .... ..., {III:::' :::: :::: :::: -11- :::: :::: ::::.... --at v... .II\ .,,. '"' --~» t... .... ,,,,w-1 ---» -... ..., .... -:3; ---- .... .... ....:::; :::‘ :.:. ..:i :::: -... up /........ .-‘l .Z.§ Z: I “' I--~ 1-. "1' ,,,j '"':::: ::: :::' ===- :::.:::: ::: :::: :::; :::‘ =11! :::. :::: :::: /SEES55:; :3; :::; 23- -~. :22; ;;; »-- .... /__..:::: ::: ;;;; :::: ;;;» /:::!"=1 I1! ::: "-1 =11 ::::3;"" "' 001- I" nun UIUI0 2:2‘ :2! ---- Z111 :::‘5 6 10Number of reuse
Fig. 4.4.11 Reusabilily of a-amylase immobilized on TS.
l O6
Chapter 4 Immobilization of Diastase a-amylase
4.5 Immobilization of a-amylase on Poly(o-toluidine) Base
4.5.1 Optimal parameters for a-amylase immobilization
The effects of the following parameters on the immobilization process were
investigated: pH of the solution, reaction time and concentrations of enzyme. As expected
from our previous studies, it was found that the immobilized enzymes presented a relatively
high activity when the pH value of the enzyme solution was 5-5.5 (fig. 4.5.1).
1 00 — .. . .53:" I | B 1.Q;Q;Q 1.I.l:I 4I I 9 - 0 v I:-:-:-. ;-:-;- . ...... . .-:-:-:-:-:-:-:-:-:- I B 2Q.fi.Q.\ >,°,',', _Q.Q I I_I_O‘O Q QQ'Q'< ' 9 ' - 0 Q 0Q Q "5'; Q Q QP I ‘ 0 0 0 < ' ' ' ‘.Q n Q Q Q >‘Q.‘.I
Re at've actvty %2325.2Z &\\\\\\\‘
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I‘O.Q.v0 Q I 0 1 Q . 0 0 I O 0 Qv'0 I, Q Q Q . Q Q Q Q',','. O‘O.I.‘ Q'Q'Q‘.-1-;-;~ Q:Q:O:— ~.~.~.-. .-,-,-.» ;.;.;.- ;.;Q;.00I~ OOO— i.Q_Q'-_ QQQ- QQQ‘ viii‘I-0 ‘CID .QQ "*° QQQ- III!IQO~ QQQ. ,5, ~-~- QQQ QQQ-:-:-zl »-:-:-; :-:-:-.0" 000‘ ‘QQQ. ‘V’ one IIIQQQ QQQ VII. ‘Q"'- QQQ‘ QQQ. ,,,:-:-:~. :-:-:-* t-3-2' -:-:-:~ -:-:~:~ .-:-:-:- e-:-:-:- t-2-2-2‘ -.-:-t :-:-:- :-:-:- //4 .e.s.:.
pH
Fig. 4. 5.] Influence of immobilization pH on theretained activity of a-amylase.
As shown in fig. 4.5.2 prolonging the immobilization time more than 45, 30 and 60
minutes did not improve the activity of a-amylase immobilized on TB, TBI, and TB2
respectively. In this case, a possible interpretation is that the reactive functional groups on
the support were saturated by the enzyme molecules after duration of 45-60 minutes, hence
extending the duration time further could not improve immobilized enzyme activity.
Instead, the activity may be reduced owing to the spatial repulsion of overcrowded enzyme
molecules immobilized onto supports. It has been reported [29] that the coupling time is
leveled after 18 hour of immobilization of a-amylase on reactive membranes with
maximum loading 83 /Jg cm'2 of support.
107
A Study on the Immobilization of Enzymes on Polymeric Supports
1TBTB1,00_ £52.;.;.;.;. . . . . . ._.~‘.'.‘.' ~ - ¢ . '-'.'- ._._._._. . . . .....
