KINETIC STUDIES ON SOLUBLE AND IMMOBILIZED ALPHA , AMYLASE AND GLUCOAMYLASE ' s

Alpha amylase and glucoamylase were coupled seperately to Sepharose4B which was activated by an electrophilic method (by using cyanogen bromide). The kinetic properties of these enzymes in immobilized form were compared with those of their soluble enzymes. The Km values of soluble and immobilized alpha amylase for starch were 0.89% (WIV) and 1.33% ( W M respectively at pH 6.9 and a t 40°c and the Km values of soluble and immobilized glucoamylase for starch were 0 0.25% (WIV) and 0.72% (WIV) respectively at pH 4.8 and a t 50 C. The optimum pH f i r so lub l~ alpha amylase =ompared with that of the immobilized enzyme shifted from 6.9 to 6.5 in .0 .02~ phosphate buffer while in the caSe of ghcoamy1,ase the pH optimum in 0.01M acetate buffer increased from 4.8 to 5.2 on immobilization. There was no shift'in optimum 'phosphate concenmation .(0.02M) for both soluble 0 and immobilized alpha amylase at pH.6.9 and at 40 C and likewise no shift in optimum acetate concentration (0.01M) for soluble and immobilized glucoamylase at pH 4.8 and at 50°c. The optimum temperature for the activities of soluble alpha amylase compared with the immobilized enzyme assayed in 0.02M phosphate buffer (pH 6.9) shifted from 4 5 ' ~ to 5 0 ' ~ and that of glucoamylase assayed in 0.0lM 0 0 acetate buffer (pH 4.8) shifted from 55 C so 58 C.


Introduction
Enzymes of industrial or analytical use could have increased applicability, if they )could be immobilized while still in an active state.Many studies have been Yeported on the preparation of immobilized enzymes.2+3*6~7~8Great interest in the use of water insoluble enzymes has been shown in industry.These enzymes can be readily recovered from the reaction mixture resulting in a considerable saving in process costs by repeatedly using the en~yrnes.~Many products are currently produced from starch through the'use of enzymes, whether soluble or immobilized .Both alpha amylase (1,4-a-D glucan glucanohydrolase; E.C. 3.2.1 .I) and glucoamylase (1,4--a-D glucan glucohydrolase; E.C.3.2.1.3)are important industrial enzymes.These are used in large scale for liquefaction and saccharification of starch.
Immobilized enzymes may exhibit slightly altered chemical and physical properties,6 l 8 Therefore while designkg chemical reactors for utilizing immobilized enzymes, certain parameters such as catalyst size, liquid f l o u r rate, temperature,' pH, ionic strength, substrate concentration etc.,must be carefull'y controlled in order to yield optimum conversion.
Our earlier study was undertaken to develop a functional immobilized alpha.amylase and g l u c ~a r n ~l a s e .~ This paper compares the kinetic proper-ties of the soluble and immobilized forms of alpha amylase and glucoamylase.A knowledge of the kinetic parameters could be helpful in the design of reactors for contin~xous production of liquid glucose from soluble starch.

Materials
Alpha amylase (Hog pancreas) was obtained from Sigma Chemical Company, U.S.A. crude glucoamylase (Aspergillus niger) was from Ceylon Institute of Scientific and Industrial Research (G.I.S.I.R.), Sri Lanka, Sepharose-4B was from Pharmacia Fine Chemicals, and starch (soluble) was from BDH, UK.
Cyanogen bromide was prepared in our laboratory.' 2.2 Imrn~,bilization of Alpha Amylase and Glucoamylase t o Sepharose -4B In the preparation of both the immobilized alpha amylase and glucoamylase the soluble enzymes used had a spcific activity of 906 u/mg protein and 370 u/mg protein respectively.They were prepared by electrophilic method." In the comparative studies equal amounts of soluble and immobilized enzyme proteins (0.96 mg protein as determined by micro-Kjeldhal method) were used.
The reducing equivalents produced by-the action'of alpha .amylaseand glucoam'ylase on starch were measured in terms of maltose and glucose respectively.4 One unit of alpha amylase will liberate 1pg of maltose from starch in 3 min at pH 6.9 and at 4 0 ' ~.One unit of glucoamylase will liberate 1pg of glucose from starch in 10 min at pH 4.8 and at 50°C.'2.3 Kinetic Properties o f .Soluble and Immobilized Alpha Amylase and
The effect of pH on the activities of zlphs amylase and glucoamylase was studied by altering the pH from 5.6 to 8.0 of 0.02M phosphate baffer at 40°C and from 3.6 to 6.0 of 0.OlM acetate buffer at 50°C respectively.

