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Caseinolytic Activity of Immobilized Cucumisin
Caseinolytic Activity of Immobilized Cucumisin M. KANEDA, S. NISHIMURA, and N. TOMINAGA Department of Chemistry Faculty of Science Kagoshima University Korimoto, Kagoshima 890, Japan ABSTRACT
The operational character of an immobilized cucumisin (EC 3.4.21.25) column was tested by passing a casein solution continuously through it. Occasionally vibrating the rubber tube was effective in preventing the depression of its hydrolyzing ability with time. The optimal temperature of the immobilized enzyme column was about 55°C. The casein molecules were hydrolyzed to smaller peptides by circulation of its solution.
because typical plant proteases so far isolated have belonged mainly to the thiol protease group. The thiol protease is able to assume an active form only in the presence of a reductant, but this is not necessary for cucumisin. We previously reported the preparation of immobilized cueumisin and its properties (5). The immobilized cucumisin had a better stability at an alkaline pH and at a higher temperature than the soluble cucumisin. In this paper we describe the properties and capability of immobilized cucumisin and the continuous hydrolysis of casein passing through the column.
INTRODUCTION
MATERIALS AND METHODS
The preparation of water-insoluble enzymes has attracted much interest during recent years. The catalytic activity and other properties of the immobilized enzyme are dependent on the nature of the insoluble carriers. For the fixation of proteases, cyanogen-bromide-activated spheres of agarose are suitable (1, 3). The immobilized proteases combine high enzymatic activity with acceptable physical properties. The high flow rates attainable with the agarose gel allow it to be used as a bed reactor. Furthermore, the agarose matrix is permeable to large molecules such as protein. A number of proteases have been already isolated from plant sources. One of the characteristics of plant proteases is the ability to gather a large supply. Actually, the typical plant proteases, such as papain, ficin, and bromelain, are readily available (2, 7, 8). Cucumisin (EC 3.4.21.25) is a serine endopeptidase isolated from the sarcocarp of melon, Cucumis melo L. var. Prince (4). Among plant proteases, cucumisin is unique
Received December 30, 1986. Accepted December 28, 1987. 1988 J Dairy Sci 71:1132-1134
Immobilized Cucumisin Column
Cucumisin used in this work was a serine protease from the melon, Cucumis melo L. var. Prince, purified according to the method of Kaneda and Tominaga (4). The enzyme was coupled to Sepharose 4B according to Porath et al. (9). The coupled product contained 27 mg enzyme/g of dried coupled gel. The specific activity of the immobilized cucumisin compared with that of the soluble cucumisin was 41% toward casein as a substrate. The cucumisin-Sepharose suspension was kept in 1/30 M Tris-HC1 buffer, pH 7.5 at 5°C for 12 mo without loss of activity. A chromatographic column (.9 cm diameter) with a jacket was used for continuous hydrolysis of casein by the immobilized enzyme. A filter was placed at the base of the column and the immobilized enzyme was placed on top of that filter. Assay for Proteolysis
A solution of 2.0% Hammarsten casein (pH 7.3) was then passed through the column at various flow rates. Fractions of 2 ml were collected and the extent of hydrolysis estimated by the Kunitz method (6). To each fraction 3 ml of 5% TCA were added. After standing for 30 min at room temperature, the 1132
CASEINOLYSIS ON IMMOBILIZED CUCUMISIN
1133
solution, rate of hydrolysis was measured. In all cases (Figure 2), as low flow rate increased up to about 60 ml/h extent o f hydrolysis o f casein decreased proportionally. However, hydrolysis was carried out even at the high flow rate of 250 ml/h. The casein molecule presumably has peptide bonds that are highly susceptible to cucumisin.
5O
_g .o
Effect of Temperature on Reaction Rate |
0
I
5 10 Operating time (h)
15
Figure 1. Continuous hydrolysis of casein by an immobilized cucumisin column. Symbols: A with vibration ( o - - o ) ; B without vibration ( e - - e ) .
