J. Biosci., Vol. 13, Number 1, March 1988, pp. 47–54. © Printed in India. Preparation of Concanavalin A-ß-galactosidase conjugate and itsapplication in lactose hydrolysis

SUNIL KUMAR KHARE and MUNISHWAR NATH GUPTA* Chemistry Department, Indian Institute of Technology, New Delhi 110 016, India MS received 3 March 1987; revised 17 August 1987 Abstract. A Concanavalin A-β-galactosidase conjugate was prepared using glutaral-dehyde as the crosslmking reagent. The conjugate bound to Sephadex G-50 beads was more thermostable and hydrolyzed lactose faster than the free enzyme. The immobilized enzyme may prove useful in the preparation of low lactose milk which is required by persons suffering from lactose intolerance. Keywords. Escherichia coli β-galactosidase; ConA-ß-galactosidase conjugate; lactose; lactose hydrolysis; low lactose milk.

Introduction ß-Galactosidase activity has been extensively studied in a large number of sources (Wellenfels and Weil, 1972). Undoubtedly, the main reason for this has been the use of this enzyme in the hydrolysis of whey (Kosaric and Asher, 1985) and hydrolysis of milk lactose for producing low lactose milk which is required by persons afflicted with lactose intolerance (Gekas and Lopez-Leiva, 1986). Both of these applications have encouraged the immobilization of β-galactosidase from various sources on a variety of matrices (Richmond et al., 1981). in some cases, immobilized lactases have been used in commercial processes for hydrolysis of lactose in whey and milk (Pastore and Morisi, 1976). Nevertheless, the search for better enzyme derivatives continues (Makkar et al., 1981; Friend and Shahani, 1982; Nakanishi et al., 1983). Crosslinking an enzyme with a lectin to create a reusable enzyme derivative has been suggested as a possible alternative (Shier, 1985). In this paper, we describe the preparation of a conjugate of β-galactosidase with Concanavalin A (ConA) and consider its possible use in enzyme based bioreactors. Materials and methods

Escherichia coli ß-galactosidase was obtained from Sigma Chemical Co., St. Louis, Missouri, USA. ConA and o-nitrophenyl -ß-D-galactopyranoside (ONGP) were procured from CSIR Centre for Biochemicals, Delhi. Commercial glutaraldehyde (25%) was a product of Riedel. All other reagents used were of analytical grade. Assay of β-galactosidase activity Enzyme activity towards ONGP was determined following the method described by Craven et al. (1965). A 200 µl enzyme sample was incubated at 25°C, in a reaction *To whom all correspondance should be addressed. Abbreviations used: ConA, Concanavalin A; ONGP, o-nitrophenyl -ß-D-galactopyranoside.

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48 Khare and Gupta mixture consisting of 1·50 ml sodium phosphate buffer (0·3 M, pH 7·5) containing 0·003 Μ MgCl2; 1·65 ml double distilled water; 0·45 ml ONGP solution (0·014 M, in0·01 Μ Tris-acetate buffer, pH 7·5, containing 0·01 Μ MgCl2); 0·75 ml β-mercapto- ethanol (1 M). The reaction was stopped after 5 min by adding 4 ml of 1 Μ Na2CO3 and the liberated o-nitrophenol was measured by reading the absorbance at 405 nm.

Enzyme activity of Sephadex-bound enzyme was also determined in a similar way: 200 µl sample i.e., 100 µl beads in 100 µl buffer (0·1 Μ sodium phosphate, pH 6·5, containing 0·003 Μ MgCl2), was incubated with assay mixture with constant shaking.

Preparation of ConA-β-galactosidase conjugate ConA -ß-galactosidase conjugate was prepared using glutaraldehyde as bifunctional cross linking reagent. Solutions of ConA (4 mg/ml) and β-galactosidase (1 mg/ml) were prepared in sodium phosphate buffer (0·1 M, pH 6·5) containing 1 Μ NaCl and 0·003 Μ MgCl2. ConA (500 µl) and β-galactosidase (100 µl) were mixed and were cooled to 4°C. A cold 25 % aqueous glutaraldehyde solution (20 µl) was slowly added with constant mixing. The mixture was allowed to stand for 30 min at 4°C, after which it was directly loaded on a Sephadex G-50 column (1 × 15 cm) of 10 ml bed volume equilibrated with sodium phosphate buffer (0·1 M, pH 6·5) containing 0·003 Μ MgCl2. The elution was carried out with 0·1 Μ NaCl in the same buffer. Flow rate was maintained at 22 ml/h and fractions of 1 ml were collected. Fractions containing protein and β-galactosidase activity were pooled and total protein and enzyme activity were determined.

