Noncovalent immobilization of enzymes on an enteric polymer Eudragit S-100

Noncovalent immobilization of enzymes on an enteric polymer Eudragit S-100 M. Sardar, R. Agarwal, A. Kumar, and M. N. Gupta Chemistry Department, Indian Institute of Technology, New Delhi, India The noncovalent immobilization of enzymes such as alpha-amylase, beta-glucosidase, trypsin, and alkaline phosphatase was performed by adsorption on the water-soluble polymer Eudragit S-100. The strength of the binding with enzymes in some cases was critically dependent upon the initial polymer concentration used during binding. In all the cases tried, a moderate increase in polymer concentration ensured adequate immobilization of enzymes. The immobilized enzymes retained different activities: 87, 59, 49, and 24% for beta-glucosidase. alpha-amylase, trypsin, and alkaline phosphatase, respectively. The K, value of immobilized enzyme was the same as that of native enzyme for beta-glucosidase (3.8 x lo-’ M) and alpha-amylase (6 mg ml-‘) whereas the K, value decreased in the case of trypsin (from 1 x 10m3 M to 0.6 x lo-’ M) upon immobilization, The immobilized trypsin showed improved stability to autolysis at 35°C whereas immobilization resulted in a decrease in the thermal stability of alpha-amylase at 5O’C. No significant changes were observed in pH optimum of the enzymes upon immobilization. U.V. and fluorescence emission spectra of immobilized trypsin reflected the con0 1997 by Elsevier Science Inc. formational changes which enzymes undergo upon adsorption on the polymer. Keywords: Alkaline phosphatase; alpha-amylase; beta-glucosidase; Eudragit; fluorescence/U.V. proteins; noncovalent immobilization of enzymes; trypsin; water-soluble polymers

Introduction Immobilization of enzymes has become an established at; preach for enhancing their usefulness in biotechnology. Broadly, the various methods of immobilization may be divided into two categories such as covalent coupling methods and noncovalent methods. The latter includes adsorption and entrapment. Noncovalent methods of immobilization have been often preferredze5 over harsher covalent methods although quite often the enzyme slowly comes off the matrix in the former method. The origin of the present work lies in our earlier observation6 that recovery of the enzymes while using the polymer Eudragit S-100 for precipitation was dependent upon the concentration of the polymer at the precipitation stage. Eudragit S-100 is an enteric methacrylate polymer which is commercially available. It belongs to the class of reversibly soluble/insoluble polymer,7 i.e., its solubility can be controlled by altering the

spectra of immobilized

pH. It is soluble in aqueous solutions above pH 5.5 and precipitates below this pH. This property has made it a valuable polymer in the separation of proteins7-9 as well as a soluble matrix for covalent immobilization of enzymes. 7,10-12Immobilization on soluble matrices is preferred over insoluble matrices when the immobilized enzymes are to be used with macromolecular or insoluble substrates.7’1C’3 This work shows that a variety of enzymes absorb to Eudragit S-100. The strength of this binding is dependent upon the initial polymer concentration used during binding. In all cases tried, a moderate increase in polymer concentration ensured adequate immobilization of the enzymes. The immobilized enzymes were characterized in terms of their catalytic activity, kinetic parameters, and thermal stability.

Materials and methods Materials Address reprint requests to Prof. M. N. Gupta, Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi-110016, India Received 1 February 1996; accepted 13 June 1996

Enzyme and Microbial Technology 20:361-367, 1997 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

Trypsin, beta-glucosidase (from almond), alpha-amylase (from porcine pancreas), N-benzoyl-nkarginine-p-nitroanilide (PNGP) were (BAPNA): p-nitro~henyl-betz-kglucopfranoside

0141-0229/97/$17.00 PII SO141-0229(96)00152-4

Papers from Sigma Chemical, St. Louis, MO. Alkaline phosphatase (chicken intestine) and p-nitrophenyl phosphate were obtained from Sisco Research Laboratories (Bombay, India). Eudragit (S100 type) was a product from Rohm Pharma Gmbh (Weiter Stadt, Germany). All other chemicals used were analytical grade.

Preparation of Eudragit S-I 00 solution Eudragit S-100 (5 gm) was dissolved with constant stirring in 90 ml of distilled water by adding 3 M NaOH solution dropwise until the pH reached 11. After the polymer was fully solubilized, the pH of the solution was decreased to 7.0 by adding 3 M HCl. Volume was made up to 100 ml with distilled water. The solution was stored at 4°C for further use and transferred to appropriate buffer by precipitating at pH 4.5 and redissolving at pH 7.0.