precipitate was removed by filtration through Toyo filter paper number 5C and absorbancy at 280 nm of the TCA-soluble peptides formed was determined with a spectrophotometer. A reference sample was hydrolyzed completely with the soluble cucumisin for 12 h at 30°C. This value was taken as 100% hydrolysis. RESULTS A N D DISCUSSION
A solution of 2.0% casein was passed through an immobilized enzyme column at various temperatures. The rate of hydrolysis of casein by immobilized cucumisin rose with increasing temperatures. The optimal temperature was around 55°C. However, in the case o f the suspended immobilized enzyme, it was about 85-(3 (5). A softening of the gel spheres at higher temperatures could result in a certain amount of loss of shape, increasing the area of sphere-to-sphere contact, and closing the openings between them. This could result in decreasing the effective area for hydrolyzing. Degradation of Casein by Immobilized Cucumisin Column
The use of the immobilized enzyme in columns permits very high enzyme and sub-
Continuous Digestion of Casein by Immobilized Cucumisin
The immobilized cucumisin column produced hydrolysis of casein, but with time this ability decreased (Figure 1, curve B). However, vibration induced into the cucumisin several times per hour by vibration of the rubber tube joint below the cucumisin kept the curve flat for 12 h (Figure 1, curve A). This occasional vibrating of the rubber tube brought the desirable effect on the hydrolysis o f casein. It is thought that some covering, insoluble materials were removed from the surface of the immobilized enzyme spheres by the impact of vibration. In all the subsequent experiments, the rubber tube was occasionally vibrated. Relationship of Flow Rate and Hydrolysis of Casein
A solution of 2.0% casein was passed through an immobilized enzyme column at various flow rates. After the flow rate reached the steady state by passing 20 bed volumes o f the casein
75
5o
25
0
100 200 Flow rate ( m l / h )
300
Figure 2. Extent of hydrolysis of casein by immobilized cucumisin symbols: 2.4 cm height of immobilized cucumisin (o o); .6 cm (e e); .3 cm (D--~). Journal of Dairy Science Vol. 71, No. 5, 1988
1134
KANEDA ET AL. arose w h e n casein was h y d r o l y z e d w i t h t h e i m m o b i l i z e d cucumisin. Figure 3 s h o w s t h e distribution of ultraviolet-absorbing material f r o m h y d r o l y z e d casein o n Bio-Gel P-30. Even in t h e first cycle w i t h a flow rate o f 257 m l / h , casein was partially b r o k e n d o w n to smaller p e p t i d e s (Figure 3B). P e p t i d e b o n d s highly s u s c e p t i b l e to c u c u m i s i n exist in casein molecules, as described in t h e s e c t i o n o n t h e r e l a t i o n s h i p of flow rate a n d h y d r o l y s i s of casein. Curve D in Figure 3 s h o w s t h a t t h e p e p t i d e s o b t a i n e d a f t e r o n e cycle (curve C) were h y d r o l y z e d f u r t h e r to smaller p e p t i d e s d u r i n g s u b s e q u e n t cycles, and finally, t h e original p r o t e i n peak disappeared. I m m o b i l i z e d c u c u m i s i n m i g h t be a p o w e r f u l t o o l to e l u c i d a t e t h e d i f f e r e n t steps o f multistage r e a c t i o n s a n d also for t h e c o n t i n u o u s industrial d i g e s t i o n o f proteins.
15.0 1.0 =~ .5 0
10.0 1.0
~
B
.5 10.0 to .5
0 1.0
REFERENCES
.5 0
250
500
750
Elution volume (ml) Figure 3. Gel filtration of casein hydrolyzates on Bio-Gel P-30. The column (2.5 X 145 cm) was equilibrated with 1/15 M phosphate buffer, pH 7.0 containing .5 M NaCI, and eluted with the same solution. A) Starting material, B) 1-cycle-hydrolyzate at a flow rate of 257 ml/h, C) 1-cycle-hydrolyzate at a flow rate of 11 ml/h, D) lO-h hydrolyzate at a flow rate of 11 ml/h. About 1.0 cm of immobilized cucumisin was placed on top of a filter in a column (.9 cm diameter at 40 °C).
s t r a t e ratios. T h e desired e x t e n t o f e n z y m a t i c r e a c t i o n can t h u s be achieved w i t h s h o r t c o n t a c t times. S o m e i n t e r m e d i a t e p e p t i d e s
Journal of Dairy Science Vol. 71, No. 5, 1988
1 Affinity Chromatography Principles and Methods. 1979. Pharmacia Fine Chem., Uppsala, Swed. p. 15. 2 Arnon, R. 1970. Papain. Vol. 19. Page 226 in Methods in enzymology. Academic Press, New York, NY. 3 Axen, R., J. Porath, and S. Ernback. 1967. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214:1302. 4 Kaneda, M., and N. Tominaga. 1975. Isolation and characterization of a proteinase from the sarcocarp of melon fruit. J. Biochem. 78:1287. 5 Kaneda, M., and N. Tominaga. 1986. Properties of a new plant serine protease cucumisin. Agric. Biol. Chem. 51:489. 6 Kunitz, M. 1947. Crystalline soybean trypsin inhibitor. J. Gen. Physiol. 30:291. 7 Liener, I. E., and B. Friedenson. 1970. Ficin. Vol. 19. Page 261 in Methods in enzymology. Academic Press, New York, NY. 8 Murachi, T. 1970. Bromelain enzymes. Vol. 19. Page 273 in Methods in enzymology. Academic Press, New York, NY. 9 Porath, J., R. Axen, and S. Ernback. 1967. Chemical coupling of proteins to agarose. Nature 215:1491.