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»_-_._._‘J. 5'-,-,-,-, -.-.-.-. ;.;.;.;. . . . .*,',',', '-'.'.'. ¢ - . . -'~‘-‘J .'-5‘-'._._.'._ ._._._._ ._._._., _.‘._.‘. _._._._..... .... .... .... ..... . . . _._._._. _-_-_.,. ._._._._ ._._._._',‘.',', D Q - 4 .0--‘Q-1 1 0 | I 9.0-0.!‘~,~,~,-, ‘.'.'-'- ~ - . . -2'-‘J . . Q *,°,*,', '.'-‘.'. '-'-'¢‘- 0'.’-'¢' -‘-‘-'-'. . . . ._.'._._ -_._-_._ _._._._. _._._._..*,',',' e... »<¢- --v- ---. . . . _._._._. _._._._. ._._._._ ._._._._'.'.'.'. - . . . _~_¢_»_- ._._._._ ~_-,~_-.*,'_',', '.'.'.’- - - O - 1 ~ . 0 - - - . . . . . . . . ._._._._ _._._._. . . . ._¢,I,~,- .'.'-5‘ ¢ 1 - - . - ¢ .._._._. _._._._.,',.,_, _._._._. _._._._. ._._._._-,1,-,' ' ' ' ' JR.‘-‘ ‘-'-'.‘- - . . .. . . . . . . . . . . .'»'.'.'.< - . - . . . . . . .. - - - . ~ . . - J. .‘ . ._. .-_. . . _ ,-,-,' -'. .'.' ._._~_-_ . - » - _._ _.'. -‘J. ~_-,~_~ -'.:.'.' . . ._- '.:-'.:- . . - .._-_-_._ - - . . . ._. -_-_ . . . _ _ _ _-_._._._<_ '.'.'. . '. . .'. ' ' ‘ . - .0 -'-’-H . . . . ~ . - - '.‘.‘.'.45 60 90Time (minutes)
Fig. 4.5.2 Influence of coupling time on the activityof bound a-amylase.
The activities of a-amylase preparations related to the protein loading on supports,
the amounts of immobilized protein per g of different carriers as well as the immobilization
efficiency are summarized in table 4.7. The data shows that, all types of the prepared
support particles are suitable for enzyme immobilization, especially enzyme on TBl
obtained with glutaraldehyde spacer exhibiting the highest activity. Although highest
amount of protein bound on the TB2 particles, the highest activity was obtained by using
the TBI support activated with glutaraldehyde. Considerably less activity was reached after
a-amylase immobilization on TB2 than on TBl (table 4. 7). As resulted from the enzyme
immobilization experiments, the highest amount of protein was bound by TB2, but the
lowest specific activity of the immobilized enzyme was obtained by this support.
Considering the rather high specific surface area of TB2, compared to other supports the
reason for the low activity can be due to the steric hindrance caused by the enzyme
molecules forming protein multilayer on the supports‘ surface. The figure shown below (fig.
4.5.3) is the influence of initial enzyme concentration on the loading of enzyme on different
supports. Fig. 4.5.4 shows the effect of amylase concentration (1-5 mg) on the activity of
enzyme immobilized per g of supports. The activity was attained maximum when the
108
Chapter 4 Immobilization of Diastase a amylase
concentrations of amylase were 4.1 mg g" of activated TB and 2.1 mg g for non activated
TB. When the concentration of enzyme was increased further, the relative activity
decreased. This may be caused by the jostling of too many enzymes on the supports as
described earlier.
"9 m9)
i-1
1
enoadProt
IQ
Enzyme act v ty (EU
l
3- /‘2__ __i-1-ii-O
1...—I—TB—0—TB1- * —A- TB2I I I I I1 2 3 4 5
Amout of protein (mg)
Fig. 4.5.3 Variation of protein loading withrespect to the initial protein amount.
.\.‘mt8
A0’/////, -l—TB2 " -0- TB1—l— TB2| l | I I1 2 3 4 5
Initial protein (mg)
Fig. 4.5.4 Variation of activity of immobilized enzymerelative to the initial amount of protein.
109
Q3 gm“ 3 MES ‘S ©N 3“ Q;__©m is‘ 0'0 % gm fig ON 1“ ::l_ W __ j _Ev __$ Q6 3: aw M: __N H M:_>_\>< " M: A >1‘ >_Acé 1 SH: TX; M AWE W Aug~$__°_UEm_ A__\__V _ _§>_t_“ o=_%N_; S5 Eu; __Eo_:_ _ __E°_:_=o=_WN__EoEE_ M Ev; %__>E< __QN_____oEE_ _£>_ga_“___=_ 1 __o=~N__E°E____ Egg __w_=___ W __0E%_oA_Mm to a_E_#__:q|c Qs\'_@§___vm,\\m =c__Eu_i$:=E\ N _____ NEQINI ‘ ‘ II“; I I v|_
Qhyapter 4 _ iy ___ fly mpmobilizationpof Diastase a-amylase_
4.5.2 Effect of pH on activity
The initial activities of free and immobilized 0:-amylase were determined for
different pH values in the range of 4-7. lndividual relative activities for both states are
presented in fig. 4.5.5. Maximum activities were obtained at pH 5.5 for all immobilized
enzymes. As already evident, or-amylase is very stable at pH 5.5. The close similarity of pH
profiles for both states may show that no important conformational change occurs in the
immobilization of the amylase. The only difference was that for the glutaraldehyde coupled
amylase pH profile more broadened in the alkaline range whereas for the enzyme in
solution, the alkaline pH not favored the activity of enzyme.
