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Effect of presence of concanavalin a on enzymatic hydrolysis of lactose and sucrose
Bioresource Technology 49 (1994) 223-225
~-~7 ~_
© 1994 Elsevier Science Limited Printed in Great Britain. All fights reserved 0260-8774/94/$7.00 0960-8524(94)00033-6
ELSEVIER
EFFECT OF PRESENCE OF CONCANAVALIN A ON ENZYMATIC HYDROLYSIS OF LACTOSE A N D SUCROSE Rim Agarwal & M. N. Gupta* Department of Chemistry, Indian Institute of Technology, Delhi, New Delhi-l lO 016, India (Received 3 May 1994; revised version received and accepted 14 June 1994)
rates of hydrolysis of beta-galactosidase and invertase by removing a monosaccharide product of the reaction.
Abstract
It was found that Concanavalin A (Con A) accelerates the rates of hydrolysis of E. coli beta-galactosidase and yeast invertase by binding to the product (glucose) formed in the reaction. The effect of Con A can be made more significant by adding fresh Con A, as the initially added Con A becomes saturated. In this fashion, 38% of sucrose could be hydrolysed in the presence of Con A, instead of 22% hydrolysis observed without the presence of lectin.
METHODS Materials
E. coil beta-galactosidase (catalogue No. 6008) and yeast invertatase (catalogue No. 1-4504) were obtained from Sigma Chemical Co. (St. Louis, MO). Lactose was obtained from Sisco Research Laboratories (India). Sucrose was obtained from S.D. Fine Chemicals Pvt. Ltd (India). Con A was obtained from Worthington biochemical corporation (New Jersey). All other chemicals used were of analytical grade quality.
Key words: Con A, beta-galactosidase, invertase, lactose hydrolysis, sucrose hydrolysis.
INTRODUCTION Beta-galactosidase and invertase are considered to be biotechnologically important enzymes (Gekas & Lopez, 1985; Khare & Gupta, 1988a,; Wasserman, 1990; Gupta, 1991). In recent years, considerable efforts have been directed towards increasing the stability of these enzymes (Khare & Gupta, 1988b,; Nosoh & Sekiguchi, 1988; Khare & Gupta, 1990; Gupta, 1991). Another aspect, which has received less attention, is development of strategies for increasing the rate of biocatalysis. While bioconversion rates may be increased by site directed mutagenesis, one simple classical approach would be to shift the equilibrium by continuous removal of one of the products. The latter approach is especially valuable if the product also happens to be an inhibitor for the enzyme involved. Lectins have found numerous applications in bioseparation (Steinemann & Stryer, 1973; Carter & Sharon, 1977; Heyworth & Wynn, 1982). Endowed with the capacity of binding specific sugars, these proteins should be able to bind and remove products in the cases in which specific sugars are being produced by hydrolysis of disaccharides, polysaccharides and other complex carbohydrates. In this work, it was shown that Concanavalin A (Con A) accelerates the
Estimation of lactose
Lactose was estimated using the method by Nickerson et al. (1975). Lactose (250 mg) was dissolved in 4"8 ml of phosphate buffer (0"1 M, pH 7"3, containing 0"003 M MgC12 and 1 M NaC1). An aliquot of 200/~1 (62.5/~g) of beta-galactosidase in phosphate buffer was added to the above for initiating the reaction. This assay mixture was incubated at 37°C. An aliquot of 150/~1 was drawn at different time intervals and 50 kd of perchloric acid (4.2% w/v in water) was added to stop the reaction. In addition, 2.3 ml of phosphate buffer, 2.5 ml of glycine-NaOH buffer (150 ml of glycine solution containing 2.476 g glycine and 1.935 g NaCI was added to 850 ml of 0"385 MNaOH to pH 12.8), 0"25 ml methylamine solution (5% w/v of methylamine HC1 in water) and 0.25 ml of sodium sulphite solution (1% w/v) were added to the above aliquots. The tubes were mixed thoroughly and heated in a water bath at 65°C for 25 min, then they were cooled immediately in an ice-cold water bath for 2 min to stop the reaction. The absorbance was read against a blank at 540 nm. The lactose concentration was read from the standard curve prepared by estimating different concentrations of lactose by the above method. In the experiments carried out in the presence of Con A, 4 mg/ml of Con A was dissolved along with lactose in phosphate buffer.