The operational character of an immobilized cucumisin (EC 3.4.21.25) column was tested by passing a casein solution continuously through it. Occasionally vibrating the rubber tube was effective in preventing the depression of its hydrolyzing ability with time. The optimal temperature of the immobilized enzyme column was about 55°C. The casein molecules were hydrolyzed to smaller peptides by circulation of its solution.
because typical plant proteases so far isolated have belonged mainly to the thiol protease group. The thiol protease is able to assume an active form only in the presence of a reductant, but this is not necessary for cucumisin. We previously reported the preparation of immobilized cueumisin and its properties (5). The immobilized cucumisin had a better stability at an alkaline pH and at a higher temperature than the soluble cucumisin. In this paper we describe the properties and capability of immobilized cucumisin and the continuous hydrolysis of casein passing through the column.
INTRODUCTION
MATERIALS AND METHODS
The preparation of water-insoluble enzymes has attracted much interest during recent years. The catalytic activity and other properties of the immobilized enzyme are dependent on the nature of the insoluble carriers. For the fixation of proteases, cyanogen-bromide-activated spheres of agarose are suitable (1, 3). The immobilized proteases combine high enzymatic activity with acceptable physical properties. The high flow rates attainable with the agarose gel allow it to be used as a bed reactor. Furthermore, the agarose matrix is permeable to large molecules such as protein. A number of proteases have been already isolated from plant sources. One of the characteristics of plant proteases is the ability to gather a large supply. Actually, the typical plant proteases, such as papain, ficin, and bromelain, are readily available (2, 7, 8). Cucumisin (EC 3.4.21.25) is a serine endopeptidase isolated from the sarcocarp of melon, Cucumis melo L. var. Prince (4). Among plant proteases, cucumisin is unique
Received December 30, 1986. Accepted December 28, 1987. 1988 J Dairy Sci 71:1132-1134
Immobilized Cucumisin Column
Cucumisin used in this work was a serine protease from the melon, Cucumis melo L. var. Prince, purified according to the method of Kaneda and Tominaga (4). The enzyme was coupled to Sepharose 4B according to Porath et al. (9). The coupled product contained 27 mg enzyme/g of dried coupled gel. The specific activity of the immobilized cucumisin compared with that of the soluble cucumisin was 41% toward casein as a substrate. The cucumisin-Sepharose suspension was kept in 1/30 M Tris-HC1 buffer, pH 7.5 at 5°C for 12 mo without loss of activity. A chromatographic column (.9 cm diameter) with a jacket was used for continuous hydrolysis of casein by the immobilized enzyme. A filter was placed at the base of the column and the immobilized enzyme was placed on top of that filter. Assay for Proteolysis
A solution of 2.0% Hammarsten casein (pH 7.3) was then passed through the column at various flow rates. Fractions of 2 ml were collected and the extent of hydrolysis estimated by the Kunitz method (6). To each fraction 3 ml of 5% TCA were added. After standing for 30 min at room temperature, the 1132
CASEINOLYSIS ON IMMOBILIZED CUCUMISIN
1133
solution, rate of hydrolysis was measured. In all cases (Figure 2), as low flow rate increased up to about 60 ml/h extent o f hydrolysis o f casein decreased proportionally. However, hydrolysis was carried out even at the high flow rate of 250 ml/h. The casein molecule presumably has peptide bonds that are highly susceptible to cucumisin.
5O
_g .o
Effect of Temperature on Reaction Rate |
0
I
5 10 Operating time (h)
15
Figure 1. Continuous hydrolysis of casein by an immobilized cucumisin column. Symbols: A with vibration ( o - - o ) ; B without vibration ( e - - e ) .