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Phosphate buffer (pH 6;9) in the range of 0.01 to 0.12M for a alpha amylase'at 4 0 ' ~ and acetate buffer (pH 4.8) in the range of 0.004 to 0.05M for glucoamylase at 50'C were used to study the effect of ionic stren,@hs on these enzymes.
The temperature optimum for alpha amylase in 0.02M phosphate buffer (pH 6.9) and glucoamylase in 0.01M acetate buffer (pH 4.8) were ' studied in the range of 4 ' C to 8 0 ' ~.
The percentage activity was calculated in all cases (Figunes 1 & 2 :, Table I, 1

Results and Discussion
The Km values o~f soluble and immobilized alpha amylase for starch (calculated from Lineweaver -Burk plots) wefe 0.89% and 1.33% respective1.yand that of .solubleand immobilized glucoamylase for starch were 0.25% and 0.72% respectively.Krakowick et.i l k 6 have immobilized glucoaniylase (type 150, Ncvo) on aluminium oxide and on iiluminium oxide activated by TiC1,.The Km values of soluble glucoamylase, glucoamylase immobilized to aluminium oxide and glucoamylase coupled to activated aluminium oxide for starch.werefound to be 0.5%, 6.69% and 28.15% respectively.The increase in Km value of immobilized enzymes t a that of the soluble enzyme may be due to the limitations of diffusien caused by unstirred-layen: which surrounds the water insoluble particles.This causes a decrease in the affinity or an kzrease in the Km of the enzyme for the substrate.
The optimum pH values for soluble and immobilized alpha amylase L were 6.9 and 8.5 in 0.32M phosphate buffer (Figure 1) and for spluble and + immobhzed glucoamylase were 4.8 and 5.2 in 0.01M acetate buffer (Figure 2).Purified glucoamylase from Aspergillus niger showed the pH optimum at 4.3 with relatively high activity in the pH range between 3.6 and 4.6.5But in our experiment the crude enzyme used showed a pH optimum of 4.8 which could be due to the presence of isoenzymes.Wykes et.aL8 showed that the optimum pH values for soluble and CM-cellulose bound alpha amylase .were6.0 and 6.5 respectively.In that study CM-cellulose which has a negatively charged matrix vas used while in this study the matrix (sepharose-4B) was neutral and hence, the change in pH optimum could not be due to the charge effect s f the matrix.It is possibly due to the changes m conformation of the enzymes resulting from immobilization.
The concentration of phosphate buffer had a marked effect on soluble alpha amylase compared t o the immobilized enzyme (Table 1).Although the soluble and the immobilized alpha amylase showed optimal activity at 0.02M phosphate concentration, the immobilized alpha amylase had considerably higher activities than the soluble alpha amylase at dl other phosphate concentrations.Likewise the immobilized glucoamylase at all acetate concentrations except at tne optimal 0.01M acetate concentration (Table 2).
Table 1.Effect of phosphate concentration cn soluble and immobilized alpha mylase activities at ?H 6.9 and at 40°C.Absolute activities of soluble and immobilized glucoamylase were 355 ulml and 390 ulml respectively.
The optimum temperature for soluble and immobilized alpha amylase were 45'C and 5 0 ' ~ respectively and that of soluble and immobilized glucoamylase were 5 5 ' ~ and 5 8 ' ~ (Table 3) showing that immobilization had increased the optimum temperature of both enzymes slightly.
.1 & 111) for both alpha amylase and glucoamylase as the activity at a particular condition / the .maximumactivity obtained in that set of experimental conditions x 100.
e of 3 experiments as described in section 2.3.Absolute activities for soluble and immobilized alpha amylase were 870 u/ml and 1430 ulml respectively.

Table 2 .
Effect of acetate concentration on soluble and immobilized glucoamylase activities at pH 4.8 and at 50' C.Mean values of 3 experiments as described in section 2.3.

Table 3 . Effect of temperature on the activities of soluble and immobi- lized alpha amylase (in 0.02M phosphate buffer pH 6.9) and glucoamylase (in 0.01M acetate buffer pH 4.8).
v Temperature % Activity i (OC).