*To whom correspondence should be addressed. 223
224
R. Agarwal, M. N. Gupta
Estimation of sucrose
Sucrose was estimated using the method of Van Handel (1968). The use of this procedure enables us to measure the concentration of sucrose in the presence of glucose and fructose. Sucrose (5 mg) was dissolved in 0"95 ml of phosphate buffer (0.02 M, pH 5.2). An aliquot of 50/~1 (0.5 mg) of invertase in phosphate buffer was added to the above solution. This assay mixture was incubated at 25°C. Aliquots of 20/zl were drawn at various time intervals. To the above aliquots, 80/tl of phosphate buffer and 100/~1 of 30% K O H was added. The mixtures were heated in a boilingwater bath for 10 min. The tubes were cooled to room temperature, 3 ml of anthrone reagent was added and tubes were kept at 40°C for 10 min. Absorbance was read against a blank at 620 nm. The concentration of sucrose was read from the standard curve obtained by using known concentrations of sucrose solution. In the experiments with Con A, 12 mg of Con A was initially dissolved with sucrose in phosphate buffer. An aliquot of 20 ~l was drawn at 15 min. Then the solution was centrifuged at 5000 rpm (1800 g) for 10 min to remove the precipitated Con A. Fresh Con A (12 mg) was added at 30 min and an aliquot of 20/zl was drawn at 40 min. Again, the solution was centrifuged at 5000 rpm (1800 g) for 10 min and fresh Con A (12 mg) was added at 55 min. An aliquot of 20/~l was drawn at 65 min. Sucrose was estimated in the above aliquots in the manner already described.
RESULTS AND DISCUSSION Beta-galactosidase catalysed lactose-hydrolysis in the presence and absence of Con A is shown in Fig. 1. The total amount of lactose hydrolysed by the enzyme was 66% in 4 h. In the presence of Con A, this amount increased to 75%. This increase was expected because Con A binds to the glucose formed when lactose is hydrolysed by beta-galactosidase to glucose and galactose. The increase (10%) is small, however, because after Con A gets saturated with glucose, its presence has no further effect. This observation was replicated in the next experiment where hydrolysis of sucrose was measured in the presence of Con A. Hydrolysis of sucrose by invertase was measured in the presence and absence of Con A as shown in Fig. 2. It can be seen that the rate of enzymatic hydrolysis of sucrose was increased in the presence of Con A when fresh Con A was used. Thus, the data reported here show the viability of this approach. However, it cannot be used beyond laboratory scale for a number of reasons. One constraint is the cost of Con A. However, as more efficient bioseparation procedures for lectins are developed (Senstad & Mattiasson, 1989) this factor may not be an unsurmountable obstacle. One possibility may be to use immobilized Con A. In that case the Con A saturated with the sugars (glucose and galactose) can be
~k l.aoloee hydrolynd
80
6O
4O
2O
0
1
2 Time (hre)
3
4
Fig. 1. Percent hydrolysis of lactose in the presence and absence of Con A: (e) lactose; ( + ) lactose with Con A.
40
Hydrotyllle of muorose
J/
,o
6o
f
10
10
20
30
40
8O
6O
7O
Time Imin)
Fig. 2. Percent hydrolysis of sucrose in the presence and absence of Con A: (e) sucrose; ( + ) sucrose with Con A; ( ~ fresh Con A added.
removed from the reaction mixture simply by centnfugation. The sugars bound to a Con A sample may be removed by washing the Con A sample with low pH ( - 3 ) buffer. The regenerated Con A sample may be reused (Montreuil et al., 1986). Another development that may render the approach reported here a realistic one is the design of abiotic receptors for small organic molecules (Braco et al., 1990). In fact, in the latter eventuality, it should be possible to accelerate the rate of any reaction in general and the strategy reported here would find general applications. ACKNOWLEDGEMENT The authors are grateful to CSIR, India, for providing JRF/SRF to one of us (Ritu Agarwal) and to DST, Government of India, for a research grant to M. N. Gupta. REFERENCES
Braco, L., Dabulis, K. & Klibanov, A. M. (1990). Production of abiotic receptors by molecular imprinting of proteins. Proc. NatlAcad. Sci. USA, 87, 274-7. Carter, W. G. & Sharon, N. (1977). Properties of the human Erythrocyte membrane receptors for peanut and Dolichos biflorus lectins. Arch. Biochem. Biophys., 180, 570-82.