100- 0v/7 X‘
at ~\. ;\;\\\\../’2///%
so
. — —Free1. — —TB
— —TB1— —TB2
PG.4
6,
oi.
pH
Fig. 4. 5.5 Variation Qfaczivity with pHf0z-freeand immobilized 0:-amy/use.
4.5.3 Effect of temperature on the activity
Immobilization produced remarkable change on the temperature dependence on the
activity of the enzyme. It is evident from the fig. 4.5.6 that the optimum temperature for
starch hydrolysis decreased by 10 °C compared to free enzyme. After 40 "C, a sharp
decrease in activity of immobilized enzymes was observed. The decline in activity of cz
lll
A Study on the Immobilization of Enzymes on Po_ly_n_1£ric Supports _
amylase at higher temperature as a result of desorption of enzyme from support was
reported [12] by Dey er al.
%§:”'\.
ve act v ty %8
-i
.//
—o— B—A—— TB1 mg0 — —TB‘IL: - t ~ 1 -‘ z 130 40 50 60
—0— FreeT
V
Temperature (°C)
Fig. 4.5.6 Influence Qfremperature on theacrz'w't_1' offree and hound a-amylase.
4.5.4 Thermal stability
Utilization of enzymes in processes often encounters the problem of thermal
inactivation of enzyme. At high temperature, enzyme mtdergoes partial unfolding by heat
indueed destruction of non-covalent interaction [28, 29]. The themial stability of free and
immobilized enzyme was investigated with in the temperature range of 30-60 "C (fig/1.5. 7)
and thermal inactivation checked at definite time intervals (fig. 4.5.8) within 2 hour, by
measuring residual activity. It is clear from the graph that the enzyme on TB is less stabile
than other forms. In TBI and TB2 immobilization improved the thermal stability of the
protein structure through a protection against heat. In all forms immobilized 0z—amylase
showed better thermal stability than free form even after prolonged incubation in the
absence of substrate. The reason may be that the polymer matrix is supposed to preserve the
enzyme structure. lt has been reported that polymeric carriers such as poly(2-hydroxyethyl
methaciylate) [5] poly(methyl methacrylate—acrylic acid) [41] and poly(alIyl glycidyl ether
112
Chapter 4 Immobilization of Dlastase a amvlase
co-ethylene glycol dimethacrylatc) [50] protect enzyme from thermal 1nact1vat10n and y1eld
__ _i_~_—_——_‘_— __ —
highcr thcnnal stabilities.
'ty (°/0eactv
u—
1
Re at'v
100-1
Q
'ty %
1:1-1
Re afve act v
50 .\o
U1O
\POO
— Free A-- TB— TB‘! 0— TB2
giq
| —— e — ~ 1 ” f—' ' |40 so 60Temperature (°C)
Fig. 4. 5. 7 Thermal stability Q/‘free and immobilizeda-amylase.
I °§Q§Q="'=Y%¢._._--v-;$--—A%A at40qo\\\0&0
\ X\ \. .1- 0. 400\
POO
Free \_ IN- TB1
—TB2 \ at so "<1
<1
. | . e | - r 30 60 90 T20Time (minutes)
Fig. 4. 5.8 Varialion qf enzyme acI:'vi1y reiative tothe prer'nc*ubaIi0n time.
113
A Studyon the Immobilization of Enzymes on Polymeric Supports i
4.5.5 Kinetic parameters
The effect of immobilization on the kinetic parameters Km and V,m,_. was also
studied. Results obtained using starch as substrate is shown in table 4.8. The relationship
between the velocity of product formation and substrate concentration described a typical
quadratic hyperbole that was converted in the double reciprocal plot of Lineweaver-Burk
and Hanes-Woolf plots (fig. 4.5.9).
Table 4.8 Kinetic paranzefersfor rhefiee and immobilized a—an7yla.s'e.— 7 ' 7 * ‘ 4- 7 — * ‘T 1T‘ Fm TB TBI TB2
enzyme. n - -. 1- .-_’ - . . . T Km (mg mu‘) 1 0.46 4 0.01 7.74 4. 0.13 5.09 1 0.00 4.11 4 0.04t -l_ .l T 11/,,,.,. (EU pg“ protein) A 7.95 i 0.05 4.33 1 0.07 A 4.s2 4 0.08 3.28 4: 0.09 A
There was 9-17 fold increase in Km values presented for the immobilized enzymes.