Immobilization of enzyme on Eudragit S-l 00 To 4 ml of Eudragit S-100 (concentrations in different experiments varied in the range of O.lA%), different enzyme solutions such as 1 ml alpha-amylase (5 mg ml-’ in 0.02 M phosphate buffer containing 6 rrt~ NaCl pH 6.9), 1 ml alkaline phosphatase (5 mg ml-’ in 0.1 M Tris-HCI pH 8.0), 1 ml of trypsin (2.5 mg ml-’ in 0.05 M Tris-HCI containing 0.02 M CaCl, pH 8.0) and 0.2 ml of beta-glucosidase (1 mg ml-’ in 0.01 M acetate buffer pH 5.8) were

Table 1

added separately. After incubation for 1 h at room temperature, polymer was precipitated by adjusting pH to 4.5 by adding 0.1 M acetic acid. After 20 min, the suspension was centrifuged at 12,000 g for 15 min. The enzyme activity and protein were measured in the supematant. The precipitate obtained was dissolved in appropriate buffers as above so that the final volume was 5 ml. An appropriate aliquot of the dissolved precipitate was taken to calculate the expressed activity (Table I).

Optimization of binding of enzymes on Eudragit S-I 00 To 4 ml of Eudragit S-100 (l%), varying amounts of betaglucosidase, trypsin, alkaline phosphatase, and the alpha-amylase dissolved in 1 ml of appropriate buffer were added separately (Table 2). In each case, the following procedure was performed. After incubation for 1 h at room temperature, the polymer was precipitated by adjusting the pH to 4.5 by adding 0.1 M acetic acid. After 20 min, the suspension was centrifuged at 12,000 g for 15 min. The precipitate was washed with the acetate buffer until no enzyme activity could be detected in the supematant. Enzyme activity and protein were measured in the supematant. The precipitate was dissolved in their respective buffers so that the final volume was 5 ml. The yield of the immobilized preparation was

Binding and recovery of enzymes on Eudragit S-100 at different polymer concentrationa

Polymer concentration (%I A-Binding 0.1 0.5 1.0 2.0 3.0 4.0 B-Binding 0.1 0.5 1.0 2.0 3.0 4.0 C-Binding 0.1 0.5 1.0 2.0 3.0 4.0 D-Binding 0.1 0.5 1.0 2.0 3.0 4.0

Unbound activity in supernatant (%I

Bound enzyme theoretical (%I

Expressed activity (%)

1

M

NaClb (%I

Leaching of activity with 50% EG” (%I

of beta-glucosidase 38 25 11 6 3 2

10

99

83 85 87 87 87 86

90 96 98 99 99 99

41 45 48 48 47 49

30 27 25 18 12 15

19 18 15 10 10 8

100 100 100 100 100 100

48 57 59 58 58 58

80 90 96 96 97 100

16 21 21 21 20 19

0 0 0 0 0 0

0 0 0 0 0 0

4 2

96 98

1 1

E 99

2 0.5 0.2 0.2

of trypsin 10 4 2

of alpha-amylase

of alkaline phosphatase 20 10 4 4 3 0

‘Each value is a mean result of duplicate experiments. The difference in individual readings in all cases was less than *5 bLeaching of the activity was checked by dissolving the polymer in phosphate buffer (0.1 M, pH 7.2) containing 1 M NaCl or Tris buffer (0.05 M, pH 7.0 containing 20 mM CaCi, + 1 M NaCI) in case of trypsin. After incubation for 30 min. the polymer was reprecipitated by adding 0.1 N acetic acid to pH 4.5. After centrifugation, activity was measured in the supernatant =ln this case, the precipitated pellet was just washed with 50% ethylene glycol (EG)

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Noncovalent Table 2

Optimization

of binding of enzymes on Eudragit S-100’

Enzyme addedb (X) units A-Binding of (Binding 120 240 480 720 (Binding 240 B-Binding of (Binding 3.1 6.2 18.6 31.1 43.2 62.0 (Binding 31.1 C-Binding of (Binding 1.68 2.7 5.4 10.8 (Binding 5.4 D-Binding of (Binding 5.4 10.8 21.6 43.2 (Binding 10.8

of enzymes on an enteric polymer: M. Sardar et al.