Concanavalin A on enzymatic hydrolysis of lactose and sucrose Gekas, V. & Lopez, M. (1985). Hydrolysis of lactose: a literature review. Proc. Biochem., 20, 1. Gupta, M. N. (1991 ). Thermostabilization of proteins. Biotech. Appl. Biochem., 14, 1-11. Heyworth, C. M. & "v~ynn, H. C. (1982). The binding of human liver acid beta-galactosidase to wheat-germ lectin is influenced by aggregation state of the enzyme. Biochem. J., 201,615-19. Khare, S. K. & Gupta, M. N. (1988a). Immobilization of E. coli beta-galactosidase and its derivatives by polyacrylamide gel. Biotechnol. Bioeng., 31,829-33. Khare, S. K. & Gupta, M. N. (1988b). Preparation of Concanavalin A beta-galactosidase conjugate and its application in lactose hydrolysis. J. Biosci., 13, 47-54. Khare, S. K. & Gupta, M. N. (1990). An active insoluble aggregate of E. coli beta-galactosidase. Biotech. Bioeng., 35, 94-8.
225
Montreuil, J., Bouquelet, S., Debray, H., Fournet, B., Spik, G. & Strecker, G. (1986). Glycoproteins. In Carbohydrate Analysis, ed. M. F. Chaplin & J. F. Kennedy. IRL Press, Oxford, pp. 143-204. Nickerson, T. F., Vujiue, I. F. & Liu, A.Y. (1975). Calorimetry of lactose. J. Diary Sci., 59, 386-90. Nosoh, Y. & Sekiguchi, T. (1988). Protein thermostability: mechanism and control through protein engineering. Biocatal., 1,257-73. Senstad, C. & Mattiasson, B. (1989). Affinity precipitation using chitosan as a ligand carrier. Biotech. Bioeng., 33, 216-20. Steinemann, A. & Stryer, L. (1973). Accessability of carbohydrate moiety of rhodopsin. Biochem., 12, 1499-502. Van Handel, E. (1968). Direct microdetermination of sucrose. Anal, Biochem., 22, 280-3. Wasserman, B. P. (1990). Evolution of enzyme technology: progress and prospects. Food Tech., 44(4), 118-22.
~-~7 ~_
© 1994 Elsevier Science Limited Printed in Great Britain. All fights reserved 0260-8774/94/$7.00 0960-8524(94)00033-6
ELSEVIER
EFFECT OF PRESENCE OF CONCANAVALIN A ON ENZYMATIC HYDROLYSIS OF LACTOSE A N D SUCROSE Rim Agarwal & M. N. Gupta* Department of Chemistry, Indian Institute of Technology, Delhi, New Delhi-l lO 016, India (Received 3 May 1994; revised version received and accepted 14 June 1994)
rates of hydrolysis of beta-galactosidase and invertase by removing a monosaccharide product of the reaction.
Abstract
It was found that Concanavalin A (Con A) accelerates the rates of hydrolysis of E. coli beta-galactosidase and yeast invertase by binding to the product (glucose) formed in the reaction. The effect of Con A can be made more significant by adding fresh Con A, as the initially added Con A becomes saturated. In this fashion, 38% of sucrose could be hydrolysed in the presence of Con A, instead of 22% hydrolysis observed without the presence of lectin.
METHODS Materials
E. coil beta-galactosidase (catalogue No. 6008) and yeast invertatase (catalogue No. 1-4504) were obtained from Sigma Chemical Co. (St. Louis, MO). Lactose was obtained from Sisco Research Laboratories (India). Sucrose was obtained from S.D. Fine Chemicals Pvt. Ltd (India). Con A was obtained from Worthington biochemical corporation (New Jersey). All other chemicals used were of analytical grade quality.
Key words: Con A, beta-galactosidase, invertase, lactose hydrolysis, sucrose hydrolysis.