The bound ConA -ß-galactosidase activity was eluted using 0·2 Μ glucose in sodium phosphate buffer (0·1 M, pH 6·5) containing 0·003 Μ MgCl2. Fractions of 1 ml were collected at a flow rate of 22 ml/h. The fractions containing conjugated β- galactosidase were pooled and total protein and enzyme activity were determined. Conjugation was also tried at pH 7; this resulted in precipitation of protein. The same result was obtained when β-galactosidase was increased to 200 µg in the reaction mixture and cross-linking was continued for 30 min. Hence in the latter case, attempts were made to obtain the conjugate by limiting the cross-linking time to 15 min (table 1).

Table 1. Optimization of conditions for preparation of ConA -ß-galacto- sidase conjugate.

Protein was estimated by dye binding assay (Bradford, 1976) using bovine serum

albumin as standard. Km determination The Km 's of the free native enzyme and the Sephadex-bound ConA -ß-galactosidase

ConA-ß-galactosidase conjugate 49 conjugate towards ONGP and lactose were determined. In each case enzyme acti- vities were determined at various concentrations of substrate. The Km values were calculated after plotting the data according to Lineweaver and Burk (1934). Lactose hydrolysis One ml of lactose solution (5% in potassium phosphate buffer, 0·1 M, pH 7·2, containing 0·003 Μ Mg2+) was incubated with 100 µl of the enzyme sample at 50°C. Aliquots of 100 µl were withdrawn at various times and their glucose content was measured by the PGO enzymatic method (Sigma Technical Bulletin, No. 510). These data gave the extent of lactose hydrolysis after various times of incubation. Results It has been suggested that enzyme-lectin conjugates may be useful derivatives for immobilization of enzymes (Shier, 1985). In the conjugate of β-galactosidase described here, ConA was chosen as the lectin component because this lectin is well characterized and easily available. The E. coli enzyme was chosen because its pH optimum around neutrality makes it an appropriate enzyme for milk lactose hydrolysis.

ß-Galactosidase was cross-linked to ConA using glutaraldehyde. The conjugate was expected to bind to a Sephadex because of the affinity of the lectin to Sephadex column (Sharon and Lis, 1972). The binding and subsequent elution of the bound protein with 0·2 Μ glucose are shown in figure 1. The eluted protein consists of unreacted ConA and the conjugate of ConA with the enzyme. When enzyme con- centration was varied, the best results were obtained at an enzyme concentration of 100 µg/620 µl of reaction volume (table 1). The quantitative details of the recovery of enzyme activity at various stages are summarized in table 2. Thus 10% of the enzyme activity was recovered in the conjugate. The actual enzyme activity may in fact be

Figure 1. Purification of ConA -ß-galactosidase conjugate by affinity chromatography on Sephadex G-50. Arrow indicates point of start of elution with 0·2 Μ glucose. (●), Absorbance at 595 run (protein); (○), absorbance at 405 nm (enzyme activity).

50 Khare and Gupta Table 2. Recovery of enzyme activity at various stages of preparation of the conjugate.

slightly more since the measurement was made in the presence of glucose which is an inhibitor of β-galactosidase (Deschawanne et al., 1978). Dialysis to remove the glucose resulted in loss of enzyme activity. Such loss in enzyme activity on prolonged dialysis in the case of β-galactosidase has also been reported by Rickenberg (1959), Since most of the activity was recovered in the initial washings with 0·1 Μ NaCl, the conjugation was not very efficient. There was also about 13% loss in enzyme activity upon conjugation. A similar loss in activity upon conjugation was observed using lactoseas substrate (Khare, S. K. and Gupta, M. N., unpublished results).

