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Purification and some properties of thermostable invertase from wine
JOURNALOF FERMENTATIONAND BIOENGINEERING Vol. 71, No. 1, 66-68. 1991
Purification and Some Properties of Thermostable Invertase from Wine K O T O Y O S H I N A K A N I S H I , * W E N WU, AND KOKI Y O K O T S U K A
Institute of Enology and Viticulture, Yamanashi University, Kofu 400, Japan Received 2 July 1990/Accepted 90clober 1990 Invertase from a white table wine made from Semillon grapes was purified to homogeneity on polyacrylamide gel electrophoresis. The enzymatic and physicochemical properties of the enzyme were compared with those of invertase purified from Semillon grape juice. The invertases from the two sources showed similar properties, suggesting that the wine invertase originated from the juice and was stable during the vinification and aging processes.
In a previous paper (1), we examined the characteristics of invertases collected from 14 varieties o f wine grapes. Although the activities o f the invertases showed variation among the grape varieties, all the enzyme preparations exhibited a high degree of thermostability, and were stable toward acidic p H and high concentrations of alcohol or SO2. Significant levels of invertase activity were also detected in several white table wines. The enzymatic properties of these wine invertases suggested they originated from the grapes rather than from the yeast cells. Usually, vinification and aging processes involve many biological, chemical, and physical reactions, which influence the activity level of various enzymes originating from grape juice. However, any enzyme which originates from grape juice has not been purified from fermenting must or the resultant wine. It is, therefore, o f interest to elucidate whether wine invertases maintain their original properties even after vinification and aging. As far as we know, there have been no reports on the purification of invertase from wines until now, although a few studies (1-3) have been published on the purification and properties of invertase from grapes. The present paper deals with the purification and some properties of invertase from Semillon wine. The enzymatic and physicochemical properties of the enzyme were compared with those of the purified invertase from Semillon grape juice. Semillon grapes were harvested from our lnstitute's vineyard in 1986, and wine was produced by conventional vinification procedures at our winery. The wine was stored at 15°C for two years before use. The activity o f invertase was measured by following the release of reducing sugar from sucrose, as described previously (1). One unit of enzyme activity was defined as the a m o u n t o f enzyme which was capable of liberating 1 p m o l o f glucose in 1 min. Specific activity (units/rag) was represented as the enzyme activity to 1 mg of protein. Protein concentration was measured by a Bio-Rad protein assay kit (Bio-Rad Laboratories, New York, USA) with bovine serum albumin as the standard. Polyacrylamide disc gel electrophoresis was done on a 12.5% acrylamide gel with 0.05-M Tris0.38 M glycine buffer (pH 8.3) (4). Isoelectric focusing was performed using polyacrylamide gel (5.3%) containing 2.59/00 p h a r m a l a t e (pH 2.5-5). The p h a r m a l a t e and a pI m a r k e r protein kit were purchased from P h a r m a c i a Fine Chemical Corp. (Uppsala, Sweden). A b o u t 20/Jg of the
purified enzyme was charged and the focusing was done at 200V for 12h, and then at 400V for 1 h. The molecular weight of the purified enzyme was estimated by sodium dodecyl sulfate slab gel electrophoresis (5). M W - M a r k e r containing five kinds of proteins (Oriental Yeast Co., Ltd., Tokyo) was used as the m a r k e r proteins. The amino acid composition was analyzed with an amino acid analyzer (Kyowa KTC-104, Tokyo) after hydrolysis of the enzyme with 6 N HCI for 24 h at 110°C in a sealed evacuated tube. The c a r b o h y d r a t e content o f the enzyme was measured by the phenol-sulfuric acid method (6). Results of the enzyme purification are summarized in Table 1. Two liters of wine were sufficiently dialyzed against distilled water at 4°C. The dialyzed wine (2.53/) was concentrated to 490 ml by a flash evaporator at 40°C, and filtered by filter paper (Toyo Roshi no. 5C) to remove insoluble materials. A m m o n i u m sulfate was added to the filtrate to 80% saturation and the mixture was stored overnight at 4°C. The precipitate obtained by centrifugation was dissolved in 0.1 M acetate buffer (pH 5.0) and the solution was dialyzed against the same buffer. The dialyzed solution was applied on a DEAE-Sephadex A-25 column (2.1 ~ 55 cm) equilibrated with the same buffer. Gradient elution was performed with increasing NaCI concentration from 0 to 0.35 M. The elution profile is shown in Fig. 1. The active fractions (fraction nos. 130-170) were collected and concentrated by a rotary evaporator. Ammonium sulfate was added to the concentrated enzyme
lOO
E iO
e-
1.0 , D
>
50~ ~0.5 ..Q <
70
100
150
200
Fraction no.(Sml/tube) FIG. 1.