Similar to this observations A/{soy er al. observed [41] a l2-fold increase in Km on
immobilization to poly(1nethyl methacrylate aciylie acid) microspheres and noted that
immobilized form possessed between 68-80 % of the activity of the soluble enzyme
depending on the coupling method used. The restricted mobility of the enzyme caused by
the immobilization process, as well as the presence of factors such as the limited rate of
mass transfer of the substrate from the bulk of the solution to the surface of the support
material, were expected to affect the reaction rate as well the affinity of the enzyme [64].
Hence the I/,,m is found to be always low.
ll4
Imm%Qbil?izzItig_II??Qf Di?2_Is?ta_s?e a-amylaseChaI2¥¢r:L_:I
8
5
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an'_LQ
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5.~l . ‘. €A 2ms;
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I '0'0 ( 0'4 I — W KTTZ ,4 0 21r[sn1 [8,]I llFig. 4.5.9 Linewcaver-Burk plols (I) and Hanes- W00/fplors _(II)jbr a-anzylase
inrnzobi/ized on TB (0) on TB] (b) and on TB2 (c).
1 15
A Study on the Immobilization of Enzymes on Polymeric Supports
4.5.6 Storage stability
Long term storage for 6 months of itmnobilized amylase on poly(0-toluidine) is
shown in fig. 4.5.10. As expected, immobilization improved the storage stability of the
enzyme. Highest stability was showed by covalently bonded enzyme using glutaraldehyde
spacer. Adsorbed enzyme showed least stability but better than free enzyme when free
enzyme lost all its activity within 2 days. Immobilized enzyme retained more than 50 % of
its initial activity during the storage period of 4 months. This decrease in activity was
explained as the time-dependent natural loss in enzyme activity.
I TB2,... V//A TB1% T B2
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Month
Fig. 4.5. I 0 Influence of storage time on percentageresidual activity of a-amylase immobilized on T B.
4.5.7 Reusability
The reusability of the immobilized enzyme is another very important factor from the
point of view of reducing the cost of the enzyme, which is an important aspect when taking
into account its suitability for commercial applications. It was observed that the covalently
immobilized enzymes retained 70 % of its initial activity even after the reaction was
repeated nine times (fig. 4.5.11).
116
Chapter 4 Immobilization of Diastase a-amylase
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Number of reuse
Fig. 4. 5.1 I Reusability of a-amylase immobilized on TB.
4.6 Conclusion
We have studied various carriers polyaniline and poly(o-toluidine) in acidic and
basic form that are applicable for immobilization of a/-amylase and determined several
parameters essential for the practical application of the immobilized enzyme. Initial work
using amylase evaluated the binding parameters for the various carriers tested. Taking into
account the main parameters, such as immobilization efficiency, pH and thermal stability,
reusability, and storage stability we found that covalent binding with glutaraldehyde spacer
are the most efficient methods of irmnobilization for all of the supports selected. The
tendency to form dimeric form structure by this activator results in creation of long spacer
between matrix and protein surfaces, which keep the enzyme not so close to the polymers
surface and the access of large starch molecules to a-amylase not so restricted [65]. The
main observations of the study on immobilized a-amylase are:
It Free diastase a-amylase hydrolyzed starch when the optimum conditions of pH
5-5.5 and temperature 50 "C. The Michaelis-Menten constant for starch
hydrolysis was evaluated as 0.46 mg mL".
117
A Study on the Immobilization of Enzymes on Polymeric Supports
The enzyme a-amylase immobilized on various supports in the pH range of 4. 5
5.5 within the duration of 30-60 minutes irrespective of the supports andmethods used.
After immobilization the pH for optimum enzyme activity was changed to either
5 or 5.5, depends upon supports and method used. The glutaraldehyde activated
supports showed better resistance to pH changes.
The optimum temperature for starch hydrolysis was lowered by I0-15 "C after
immobilization. The immobilized enzymes showed better thermostability and
retained 80-90 % activity afler 2 hour incubation at their optimum temperature.
The kinetic parameter Km showed a 2-l 7 fold increase after immobilization and
Vmx was found to be low for immobilized enzyme.
The immobilized enzyme could be reused efficiently in batch reactor. Covalently
immobilized retained more than 50% activity even afier 10 cycles of use.
The immobilization led to an improvement in storage stability. Enzyme could be
stored for 6 months with very little change in activity.
118
Chapterfl lmmolglization of Digstase 0:-amylase
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