INTRODUCTION Beta-galactosidase and invertase are considered to be biotechnologically important enzymes (Gekas & Lopez, 1985; Khare & Gupta, 1988a,; Wasserman, 1990; Gupta, 1991). In recent years, considerable efforts have been directed towards increasing the stability of these enzymes (Khare & Gupta, 1988b,; Nosoh & Sekiguchi, 1988; Khare & Gupta, 1990; Gupta, 1991). Another aspect, which has received less attention, is development of strategies for increasing the rate of biocatalysis. While bioconversion rates may be increased by site directed mutagenesis, one simple classical approach would be to shift the equilibrium by continuous removal of one of the products. The latter approach is especially valuable if the product also happens to be an inhibitor for the enzyme involved. Lectins have found numerous applications in bioseparation (Steinemann & Stryer, 1973; Carter & Sharon, 1977; Heyworth & Wynn, 1982). Endowed with the capacity of binding specific sugars, these proteins should be able to bind and remove products in the cases in which specific sugars are being produced by hydrolysis of disaccharides, polysaccharides and other complex carbohydrates. In this work, it was shown that Concanavalin A (Con A) accelerates the
Estimation of lactose
Lactose was estimated using the method by Nickerson et al. (1975). Lactose (250 mg) was dissolved in 4"8 ml of phosphate buffer (0"1 M, pH 7"3, containing 0"003 M MgC12 and 1 M NaC1). An aliquot of 200/~1 (62.5/~g) of beta-galactosidase in phosphate buffer was added to the above for initiating the reaction. This assay mixture was incubated at 37°C. An aliquot of 150/~1 was drawn at different time intervals and 50 kd of perchloric acid (4.2% w/v in water) was added to stop the reaction. In addition, 2.3 ml of phosphate buffer, 2.5 ml of glycine-NaOH buffer (150 ml of glycine solution containing 2.476 g glycine and 1.935 g NaCI was added to 850 ml of 0"385 MNaOH to pH 12.8), 0"25 ml methylamine solution (5% w/v of methylamine HC1 in water) and 0.25 ml of sodium sulphite solution (1% w/v) were added to the above aliquots. The tubes were mixed thoroughly and heated in a water bath at 65°C for 25 min, then they were cooled immediately in an ice-cold water bath for 2 min to stop the reaction. The absorbance was read against a blank at 540 nm. The lactose concentration was read from the standard curve prepared by estimating different concentrations of lactose by the above method. In the experiments carried out in the presence of Con A, 4 mg/ml of Con A was dissolved along with lactose in phosphate buffer.
*To whom correspondence should be addressed. 223
224
R. Agarwal, M. N. Gupta
Estimation of sucrose
Sucrose was estimated using the method of Van Handel (1968). The use of this procedure enables us to measure the concentration of sucrose in the presence of glucose and fructose. Sucrose (5 mg) was dissolved in 0"95 ml of phosphate buffer (0.02 M, pH 5.2). An aliquot of 50/~1 (0.5 mg) of invertase in phosphate buffer was added to the above solution. This assay mixture was incubated at 25°C. Aliquots of 20/zl were drawn at various time intervals. To the above aliquots, 80/tl of phosphate buffer and 100/~1 of 30% K O H was added. The mixtures were heated in a boilingwater bath for 10 min. The tubes were cooled to room temperature, 3 ml of anthrone reagent was added and tubes were kept at 40°C for 10 min. Absorbance was read against a blank at 620 nm. The concentration of sucrose was read from the standard curve obtained by using known concentrations of sucrose solution. In the experiments with Con A, 12 mg of Con A was initially dissolved with sucrose in phosphate buffer. An aliquot of 20 ~l was drawn at 15 min. Then the solution was centrifuged at 5000 rpm (1800 g) for 10 min to remove the precipitated Con A. Fresh Con A (12 mg) was added at 30 min and an aliquot of 20/zl was drawn at 40 min. Again, the solution was centrifuged at 5000 rpm (1800 g) for 10 min and fresh Con A (12 mg) was added at 55 min. An aliquot of 20/~l was drawn at 65 min. Sucrose was estimated in the above aliquots in the manner already described.
