A Smart Bioconjugate of Chymotrypsin - ACS Publications

Biomacromolecules 2003, 4, 330-336

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A Smart Bioconjugate of Chymotrypsin Shweta Sharma,† Prabhjot Kaur,† Aklank Jain,‡ Moganty R. Rajeswari,‡ and Munishwar N Gupta*,† Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India, and Biochemistry Department, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India Received September 17, 2002; Revised Manuscript Received December 7, 2002

R-Chymotrypsin was immobilized on Eudragit S-100 via covalent coupling with 93% retention of proteolytic activity. The conjugate behaved as a smart biocatalyst and functioned as a pH-dependent reversibly solubleinsoluble biocatalyst. The pH optimum of chymotrypsin broadened on immobilization, and the immobilized preparation showed better stability at and above pH 6.5 as compared to the free enzyme. The immobilized enzyme showed a slight shift in the temperature optimum and enhanced thermal stability retaining 70% of its original activity after 1 h of exposure to 40 °C as compared to the 25% residual activity for the free enzyme under identical conditions. Km and Vmax values did not change on immobilization. Also, the immobilized preparation was quite stable to reuse, it retained almost 85% of its original activity even after a fifth precipitation cycle. UV spectroscopy and circular dichroism were used to probe structural changes in the enzyme upon immobilization. Introduction Immobilized enzymes have found numerous applications in synthesis, analysis, and bioconversions.1-4 The main advantages of immobilized enzymes are reusability and (quite often) enhanced storage and operational stability. Conventionally, solid and porous matrixes such as agarose, cellulose, silica, etc., are used for adsorbing or covalently linking enzymes. One major drawback with such immobilized systems is the mass transfer limitation, which contributes significantly to decrease in “effectiveness factor”. With solid matrixes, diffusional resistance to internal transport of reactants and products inside the porous matrix adds on to this mass transfer limitation.5,6 Thus, such immobilized enzymes perform poorly when the substrate is macromolecular or insoluble in nature. Use of smart polymers as matrixes has been suggested as a solution to this problem.7-10 Thus, an enzyme linked to reversibly soluble-insoluble polymer can be used in soluble form as a homogeneous catalyst. After the biotransformation is over, the biocatalyst conjugate can be separated from the unreacted substrate(s) and products and reused. The present work describes covalent coupling of R-chymotrypsin to Eudragit S-100. Eudragit S-100 is a methyl methacrylate polymer which is completely soluble above pH 4.5.6,11-13 This polymer has been extensively used for separation of enzymes by affinity precipitation.11-15 Thus, its behavior as a smart polymer is well documented. The similar bioconjugates with eudragit class of polymers have * To whom correspondence should be addressed at Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110 016, India. Tel.: 91-11-26591503, 91-11-26596568. Fax: 91-11-26581073. Email: [email protected]. † Indian Institute of Technology. ‡ All India Institute of Medical Sciences.

been described for hydrolysis of starch,7 pectin,16 xylan,10 and cellulose.8 The data reported here include evaluation of the bioconjugate for casein hydrolysis. Use of the water-soluble matrix also allows one to probe structural changes (spectroscopically) which an enzyme undergoes upon covalent coupling. The results on characterization of the bioconjugate using fluorescence and circular dichroism are also described. Materials and Methods Materials. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was purchased from Sigma Chemical Co. (St. Louis, MO). Eudragit S-100 was purchased from Ro¨hm Pharma GmbH, Darmstadt, Germany. R-Chymotrypsin was procured from SRL (Sisco Research Laboratories), Mumbai, India. All other chemicals were of analytical grade. Enzyme Assay. The proteolytic activity of chymotrypsin activity was measured using casein as the substrate.17 A 1.0 mL portion of the enzyme solution in 10 mM phosphate buffer, pH 7.6, was incubated with 1.0 mL of the 1% casein solution at 37 °C. After 30 min, 3.0 mL of 5% TCA solution was added and allowed to stand for 15 min at the assay temperature. This was followed by centrifugation at 5000 rpm for 10 min. Supernatant was collected for the estimation of amount of product formed. To 1.5 mL of the supernatant, 3.0 mL of 0.5 M NaOH and 0.5 mL of Folin-Ciocalteau reagent (diluted in the ratio of 1:1 with distilled water) were added. The mixture was kept at room temperature for 10 min, and the absorbance was read at 578 nm. All assays were carried out under conditions of linearity vs amount of enzyme. The enzyme unit is an absorbance unit which is defined as the increase of one in absorbance as measured by Lowry’s method at 578 nm (which is directly proportional to the increase in the amount of TCA-soluble peptides).18