In this work, the unreacted ConA was not separated from the conjugate as theenzyme is known to lose activity at low concentration and normally inert proteinsare added in order to obtain a stable enzyme solution (Palmieri and Koldovsky, 1972). However, for further characterization, it should be easy to separate the conjugate from unreacted ConA with the help of a β-galactosidase affinity material (Wellenfels and Weil, 1972).

The conjugate showed a marginal increase in thermal stability compared to the native enzyme (figure 2).

However, the Sephadex-bound galactosidase conjugate showed considerable enhancement in thermal stability (figure 3). Many workers have studied the hydro- lysis of milk lactose by immobilized enzyme at 50°C (Friend and Shahani, 1982; Nakanishi et al., 1983). The conjugate bound to the Sephadex beads retained enzyme activity even after 12 h at 50°C (figure 3). At higher temperature (55°C) though the bound conjugate showed enhanced thermal stability it gradually lost its activity with time (figure 3).

At low temperature (4°C), the bound conjugate again showed enhanced stability (figure 4). While there was no loss in enzyme activity after 12 days, only 10% enzyme activity was lost even after 30 days.

The bound conjugate had the same pH optimum (figure 5) and temperature optimum (figure 6) as the native free β-galactosidase.

The Km's of the free enzyme and the conjugate towards ONGP and lactose were determined (table 3). With both substrates Km values were higher for the conjugate. The conjugation seems to have affected the binding of both ONGP and lactose to the enzyme. This may be due to decreased access of the substrate to the active site of the enzyme in the conjugate.

ConA-β-galactosidase conjugate 51

Figure 2. Thermal stability profiles of free and ConA-conjugated β-galactosidase. Samplesof free native β-galactosidase and ConA -ß-galactosidase conjugate with identical activities were tested at 550C; 2 mg ConA was added to the free enzyme solution. Aliquots of 200 µl were tested for activity after various times of incubation. (○), Free enzyme; (●), conjugate.

Figure 3. Thermal stability of Sephadex-bound ConA -ß-galactosidase at 55°C (A) and50°C (B). In each case, a free enzyme control with 2 mg ConA was also tested. (○), Control; (●), ConA -ß-galactosidase conjugate.

The bound conjugate was used for lactose hydrolysis at 50°C (figure 7). It was

found to be a more efficient biocatalyst compared to the free enzyme. Increased lactose hydrolysis by the bound conjugate is understandable since it was found to be a more thermally stable enzyme preparation compared to the free enzyme. Discussion In principle, lectin-enzyme conjugates may prove useful since the enzyme can be recovered by using an appropriate affinity matrix (or Sephadex, if the lectin used is

52 Khare and Gupta

Figure 4. Effect of storage at 40C on the. β-galaclosidase activity of Sephadex-bound ConA-ß-galactosidase. (○), Control (as in figure 3); (●), Sephadex-bound ConA-ß-galacto- sidase.

Figure 5. Effect of pH on enzymatic activity of Sephadex-bound ConA-ß-galactosidaseconjugate. The conjugate preparation and a free native enzyme control (as described in figure 3) were incubated at 25°C in sodium phosphate buffer (0·3 M) at different pH values. After 15 min of incubation, enzyme activity was determined using ONGP as substrate. (○), Free native enzyme; (●), Sephadex-bound ConA-ß--galactosidase conjugate.

ConA). As the ConA-β-galactosidase conjugate is not very stable, it can not be used as a reusable enzyme derivative. However, the conjugate bound to Sephadex beads constitutes a useful reversibly immobilized lactase system. One possible disadvantage lies in the choice of ConA as a lectin (this choice was made because ConA is easily available in pure form), since when sufficient lactose is hydrolyzed the product glucose may reach sufficient concentration to dissociate the conjugate from the Sephadex beads. We are investigating this by using the Sephadex-bound enzyme in a column-type reactor since it is possible that under optimized conditions in a flow- type reactor, flow of the product glucose down the column may prevent the build-up of glucose concentration to a level where it can compete with the dextran matrix of Sephadex for binding. Nevertheless, the exceptionally good stability of Sephadex- bound enzyme indicates two possible approaches:

ConA-β-galactosidase conjugate

Figure 6. Effect of temperature on the enzymatic activity of Sephadex-bound ConA-ß- galactosidase conjugate. The conjugate and free native enzyme (as described in figure 3) were incubated with the assay mixture (containing ONGP as substrate) at various temperatures. The enzyme activity was determined by estimating the liberated o-nitrophenol spectrophotometrically at 405 nm. (○), Free native ß-galactosidase; (●), Sephadex-bound ConA -ß-galactosidase.