immobilization

Enzyme remaining unbound (Y) units

beta-glucosidase in soluble phase at pH 7.5) 0.025 0.025 0.030 0.040 in solid phase at pH 4.5) 0.02 trypsin in soluble phase at pH 7.5) 0.03 0.08 0.15 0.19 0.40 0.76 in solid phase at pH 4.5) 0.21 alpha-amylase in soluble phase at pH 7.5) 0 0 0

Bound enzyme theoretical (X - Y = A)

Activity of immobilized enzyme (B) units

Activity yield B/A x 100 (%)

119.9 239.9 479.9 719.9

104 206 384 576

87 86 80 80

239.9

211

88

in solid phase at pH 40.5) 0 alkaline phosphatase in soluble phase at pH 7.5) 0.15 0.33 0.7 in solid phase at pH :I:) 0.29

3.06 6.11 18.4 30.9 42.8 61.2

1.5 2.9 9.0 14.9 18.0 22.6

49.0 48.2 48.8 48.2 42 37

30.89

14.5

47

1.68 2.7 5.4 10.8

1.0 1.58 3.12 6.0

59 58 58 56

5.4

3.0

56

5.25 10.5 20.9 41.7

1.2 2.25 4.5 8

23 21 22 19

10.51

2.5

24

“Each value is a mean result of duplicate experiments. The difference in individual readings in all cases was less than i5 bVarying amount of enzymes (O.D. units given above) dissolved in 1 ml of appropriate assay buffer was added to 4 ml of Eudragit S-100 solution (containing 40 mg of Eudragit S-100

calculated in terms of expected and expressed activities (Table 2). The binding of the enzyme to the polymer was also studied by directly adding enzyme solutions to the insoluble polymer at pH 4.5.

Effect of polymer concentration on leaching of immobilized enzymes The immobilized enzyme preparations with varying polymer concentrations obtained above were dissolved separately in 4 ml of different buffers containing 1 M NaCl. After shaking for 1 h in a waterbath at 25”C, the polymer was precipitated by adjusting the pH to 4.5 by adding 0.1 M acetic acid. After 20 min, the suspension was centrifuged at 12,000 g for 18 min. The activity was measured in the supematant. This step was repeated until no enzyme activity could be detected in the supematant (Table I).

Determination of K, Km values of native and immobilized enzymes were determined by measurement of enzyme activity with various concentrations of substrate. The same amount of free and immobilized enzyme (in terms of protein) were used in these experiments. Km values were

calculated Burk.14

by plotting

the data according

to Lineweaver

and

Determination of pH optimum and thermal stability Effect of pH on native and immobilized enzymes was studied by assaying both the preparations at different pH values. Thermal stability of beta-glucosidase was studied at 55°C. An appropriate aliquot of native and immobilized enzyme was drawn at various time intervals of incubation and their activities were determined. Similarly, thermal stability of trypsin (at 35, 40 and 45°C) and alpha-amylase (at 40 and 5O’C) were studied (Figures la-lc).

Estimation of enzyme activities Activity of beta-glucosidase was measured using p-nitrophenyl beta-pglucopyranoside as substrate.15 Activities of trypsin and alkaline phosphatase were estimated using BAPNA and p-nitrophenyl phosphate as a substrate, respectively.‘6.‘7 Activity of alphaamylase was measured using starch as a substrate.” In the case of immobilized enzymes, an appropriate aliquot of the immobilized enzyme (the polymer-bound enzyme was dissolved in appropriate

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Papers buffers above pH 5.5 as Eudragit is soluble above this pH) was taken and the same assay procedure was followed as for the free enzyme. A control of polymer alone was run in each case.

Estimation of protein Protein was estimated using the dye-binding method of Bradford” using bovine serum albumin as a standard protein.

Results and discussion Eudragit S-100 has several attractive features which favors its use as an immobilization matrix for enzymes/proteins. It

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is nontoxic (safe for use in food processing industries), water soluble, recoverable from solution by altering pH, economical, and commercially available.*’ It has been extensively used for precipitation and affinity precipitation of enzymes.20721In these applications,9320it has been observed that enzymes show considerable nonspecific adsorption to this polymer. Four different enzymes (beta-glucosidase, trypsin, alphaamylase, and alkaline phosphatase) were adsorbed to the polymer Eudragit S-100 using different polymer concentrations in the range of 0.14% (w/v). In all four cases, most of the enzyme activity was bound to the polymer (Table 1). In the case of alpha-amylase and alkaline phosphatase, binding of the enzymes to the polymer was adequately strong since no activity leached off the matrix when the immobilized enzymes were precipitated in the presence of 1 M NaCl. In the cases of beta-glucosidase and trypsin, the effect of polymer concentration on the strength of the binding was reflected. In both cases, decreased leaching of the enzyme activity off the matrix was observed as the polymer con-