10.1021/bm0256799 CCC: $25.00 © 2003 American Chemical Society Published on Web 01/16/2003

A Smart Bioconjugate of Chymotrypsin

Protein Estimation. Protein content was estimated by the dye-binding method using bovine serum albumin as the standard protein.19 Immobilization Procedure. R-Chymotrypsin was covalently linked to Eudragit S-100 by carbodiimide coupling by following a protocol described by Hermanson et al.20 and Arsaratanam et al.21 A solution of Eudragit S-100 (0.5%; 5.0 mL) was prepared in distilled water.22 Lowering the pH to 4.5 (by adding 75-80 µL of 1 N HCl) precipitated the polymer.22 The precipitate was suspended in 5.0 mL of 0.1 M acetate buffer, pH 4.5, followed by addition of 0.15 g of EDC. The pH of the reaction mixture was maintained at 4.5 (a total of 20 µL of 0.01 M NaOH had to be added) for 3 h with continuous stirring at 25 °C. Thereafter, the precipitate was washed thoroughly with 0.1 M acetate buffer, pH 4.5, to remove the excess EDC.20,21 This precipitate was suspended in 1.9 mL of 0.1 M acetate buffer, pH 4.5. Solid enzyme containing 34 U (with a specific activity of 150 U mg-1) dissolved in 0.1 mL of the 0.1 M acetate buffer (pH 4.5) was added to the activated polymer and kept at 4 °C. After 1 h the reaction mixture was centrifuged at 8000g for 10 min. The supernatant was removed and tested for unbound chymotrypsin activity. The immobilized preparation (precipitate) was washed by suspending the same in 2.0 mL of 0.1 M acetate buffer, pH 4.5. The difference between the added chymotrypsin activity and the total activity in the supernatant (and washings) represented the amount of the enzyme immobilized. It was found that 17 mg of the enzyme bound per gram of the polymer. Effect of pH. The pH optimum was determined by preparing the substrate solutions in 0.01 M phosphate buffers of pH 5.7-8.0. The enzyme assays for the free and immobilized enzyme (1.0 mL containing 14 U) were carried out by the protocol given for chymotrypsin assay except that the pH of the assay was varied in the above range. The pH stability on the other hand was determined by keeping enzyme solution for 2 h at 4 °C in the buffer of a particular pH and measuring the residual enzyme activity in an aliquot under standard assay conditions. Effect of Temperature. Thermostability of the free and immobilized chymotrypsin (1.0 mL containing 14 U) was determined by exposing the enzyme preparations to various temperatures (20, 25, 30, 35, 37, 40, 45, and 50 °C for 10 min) and determining the residual activity under standard assay conditions. Further the stability of the free and immobilized preparation was tested at 40 °C for 60 min. The chymotrypsin assay was carried out after every 10 min by withdrawing an aliquot from the enzyme solutions incubated at 40 °C. Starting activity of the enzyme preparation (before incubating at 40 °C) was taken as 100%. Effect of Substrate Concentration. Various concentrations of casein (0.1, 0.2, 0.3, 0.4, 0.5, 0.7, and 1%) were used for studying the effect of substrate concentration. The enzyme activity for the free and immobilized preparations was assayed by the protocol described in chymotrypsin assay section. Reusability. The stability of the free and immobilized enzyme toward alternate exposure to pH 4.5 and 7.6 was estimated as follows. The pH of both free and immobilized