precipitate was removed by filtration through Toyo filter paper number 5C and absorbancy at 280 nm of the TCA-soluble peptides formed was determined with a spectrophotometer. A reference sample was hydrolyzed completely with the soluble cucumisin for 12 h at 30°C. This value was taken as 100% hydrolysis. RESULTS A N D DISCUSSION
A solution of 2.0% casein was passed through an immobilized enzyme column at various temperatures. The rate of hydrolysis of casein by immobilized cucumisin rose with increasing temperatures. The optimal temperature was around 55°C. However, in the case o f the suspended immobilized enzyme, it was about 85-(3 (5). A softening of the gel spheres at higher temperatures could result in a certain amount of loss of shape, increasing the area of sphere-to-sphere contact, and closing the openings between them. This could result in decreasing the effective area for hydrolyzing. Degradation of Casein by Immobilized Cucumisin Column
The use of the immobilized enzyme in columns permits very high enzyme and sub-
Continuous Digestion of Casein by Immobilized Cucumisin
The immobilized cucumisin column produced hydrolysis of casein, but with time this ability decreased (Figure 1, curve B). However, vibration induced into the cucumisin several times per hour by vibration of the rubber tube joint below the cucumisin kept the curve flat for 12 h (Figure 1, curve A). This occasional vibrating of the rubber tube brought the desirable effect on the hydrolysis o f casein. It is thought that some covering, insoluble materials were removed from the surface of the immobilized enzyme spheres by the impact of vibration. In all the subsequent experiments, the rubber tube was occasionally vibrated. Relationship of Flow Rate and Hydrolysis of Casein
A solution of 2.0% casein was passed through an immobilized enzyme column at various flow rates. After the flow rate reached the steady state by passing 20 bed volumes o f the casein
75
5o
25
0
100 200 Flow rate ( m l / h )
300
Figure 2. Extent of hydrolysis of casein by immobilized cucumisin symbols: 2.4 cm height of immobilized cucumisin (o o); .6 cm (e e); .3 cm (D--~). Journal of Dairy Science Vol. 71, No. 5, 1988
1134
KANEDA ET AL. arose w h e n casein was h y d r o l y z e d w i t h t h e i m m o b i l i z e d cucumisin. Figure 3 s h o w s t h e distribution of ultraviolet-absorbing material f r o m h y d r o l y z e d casein o n Bio-Gel P-30. Even in t h e first cycle w i t h a flow rate o f 257 m l / h , casein was partially b r o k e n d o w n to smaller p e p t i d e s (Figure 3B). P e p t i d e b o n d s highly s u s c e p t i b l e to c u c u m i s i n exist in casein molecules, as described in t h e s e c t i o n o n t h e r e l a t i o n s h i p of flow rate a n d h y d r o l y s i s of casein. Curve D in Figure 3 s h o w s t h a t t h e p e p t i d e s o b t a i n e d a f t e r o n e cycle (curve C) were h y d r o l y z e d f u r t h e r to smaller p e p t i d e s d u r i n g s u b s e q u e n t cycles, and finally, t h e original p r o t e i n peak disappeared. I m m o b i l i z e d c u c u m i s i n m i g h t be a p o w e r f u l t o o l to e l u c i d a t e t h e d i f f e r e n t steps o f multistage r e a c t i o n s a n d also for t h e c o n t i n u o u s industrial d i g e s t i o n o f proteins.
15.0 1.0 =~ .5 0
10.0 1.0
~
B
.5 10.0 to .5
0 1.0
REFERENCES
.5 0
250
500
750
Elution volume (ml) Figure 3. Gel filtration of casein hydrolyzates on Bio-Gel P-30. The column (2.5 X 145 cm) was equilibrated with 1/15 M phosphate buffer, pH 7.0 containing .5 M NaCI, and eluted with the same solution. A) Starting material, B) 1-cycle-hydrolyzate at a flow rate of 257 ml/h, C) 1-cycle-hydrolyzate at a flow rate of 11 ml/h, D) lO-h hydrolyzate at a flow rate of 11 ml/h. About 1.0 cm of immobilized cucumisin was placed on top of a filter in a column (.9 cm diameter at 40 °C).
s t r a t e ratios. T h e desired e x t e n t o f e n z y m a t i c r e a c t i o n can t h u s be achieved w i t h s h o r t c o n t a c t times. S o m e i n t e r m e d i a t e p e p t i d e s
Journal of Dairy Science Vol. 71, No. 5, 1988
1 Affinity Chromatography Principles and Methods. 1979. Pharmacia Fine Chem., Uppsala, Swed. p. 15. 2 Arnon, R. 1970. Papain. Vol. 19. Page 226 in Methods in enzymology. Academic Press, New York, NY. 3 Axen, R., J. Porath, and S. Ernback. 1967. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature 214:1302. 4 Kaneda, M., and N. Tominaga. 1975. Isolation and characterization of a proteinase from the sarcocarp of melon fruit. J. Biochem. 78:1287. 5 Kaneda, M., and N. Tominaga. 1986. Properties of a new plant serine protease cucumisin. Agric. Biol. Chem. 51:489. 6 Kunitz, M. 1947. Crystalline soybean trypsin inhibitor. J. Gen. Physiol. 30:291. 7 Liener, I. E., and B. Friedenson. 1970. Ficin. Vol. 19. Page 261 in Methods in enzymology. Academic Press, New York, NY. 8 Murachi, T. 1970. Bromelain enzymes. Vol. 19. Page 273 in Methods in enzymology. Academic Press, New York, NY. 9 Porath, J., R. Axen, and S. Ernback. 1967. Chemical coupling of proteins to agarose. Nature 215:1491.