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Figure 1 Thermal stability of free and immobilized betaglucosidase at 55’C (a). Free and immobilized beta-glucosidase in 0.05 M acetate buffer pH 5.0 were incubated at 55°C. A 50 ul aliquot was withdrawn in each case at various intervals of incubation and beta-glucosidase activity was determined using PNGP as a substrate. The activity of the free enzyme is taken as 100%. The actual expressed activity of the immobilized enzyme (i.e., when the enzyme load, protein-wise, corresponds to a protein equivalent of 100% activity for free enzyme) is plotted. Native enzyme (69); and immobilized enzyme inactivation (0) of free and immobilized trypsin (b). Free and immobilized trypsin in 0.05 M Tris-HCI pH 8.0 containing 20 mM CaCI, were incubated at different temperatures. Aliquots (0.6 ml) were withdrawn after various time intervals of incubation in each case and trypsin activity was determined using BAPNA as substrate. The activity of the free enzyme is taken as 100%. The actual expressed activity of the immobilized enzyme (i.e., when the enzyme load, protein-wise corresponds to a protein equivalent of 100% activity in the case of free enzyme) is plotted. Free trypsin at 35°C (0); immobilized trypsin at 35’C (U); 40°C (A); and 45’C (A). Thermal stability of free and immobilized alpha-amylase (cl. Free and immobilized alpha-amylase in 0.02 M phosphate buffer containing 6 mM NaCl pH 7.0 were incubated at 40°C and 5O’C. Aliquots (0.4 ml) was withdrawn at various time intervals of incubation in each case and alpha-amylase activity was determined using starch as a substrate. The activity of the free enzyme is taken as 100%. The actual expressed activity of the immobilized enzyme (i.e., when the enzyme load, protein-wise corresponds to a protein equivalent to 100% activity in the case of free enzyme) is plotted. Free enzyme at 40°C (Xl; free enzyme at 5O“C (A); immobilized enzyme at 40°C (W); and immobilized enzyme at 5O“C (0)

Noncovalent

immobilization

centration during immobilization was increased. This shows that by employing higher polymer concentration during immobilization, one could strengthen the noncovalent forces between the polymer and an enzyme. This was true of both electrostatic and hydrophobic bonds since even elution of the enzymes from the polymer with 50% ethylene glycol decreased in the cases of beta-glucosidase and trypsin on higher polymer concentration. It is also interesting to note that in all four cases, the expressed activity (i.e., the actual activity observed with polymer-enzyme conjugate in the soluble state with the respective standard assays) marginally improved with higher polymer concentration. While the variation is within experimental error range, the consistency in all four cases shows a trend. Table 2 shows how enzyme loading affects the expressed activity when a constant polymer concentration was employed. In all four cases, the polymer showed good capacity and no significant decrease was observed as a result of crowding on the matrix** even at fairly high enzyme loads. In order to determine whether adsorption or entrapment was involved here, the immobilization was also performed by adding enzymes to the polymer in solid state. This was possible by addition of the enzyme to the polymer at a low pH such as 4.5. The data shown in Table 2 indicates that in all four cases, the amounts of enzyme bound and expressed activities observed remained unchanged; thus, the results are identical whether immobilization is performed by adding enzymes to the solid matrix at lower pH or by coprecipitating enzymes and the polymer by lowering the pH. Hence, it is proper to term this as immobilization by adsorption rather than entrapment. Relatively speaking, rather low expressed activity was observed in the case of alkaline phosphatase; hence, all further work was performed with other three enzymes. The pH-dependent precipitation curves of Eudragit and Eudragit-bound enzymes are similar though not identical (Figure 2). The minor changes observed are expected to be an outcome of complex interplay between isoelectric points of the enzymes adsorbed, extent of adsorption, and the distribution of enzyme molecules on the polymer surface. From the practical point of view, it was useful that all immobilized enzymes precipitated quantitatively and could be dissolved quantitatively by raising the pH; thus, the adsorbed enzymes constituted reversibly soluble/insoluble enzyme derivatives. The catalysis in all the cases could be performed at their respective pH optimum in free solution and the enzyme derivatives could be recovered for reuse by lowering the pH. The noncovalently adsorbed enzymes were examined for thermal stability. In the case of betaglucosidase, immobilization resulted in marginal thermostabilization (Figure la). At 55’C, the native enzyme retained 32% activity after 90 min whereas the immobilized enzyme retained 50% activity under identical conditions. There was a significant decrease in inactivation of immobilized trypsin when compared to free trypsin (Figure lb). Perhaps the most interesting outcome of the immobilization is in the case of alpha-amylase. This enzyme showed enhanced thermolability at a moderate temperature of 50°C. Within 80 min, the immobilized enzyme retained no activity whereas the free enzyme sample was still 72% active under identical

of enzymes

on an enteric

polymer:

M. Sardar et al.

Figure 2 pH precipitation pattern of Eudragit and Eudragitbound enzymes. The polymer and polymer-bond enzymes were dissolved separately in 0.05 M Tris-HCI buffer pH 8.0. The pH of the solution was adjusted to a specified value by the addition of 2 M acetic acid. The solution was centrifuged and the amount of polymer-bound enzyme precipitated wes calculated by measuring the enzyme activity still present in the supernatant. (The pH of the supernatant was adjusted to the assay pH before the enzyme assay was performed). Furthermore, in another control, it was observed that trypsin and beta-glucasidase were stable at all pHs from 4 to 8 for 30 min. although in the case of alphaamylase, enzyme activity decreased by 30% after incubation at pH 4.5 for 30 min). Activity present in the solution at pH 8.0 assayed and taken as 100%. In the case of Eudragit only, absorbance at 280 nm was measured to estimate unprecipitated Eudragit. 100% was taken as the absorbance of 4% Eudragit at pH 8.0. Beta-glucosidase bound to Eudragit (x); trypsin bound to Eudragit (WI; alpha-amylase bound to Eudragit (0); and Eudragit s-100 (A)

conditions (Figure Zc). In the case of alpha-amylase, this reduced thermostability is desirable since thermolabile alpha-amylase is reported to be useful for improving both loaf volume and crumb softness of bread products without the danger of overdextrination.23 The pH optimum of the immobilized enzyme was the same as that of the native enzyme in all cases. The K, value of the immobilized enzyme was the same as that of the native enzyme for beta-glucosidase (3.8 x 10e3 M) and alpha-amylase (6 mg ml-‘) using PNGP and starch as substrates, respectively. In the case of trypsin, there was a decrease in the K,,, value of immobilized enzyme (from 1.0 x 10e3 M to 0.6 x 10m3M) using BAPNA as substrate. This decrease in K,,, value is likely to be due to the fact that the local concentration of the substrate (with its positive charge) increases because of the negatively charged matrix near the immobilized enzyme. In fact, at higher concentrations of BAPNA (1.25 mM or higher), the interaction between BAPNA and the polymer leads to the precipitation of the polymer from the solution. Working with a water-soluble polymer makes it possible for use to study the enzyme conformation with spectroEnzyme Microb.

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Papers

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$ 0.20

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300

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scopic techniques. Figures (3 and 4) show the UV and fluorescence spectra of native trypsin and the trypsin immobilized on Eudragit S-100. The decrease in the UV absorbance around the 280 run region (Figures 3a and 3b) upon immobilization indicates slight unfolding of the molecule.24*25 This is not unexpected since multivalent interaction between the protein molecule and the matrix during adsorption is likely to involve some structural “readjustment” on the part of the protein molecule. In the case of fluorescence, changes in intensity upon unfolding are unpredictable,24 however, here too, the fluorescence intensity did decrease upon immobilization which again reflects some conformational changes. For both, UV and fluorescent

Figure 4

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studies A,, absorbance and A,, emission (respectively) did not change which suggests that conformational changes are not drastic in nature.24 It is also possible that these spectral changes reflect “shielding” by the polymer rather than any significant structural changes. There is considerable interest in protein adsorption on polymeric surfaces since the phenomenon is relevant to biomedical and process engineering.26 However, there is a paucity of data using UV and fluorescent spectroscopy although some studies using li ht scattering and microcalorimetery have ellipsometry, 85 been reported. The latter have suggested that timedependent structural changes in protein conformation are observed26 after adsorption on polymeric surfaces.