Biomacromolecules, Vol. 4, No. 2, 2003 331

enzyme (2.5 mL containing 35 U) was lowered to 4.5. The enzyme preparations were kept at this pH for 30 min. The pH of the free enzyme was raised back to 7.6, and an aliquot was used for enzyme assay. This is defined as completion of one cycle. In the case of immobilized enzyme, lowering the pH of the conjugate leads to precipitation, so in this case after 30 min of incubation at pH 4.5, the precipitated conjugate was collected by centrifugation at 8000g at 25 °C for 10 min. The precipitate was dissolved in 0.01 M phosphate buffer pH, 7.6 and was assayed as for the free enzyme. The reuse of the immobilized chymotrypsin was checked as follows. The enzyme (2.5 mL) and substrate (2.5 mL) were incubated at 37 °C. After every 30 min an aliquot (200 µL) was removed and assayed for the amount of product formed. The procedure was continued wntil saturation was obtained. Hydrolysis reaction was also carried out by the addition of fresh assay buffer solution to reaction mixture after precipitation. The enzyme (2.5 mL) and substrate (2.5 mL) were incubated at 37 °C in way similar as above. After every 30 min the mixture was precipitated by lowering the pH to 3.8 (such that both the immobilized enzyme and the unhydrolyzed substrate precipitates) and the supernatant was tested for the amount of product formed. To the precipitate, 5.0 mL of the fresh assay buffer was added and the procedure was repeated as before until no product could be detected in the supernatant.7 Spectroscopic Studies. All spectroscopic studies were carried out for the free and immobilized chymotrypsin. The immobilized preparation was dissolved in 1.5 mL of 0.01 M phosphate buffer, pH 7.6. The activated Eudragit S-100 solution was taken as blank to eliminate the contribution of the polymer. UV Spectroscopy. UV spectra were recorded for the free and immobilized chymotrypsin. The immobilized preparation was dissolved in 1.5 mL of 0.01 M phosphate buffer, pH 7.6. The amount of protein was matched in both cases. For the immobilized enzyme, solution of activated Eudragit S-100 was taken as blank to eliminate the contribution of the polymer. Thermal Melting of r-Chymotrypsin. The thermal denaturation of native and immobilized R-chymotrypsin was followed on a Cary-1E UV-vis spectrophotometer from 20 to 65 °C. Thermal transitions were followed by monitoring changes in the absorbance at 280 nm. The temperature inside the sample was maintained (within (0.1 °C) by an in-built Peltier device. The sample (free and immobilized preparation in 10 mM phosphate buffer, pH 7.6) was preincubated at 20 °C for 10 min and then heated by the Peltier up to 65 °C at a rate of 0.3 °C/min. The melting temperature (Tm) was calculated (using the Cary software) from the minima of the first derivative of thermal denaturation. Fluorescence Spectroscopy. All the fluorescence spectra were recorded on a Hitachi F-4500 spectroflorometer at 20 °C using a slit width of 5 nm with a scan speed of 60 nm/ min using a 1 cm path length cuvette. The spectra of native and immobilized R-chymotrypsin (in 10 mM phosphate buffer, pH 7.6) were recorded from 300 to 400 nm using an

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excitation wavelength of 280 nm. The spectrum of the free and immobilized enzyme was recorded after subtracting the contribution of the polymer spectrum of the immobilized enzyme. The polymer was found not to quench the fluorescence of the enzyme. Circular Dichroic Spectra. All the circular dichroic (CD) spectra were recorded from 200 to 300 nm on a Jasco-810 spectropolarimeter using 0.2 cm path length cuvette. The machine was routinely calibrated with D-camphorsulfonic acid. Data acquisition and analysis were performed on a computer, which interfaced to the spectropolarimeter. Spectra were recorded at a scanning speed of 20 nm/min at 20 ( 0.1 °C using the in-built Peltier-controlled thermostat cell holder. Results and Discussion Hydrolysis of casein is a biotechnologically important process.4,23 While smart polymers as matrix have been used for linking variety of enzymes such as amylases, cellulases, and xylanases,7,8,10 no extensive work has been reported for evaluating a smart bioconjugate for casein hydrolysis. Fujimura et al.24 have mentioned that R-chymotrypsin linked to a methyl methacrylate copolymer MPM-06 gave “high activity” with a low molecular weight substrate. The same work shows that this bioconjugate was an efficient catalyst for peptide synthesis in aqueous-organic cosolvents. The conjugation of chymotrypsin with Eudragit S-100 was carried out with the carbodiimide coupling method. An earlier report from this laboratory has investigated the effect of various parameters on the coupling of the enzymes to Eudragit S-100.25 In this work, we have essentially followed the protocol of Arsaratanam et al.21 The method basically consists of activation of free carboxyl groups present on the polymer by a water-soluble carbodiimide (EDC) and the activated matrix conjugates the proteins via free amino groups present on the protein surface. Thus, the bonding between the matrix and the enzyme is an amide bond.20 In our case, 15-20% nonspecific binding was observed, i.e., when chymotrypsin was incubated with “not activated Eudragit”. However, it must be added that during actual covalent coupling, it is expected that this number will be different, as the enzyme molecules are more likely to react with the activated matrix rather than adsorb to the matrix by physical forces. Arsaratanam et al.21 has described a method for evaluating the component of the enzyme which is noncovalently linked to the matrix after carbodiiimide coupling. This method consists of washing the immobilized enzyme with a series of buffers and additives containing buffers. In this case, it was found that the enzyme was held on the matrix mostly by covalent bonds. Only 0.12% activity could be detected in the supernatant when the immobilized preparation was incubated with various eluents.21 Lower pH (0.1 M acetate buffer, pH 4.5 containing 0.14 M NaCl), phosphate buffer, pH 7.2, containing 0.14 M NaCl and presence of 50% ethylene glycol or 0.3% Triton X-100 (in 0.15 M Tris-HCl, pH 7.6 buffer) are various eluting conditions which were tried. Table 1 shows the extent to which the enzyme retained its activity upon immobilization. The decrease in enzyme