Table 3. Km values of free and ConA-conjugated β-galactosidase.

Reactions were as described in the test. Reaction with lactose as substrate was at

Figure 7. Lactose hydrolysis by Sephadex-bound conjugate. Lactose hydrolysis was monitored by estimating liberated glucose.(○),Free native ß-galactosidase; (●), Sephadex- bound ConA -ß-galactosidase.

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37°C.

54 Khare and Gupta (i) Prepare the conjugate with another lectin in which case the recovery of theenzyme from reaction mixture would be made by using an affinity column, (ii) Covalently link the E. coli enzyme to Sephadex directly or through a spacer or covalently link the ConA -ß-galactosidase conjugate to Sephadex.

In this context, a recent paper by Solomon et al. (1986) may be mentioned where an approach somewhat similar to ours has been suggested as a novel method for immobilizing enzymes. Solomon et al. (1986) have used, instead of a lectin, an immobilized monoclonal antibody to bind the enzyme and immobilize it.

Finally, it may be mentioned that the ConA -ß-galactosidase conjugate has one more potential application. It has already been reported that ConA-peroxidase conjugate can be used for staining of sciatic nerve glycoproteins on Polyacrylamide gels (Wood and Sarinana, 1975). The product of the ß-galactosidase reaction with ONGP is o-nitrophenol which is chromogenic. Thus ConA -ß-galactosidase conjugate can be used like ConA-peroxidase to detect glycoproteins on Polyacryl- amide gels. Availability of more such conjugates may make this approach a more frequently used one for the detection and analysis of glycoproteins. Acknowledgements The financial assistance granted by the Council of Scientific and Industrial Research, and Department of Science and Technology, New Delhi is duly acknowledged. References Bradford, M. M. (1976) Anal. Biochem., 72, 248. Craven, G. R., Steers, E. and Anfinsen, C. B. (1965) J. Biol. Chem., 240, 2468. Deschawanne, P. J., Viratelle, O. M. and Jeannine, M. Y. (1978) J. Biol. Chem., 253, 833. Friend, B. A. and Shahani, K. M. (1982) Biotechnol. Bioeng., 24, 329. Gekas, V. and Lopez-Leiva, M. (1985) Process Biochem,, 20, 2. Kosaric, N. and Asher, Y. J. (1985) Adv. Biochem. Engg. 19, 25. Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Soc, 56, 658. Makkar, H. P., Sharma, O. P. and Negi, S. S. (1981) J. Biosci, 43, 7. Nakanishi, K., Matsuno, R., Torii, K., Yamamoti, K. and Kamikubo, T. (1983) Enzyme Microb. Technol,

5, 115. Palmieri, M. J. and Koldovsky, O. (1972) Biochem. J., 127, 795. Pastore, Μ. and Morisi, F. (1976) Methods Enzymol., 44, 822. Richmond, M. L., Gray, J. I. and Stine, C. M. (1981) J. Dairy Sci., 64, 1759. Rickenberg, Η. V. (1959) Biochim. Biophys. Acta 35, 122. Sharon, Ν. and Lis, H. (1972) Science 177, 949. Shier, W. Τ. (1985) Methods Enzymol, 112, 248. Sigma Technical Bulletin, No. 510 (P GO enzymatic method). Solomon, B., Koppel, R., Pines, G. and Katchalski-Katzir, E. (1986) Biotechnol. Bioeng., 28, 1213. Wellenfels, Κ. and Weil, R. (1972) Enzyme, 7, 617. Wood, J. G. and Sarinana, F. O. (1975) Anal. Biochem., 69, 320.