(a)

5 i;

Figure 3 U.V. spectra of immobilized trypsin. The spectrophotometric measurements were done at 25°C on a Beckman DU-640 (a). Eudragit S-100 (4 ml; concentrations varied in the range of O.l4%) was added to 1 ml of trypsin (2.5 mg ml-‘) in 0.05 M Tris-HCI buffer pH 8.0. The solution was precipitated by decreasing the pH to 4.5. The precipitate was centrifuged at 12,000 g for 20 min and dissolved in 0.05 M Tris-HCI buffer pH 8.0. 0.1% polymer (I); 0.5% polymer (II); 4.0% polymer (Ill); trypsin immobilized on 0.1% polymer (IV); trypsin immobilized on 0.5% polymer (V); free trypsin (VI); and trypsin immobilized on 4.0% polymer (VII). The amount of protein was same in all these cases. Difference spectra of the immobilized enzyme were recorded after subtracting the respective contribution of the polymer spectra from the spectra of the immobilized enzvmes (b). Trvosin immobilized on 0.1% polymer (VIII); trypsin immobilized on 0.5% polymer (IX); and trypsin immobilized on 4% polymer (X)

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Fluorescence studies of immobilized trypsin. Emission spectra were taken on Shimadzu RF-5000 (Spectrofluoro-photometer at an excitation wavelength of 287 nm (a). Eudragit S-100 (4 ml; concentrations varied in the range of 0.54%) was added to 1 ml of trypsin (2.5 mg ml-‘) in 0.05 M Tris-HCI buffer pH 8.0. The solution was precipitated by decreasing the pH to 4.5. The precipitate was centrifuged at 12,000 g for 20 min and dissolved in 0.05 M Tris-HCI buffer pH 8.0. Trypsin immobilized on 0.5% polymer (I); free trypsin (II); and trypsin immobilized on 4% polymer (Ill). The amount of protein was same in all these cases. Difference emission spectra of the immobilized enzyme were recorded after subtracting the respective contribution of the polymer spectra from the spectra of the immobilized enzymes (b). Free trypsin (VI); trypsin immobilized on 0.5% polymer(V); and trypsin immobilized on 4% polymer (IV)

Noncovalent

immobilization

The polymer-bound enzymes could be precipitated and redissolved repeatedly with good recovery of activity. Expressed activity of immobilized trypsin changed from 49 to 47.5% after precipitating and redissolving the immobilized enzyme. Similarly, the activity of immobilized betaglucosidase changed from 87 to 85% only; thus, these studies show that in the case of Eudragit S-100, the enzymes can be immobilized noncovalently by working with higher polymer concentrations. An additional useful feature of Eudragit S-100 in this context is that it shows good capacity for enzymes. Noncovalent methods of immobilization, wherever successful, are preferred since the approach is simple, economical, and does not employ harsh conditions.

of enzymes

9.

10.

11.

12.

13.

Acknowledgments 14.

This work was supported by the Swedish International Development Co-operation Agency (SIDA) and Department of Biotechnology, Govt. of India. The financial support provided by IIT Delhi and CSIR, India to MS and RA in the form of SRF, respectively, is also acknowledged. We thank Mr. G. K. Pandita for typing the manuscript.

15.

16.

17.

References 18. 1.

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Adlercreutz, P. Immobilized enzymes. In: Enzymes in Food Processing. (Nagodawithana, T. and Reed, G., Eds.). Academic Press, New York, 1993, 103-119 Gupta, M. N. and Mattiasson, B. Unique applications of immobilized proteins in bioanalytical systems. In: Bioanalytical Applicutions of Enzymes Vol. 36 (Suelter, C. H., Ed.). John Wiley & Sons, New York, 1992, l-34 Batra, R. and Gupta, M. N. Noncovalent immobilization of potato (Solanum tuberosum) polyphenol oxidase on chitin. Biotechnol. Appl. Biochem. 1994, 19, 209-215 Cabral, J. M. S. and Kennedy, J. F. Immobilization techniques for altering thermal stability. In: Thermostubility of Enzymes (Gupta, M. N., Ed.). Springer-Verlag, Heidelberg Germany, 1993, 162-179 Tyagi, R. and Gupta, M. N. Non-covalent and reversible immobilization of chemically modified amyloglucosidase and betaglucosidase on DEAE-cellulose. Proc. Biochem. 1994,29,443448 Kumar, A., Aganval, R., Batra, R., and Gupta, M. N. Effect of polymer concentration on recovery of the target proteins in precipitation methods. Biotechnol. Tech. 1994, 8, 651-654 Fujii, M. and Taniguchi, M. Application of reversibly soluble polymers in bioprocessing. Trends Biotechnol. 1991, 9, 191-196 Guoqiang, D., Lali, A., Kaul, R., and Mattiasson, B. Affinity thermoprecipitation of lactate dehydrogenase and pyruvate kinase from

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