Sharma et al. Table 1. Immobilization of Chymotrypsin on Eudragit S-100a enzyme enzyme enzyme bound activity of effectiveness added, not bound, (theoretical) immobilized factor, X (U) Y (U) (A ) X - Y) enzyme, B (U) η ) BA-1 14.6 34.7 74.5 125.8 252.0

1.88 4.64 12.6 24.0 62.0

12.72 30.1 62.0 102.0 190.0

8.0 28.0 52.7 72.0 98.0

0.63 0.93 0.85 0.71 0.51

a Each experiment was done in duplicate. The difference in the individual readings was less than (5%.

activity upon immobilization can be due to several factors: (a) conformational changes; (b) chemical modification of the essential residues; (c) steric hindrance by the matrix for the access of the substrate; (d) partition effects which arise since matrix may interact with substrate and products; (e) mass transfer constraints which have been explained in the Introduction.26 Thus, effectiveness factor is considered a good indicator of the extent to which these factors have been minimized in a particular immobilization strategy. This “effectiveness factor” (η) varies with the enzyme units added on a fixed amount of polymer. Initial drastic increase in the parameter perhaps reflects the better productive collision percentage between the substrate and the immobilized enzyme as the enzyme density on the polymer increases. Further increase in total enzyme units added decreases the “effectiveness factor” (η) as the enzyme crowding prevents the approach of the macromolecular substrate. This steric crowding phenomenon has been observed in many systems earlier.5,6,27 At reasonable enzyme units added, an effectiveness factor of 0.93 could be obtained. The high “effectiveness factor” observed here indicates that the immobilization has not altered the activity of the enzyme molecule in a major way. Some earlier reports on immobilization of chymotrypsin, viz., on polymerized liposome28 and poly(urethanegraft-acrylic acid)29 have led to 59 and 45% retention of protelytic activity, much lower than 93% (retention of proteolytic activity) observed by us. Bilkova et al.30 have reported retention of 100% activity of chymotrypsin by carrying out oriented immobilization on supports precoupled to suitable polyclonal antibody. Our simpler and less costly procedure with 93% retention of activity thus compares favorably with the results obtained by others. Our result is positively comparable with the latter report. Characterization of the Immobilized Preparation. In this system, the Eudragit-chymotrypsin bioconjugate was soluble under the conditions used for casein hydrolysis. The pH optimum of the free chymotrypsin (7.6) altered on immobilization, which is not unexpected (Figure 1).31 The pH optimum has become broader, and there is the expected shift toward the alkaline region in view of the anionic nature of the matrix.32 The immobilized preparation showed better stability at and above pH 6.5 as compared to the free enzyme (Figure 1). In the case of proteases, immobilization is known to reduce autolysis. A useful outcome of immobilization is thus enhanced storage stability of the enzyme. It was found that whereas free enzyme lost 18% activity in 2 h at 4 °C,


Figure 1. Effect of pH on chymotrypsin activity. The enzyme assay for free and immobilized enzyme was carried out at pH values from 0.01 M phosphate buffer of pH 5.7-8.0 for determining the pH for optimum activity. The maximum activity was taken as 100%. To the effect of pH on the stability, the free and immobilized enzyme preparations were incubated at 4 °C in 0.01 M phosphate buffers of pH varying from 5.6 to 8.0. After 2 h the pH values of both the enzyme preparations were brought to assay pH and were tested for the residual enzyme activity. The maximum activity was taken as 100%.

