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

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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)

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enoadProt

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Enzyme act v ty (EU

l

3- /‘2__ __i-1-ii-O

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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

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— —TB1— —TB2

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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.

%§:”'\.

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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

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Fig. 4. 5. 7 Thermal stability Q/‘free and immobilizeda-amylase.

I °§Q§Q="'=Y%¢._._--v-;$--—A%A at40qo\\\0&0

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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.

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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.

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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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