RESULTS AND DISCUSSION Beta-galactosidase catalysed lactose-hydrolysis in the presence and absence of Con A is shown in Fig. 1. The total amount of lactose hydrolysed by the enzyme was 66% in 4 h. In the presence of Con A, this amount increased to 75%. This increase was expected because Con A binds to the glucose formed when lactose is hydrolysed by beta-galactosidase to glucose and galactose. The increase (10%) is small, however, because after Con A gets saturated with glucose, its presence has no further effect. This observation was replicated in the next experiment where hydrolysis of sucrose was measured in the presence of Con A. Hydrolysis of sucrose by invertase was measured in the presence and absence of Con A as shown in Fig. 2. It can be seen that the rate of enzymatic hydrolysis of sucrose was increased in the presence of Con A when fresh Con A was used. Thus, the data reported here show the viability of this approach. However, it cannot be used beyond laboratory scale for a number of reasons. One constraint is the cost of Con A. However, as more efficient bioseparation procedures for lectins are developed (Senstad & Mattiasson, 1989) this factor may not be an unsurmountable obstacle. One possibility may be to use immobilized Con A. In that case the Con A saturated with the sugars (glucose and galactose) can be
~k l.aoloee hydrolynd
80
6O
4O
2O
0
1
2 Time (hre)
3
4
Fig. 1. Percent hydrolysis of lactose in the presence and absence of Con A: (e) lactose; ( + ) lactose with Con A.
40
Hydrotyllle of muorose
J/
,o
6o
f
10
10
20
30
40
8O
6O
7O
Time Imin)
Fig. 2. Percent hydrolysis of sucrose in the presence and absence of Con A: (e) sucrose; ( + ) sucrose with Con A; ( ~ fresh Con A added.
removed from the reaction mixture simply by centnfugation. The sugars bound to a Con A sample may be removed by washing the Con A sample with low pH ( - 3 ) buffer. The regenerated Con A sample may be reused (Montreuil et al., 1986). Another development that may render the approach reported here a realistic one is the design of abiotic receptors for small organic molecules (Braco et al., 1990). In fact, in the latter eventuality, it should be possible to accelerate the rate of any reaction in general and the strategy reported here would find general applications. ACKNOWLEDGEMENT The authors are grateful to CSIR, India, for providing JRF/SRF to one of us (Ritu Agarwal) and to DST, Government of India, for a research grant to M. N. Gupta. REFERENCES
Braco, L., Dabulis, K. & Klibanov, A. M. (1990). Production of abiotic receptors by molecular imprinting of proteins. Proc. NatlAcad. Sci. USA, 87, 274-7. Carter, W. G. & Sharon, N. (1977). Properties of the human Erythrocyte membrane receptors for peanut and Dolichos biflorus lectins. Arch. Biochem. Biophys., 180, 570-82.
Concanavalin A on enzymatic hydrolysis of lactose and sucrose Gekas, V. & Lopez, M. (1985). Hydrolysis of lactose: a literature review. Proc. Biochem., 20, 1. Gupta, M. N. (1991 ). Thermostabilization of proteins. Biotech. Appl. Biochem., 14, 1-11. Heyworth, C. M. & "v~ynn, H. C. (1982). The binding of human liver acid beta-galactosidase to wheat-germ lectin is influenced by aggregation state of the enzyme. Biochem. J., 201,615-19. Khare, S. K. & Gupta, M. N. (1988a). Immobilization of E. coli beta-galactosidase and its derivatives by polyacrylamide gel. Biotechnol. Bioeng., 31,829-33. Khare, S. K. & Gupta, M. N. (1988b). Preparation of Concanavalin A beta-galactosidase conjugate and its application in lactose hydrolysis. J. Biosci., 13, 47-54. Khare, S. K. & Gupta, M. N. (1990). An active insoluble aggregate of E. coli beta-galactosidase. Biotech. Bioeng., 35, 94-8.
225
Montreuil, J., Bouquelet, S., Debray, H., Fournet, B., Spik, G. & Strecker, G. (1986). Glycoproteins. In Carbohydrate Analysis, ed. M. F. Chaplin & J. F. Kennedy. IRL Press, Oxford, pp. 143-204. Nickerson, T. F., Vujiue, I. F. & Liu, A.Y. (1975). Calorimetry of lactose. J. Diary Sci., 59, 386-90. Nosoh, Y. & Sekiguchi, T. (1988). Protein thermostability: mechanism and control through protein engineering. Biocatal., 1,257-73. Senstad, C. & Mattiasson, B. (1989). Affinity precipitation using chitosan as a ligand carrier. Biotech. Bioeng., 33, 216-20. Steinemann, A. & Stryer, L. (1973). Accessability of carbohydrate moiety of rhodopsin. Biochem., 12, 1499-502. Van Handel, E. (1968). Direct microdetermination of sucrose. Anal, Biochem., 22, 280-3. Wasserman, B. P. (1990). Evolution of enzyme technology: progress and prospects. Food Tech., 44(4), 118-22.