Figure 2. Thermal unfolding curves of R-chymotrypsin free [1] and immobilized [2] from 20 to 65 °C obtained by measuring absorbance at 280 nm.

under identical conditions immobilized enzyme did not lose any activity (data not shown). Determination of Tm by ∆ absorbance (280 nm) vs temperature is often used as an indication for judging the overall stability of the protein. Figure 2 shows the melting of native and immobilized R-chymotrypsin by measuring absorbance at 280 nm as a function of temperature. The aromatic residues tyrosine and tryptophan in the native enzyme are buried and present in a nonpolar environment. The changes in absorbance on heating are due to their exposure to a relatively polar, aqueous environment.33 The exact melting temperatures (Tm) calculated by the first differential curves (within Figure 2) are 48 ( 0.5 °C and 46 ( 0.5 °C for the free and immobilized enzyme, respectively. It may be noted that Tm of the free enzyme is in the same order as that reported earlier.34,35 While calculated Tm values are very close, the thermal stabilization upon immobilization becomes obvious when we examine the thermal stability at specific temperatures over a longer period of time. Thermal


Figure 3. pH cycling. The free and the immobilized preparation were assessed for stability toward exposure cycles to pH 4.5.

stability at 40 °C showed that immobilized enzyme showed enhanced stabilization as compared to the free enzyme. The immobilized enzyme retained almost 70% of its original activity after 1 h of exposure to 40 °C as compared to the 25% residual activity for the free enzyme. The thermal stability was also studied at 45 °C, and the difference in thermal stability for both the enzyme preparations was narrower at 45 °C (data not shown). From the LineweaverBurk plot, Km values were calculated to be 0.3 and 0.29 mg mL-1 for free and immobilized enzyme, respectively. Vmax for free and immobilized enzyme was 0.25 U min-1. These values were calculated using Leonara software.36 This indicated that immobilization of chymotrypsin caused almost no change in the Km and Vmax values with casein as the substrate. These unchanged values of kinetic parameters upon immobilization reflect the advantage of using a soluble matrix. More often than not Km of the enzyme upon immobilization on solid supports increases.30,31,37,38 This confirms the fact that immobilization on a smart polymer continues to be efficient despite the macromolecular nature of the substrate. Ding et al.39 have described preparation of a bioconjugate in which each thermosensitive oligomer chain had only one end attachment of trypsin. This design was aimed at minimizing “loss of enzymatic activity due to steric hindrance”. Our result shows that such elaborate design may not be always necessary, considering the fact that the unchanged kinetic parameters reported here have been obtained with macromolecular substrate casein. The reusability of the immobilized preparation was assessed by lowering the pH to precipitate the immobilized preparation, dissolving it again by increasing the pH and assaying it. It was observed that the immobilized preparation retained almost 85% of its original activity even after five precipitation cycles (Figure 3). It is interesting to note that the free enzyme subjected to similar cycles of pH changes loses almost all (above 90%) its activity after the fifth cycle. Thus, immobilization has led to stabilization of the enzyme toward alternate exposure to pH 4.5 and 7.6. More important, this shows that the bioconjugate can be used as a soluble catalyst, recovered after use by pH precipitation and reused.

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Figure 4. Reusability: (2) hydrolysis using the immobilized enzyme by removing an aliquot (200 µL) after every 30 min until saturation was obtained; (4) hydrolysis reaction in the discontinuous mode using the immobilized preparation with the addition of fresh buffer solution as mentioned in the “Materials and Methods” section.

The reusability of the enzyme preparation was further assessed by carrying out hydrolysis of casein following the strategy used by Hoshino et al.7 It was found that casein precipitates completely at pH 3.8 (data not shown). So, in the system described here, the undigested casein precipitates along with the biocatalyst and is again present in the next cycle along with the freshly added buffer solution (Figure 4). Hence, this is one design for a reusable protease for the hydrolysis of a macromolecular protein substrate. Ultrafiltration based bioreactors constitutes another possible approach which is currently often used for obtaining protein hydrolysates.40 However, cost of the membranes and their susceptibility to fouling have always been the main problems of any membrane-based approach. In the strategy outlined here, the bioreactor design is simpler and Eudragit S-100 is a fairly inexpensive and nontoxic polymer. UV Spectra. As bulk of the work related to protein or enzyme immobilization has been carried out with solid supports, limited structural information is available regarding the changes that an enzyme molecule undergoes upon immobilization. As has been pointed out earlier,10 the use of water-soluble polymer as matrix makes it possible to use spectroscopic techniques to probe changes (if any) in the enzyme conformation upon immobilization. The UV spectra of the free and immobilized enzyme are shown in Figure 5. Insignificant changes around 280 nm indicate that the microenvironment of tryptophan/tyrosine has not perhaps changed drastically. However, drastic changes around 250 nm reflect that the enzyme did undergo some conformational changes upon conjugation. Fluorescence. R-Chymotrypsin although contains three tyrosines, the fluorescence emission spectra of chymotrypsin reflects only that of the four tryptophans present at positions 45, 191, 238, and 262.41 The fluorescence emission characteristics can be considered as a more sensitive parameter to study the structural changes involving the microenvironments of tryptophans. Fluorescence emission spectra of R-chymotrypsin excited at 280 nm of free and immobilized enzyme are shown in Figure 6. The emission maxima for native and immobilized enzyme respectively are 330 and 335 nm at 20

Sharma et al.

Figure 5. UV spectra of free and immobilized chymotrypsin. The spectrophotometric determinations were carried out using a Beckman DU-640 spectrophotometer.

Figure 6. Fluorescence emission spectra of R-chymotrypsin for free [1] and immobilized [2] at 20 °C at an excitation of 280 nm.

°C; the intensity for immobilized is lower as compared to the native enzyme. The fluorescence emission characteristics of the native R-chymotrypsin are similar to that of the reported.34 It is well-known that42-45 protein denaturation causes an abrupt change in the fluorescence intensity (I) and in the wavelength emission maximum, which results from the more intense contact of the aromatic residues of the protein with polar environment. Thus, the fluorescence data confirm the results of UV absorbance that gross changes in the microenvironment of tryptophan and tyrosine have not occurred upon immobilization. On thermal denaturation (90 °C) of R-chymotrypsin, the λmax shifted from 330 to 347 nm and intensity decreased by 10-fold. Similarly, even in the immobilized enzyme λmax shifted toward red by 17 nm (335352 nm); however the fluorescence intensity decreased only by 18% (data not shown). The latter perhaps reflects that the “residual structure” at 90 °C in the case of immobilized enzyme is greater. It has been pointed out that heat-denatured proteins still contain some residual structure, which can be further abolished by chemical denaturants.46-48 Circular Dichroism. Circular dichroism has proved to be extremely useful in understanding the various structural elements in proteins, and structural transitions from order to disorder can be well documented by CD. The far-UV changes particularly give information about the secondary



References and Notes

Figure 7. Circular dichroic spectra of free [1] and immobilized [2] at 20 °C. Table 2. The Contribution of Secondary Structural Elements in the R-Chymotrypsin Evaluated from the CD Data

free immobilized

R-helix

β-sheet and β-turn (%)

random coil

5.4 0.0

50.0 38.3

44.6 61.7

structure. Figure 7 shows the CD spectra recorded for the free and immobilized chymotrypsin in the range 200-300 nm The common feature in the spectra is that they show characteristic minima, one around 210 and other at 220 nm. These negative bands are generally considered as a “hall mark” for alpha helix, etc. The contribution from R-helix, β-sheet, random coil, etc., were calculated using the software provided by JASCO (Table 2). The free enzyme has predominantly β-sheet structure and practically no R-helix content.41 The CD shows that there have been definite changes in the secondary structure upon immobilization. The small R-helical content is totally gone and there is decrease in β-sheet/β-turn structure. The randomness in the structure has increased by about 17%. This way correlation between immobilization-secondary structure-overall tertiary structure as reflected in Tm and fluorescence is needed in larger number of cases, so that we can understand immobilization at molecular level. That in turn should help in evolving better designs for reusable biocatalysts. Conclusion The novelty of the present work lies in the fact that it describes the following: An immobilized enzyme which acts as a homogeneous catalyst. The conventionally immobilized enzymes (on solid supports) are heterogeneous catalysts. An immobilized enzyme which has nearly the same enzyme efficiency as the free enzyme (BA-1 ) 93%) and minimal loss of activity upon covalent immobilization. An enzyme derivative which was stable toward pH recycling. This is necessary for reuse of this pH-sensitive smart bioconjugate. It has been possible to gather information on structure by fluorescence and CD as the immobilized enzyme can occur in soluble form.

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