DEAE Sephadex A-25 column chromatograph}'. Ex-
perimental conditions are described in the text. Symbols: @, 280 nm;, :, enzyme activity. Line: --, NaCI concentration.
Corresponding author. 66
VOL. 71, 1991
NOTES
67
TABLE 1. Summary of purification of invertase from wine Procedure
Volume (ml)
Total activity (units)
Total protein (rag)
Specific activity (units/mg)
Yield (%)
2530 490 102 200 40
19234 18875 15452 8116 5275
185 152 72 12 2.2
104 124 215 676 2398
100 98 80 42 27
Dialyzed wine" Concentration Ammonium sulfate precipitate (80O/oo) DEAE-Sephadex A-25 Sephadex G-100 a Two liters of wine were dialyzed.
(50 ml) to a saturation of 80P/o, and the resultant precipitate was dissolved in a small a m o u n t of 0.1 M acetate buffer (pH 5.0) and dialyzed against the same buffer. The dialyzed enzyme (10 ml) was applied on a Sephadex G-100 column (2.6 × 90 cm) equilibrated with the same buffer and eluted with the same buffer at a flow rate o f 15 ml per hour. After gel filtration on Sephadex G-100, the enzyme was purified to about 25-fold. The purified enzyme gave a single protein band on polyacrylamide disc gel electrophoresis ( p h o t o g r a p h not shown). In the previous report (1), the invertase activity from Semillon grape juice was recovered in two fractions which were eluted with about 0.2 M and 0.25 M o f NaC1, on a D E A E - S e p h a d e x A-25 column. The m a j o r fraction eluted with about 0.2 M o f NaCI (the ratio of activity between the m a j o r and minor fractions was about 4 : 1) was further purified by Sephadex G-100 c h r o m a t o g r a p h y . The elution profiles o f the wine invertase by D E A E - S e p h a d e x A-25 and Sephadex G-100 c h r o m a t o g r a p h i e s were somewhat different from those of the juice invertase. The specific activity of the purified enzymes was slightly higher than that o f the purified invertase from the juice. The enzymatic and physicochemical properties of the purified wine invertase were c o m p a r e d with those o f juice invertase purified as described previously (1), and stored at 20°C until use. The optimal p H and temperature, p H and thermal stabilities, molecular weights, apparent Km values for sucrose, isoelectric points, and carbohydrate contents o f these two enzymes are summarized in Table 2. In addition, the amino acid composition o f both enzymes is given in Table 3. The two tables show that there are no significant differences between the two enzymes. They both exhibited r e m a r k a b l e thermostability and high stability toward acidic p H , as well as high concentrations of alcohol or SO2. The effects o f various metal ions and other reagents on
TABLE 2.
Some properties of invertases from wine and grape juice
Optimum pH Optimum temperature pH stability" Thermal stability b Km value for sucrose Molecular weight Isoelectric point Carbohydrate content (/~g as glucose/rag protein)
Wine
Juice
4.0 75°C 2-8 70°C 10.3 mM 65000 3.8 290
4.0 75°C 2-8 70°C 9.1 mM ~ 65000 3.9 330 ~
a The enzyme solution was maintained at various pHs at 30°C for 30 rain and the residual activity was measured at the optimum pH. b The enzyme solution was maintained at various temperatures for 30 min, followed by rapid cooling in ice-cold water, after which the residual activity was measured. See reference 1.
the activities of the wine and juice invertases were examined. The enzymes were preincubated with each ion or reagent in 0.05 M acetate buffer (pH 4.0) at 30°C for 30 min and then the residual activities were assayed. The activities o f both enzymes were almost completely inactivated or inhibited by 10 2 M o f HgCI2, sodium dodecyl sulfate, and N-bromosuccinimide, as well as 10 4 M of sodium p-chloromercuric-benzoate. Furthermore, both enzyme activities were inhibited by 10 2 M o f CdCI2, CuCI2, and FeCI2 to 50-80O/oo. The effects o f 10 2 M KC1, NaC1, MnC12, MgCI2, CaC12, and ZnC12 on the activities o f both enzymes were negligible. No significant differences between the two enzymes were observed. The substrate specificity of the purified wine invertase was examined. One ml of each 0.1 M substrate solutions was incubated with l ml o f the enzyme (1.4units for sucrose and raffinose, or 7 units for melezitose, treharose, maltose, and cellobiose) and 3 ml of 0.1 M acetate buffer (pH 4.0) for 3 0 m i n at 45°C. The enzyme activity toward the substrates was determined by measuring the increase in reducing sugars in the reaction mixture. The relative activities of the enzyme toward sucrose and raffinose were 100 and 28, respectively. The enzyme was inactive toward all other disaccharides tested. These results were also similar to those obtained with juice invertase (1). Several results obtained in this study suggested that the wine invertase was identical with the juice invertase, and that the former originated from the latter. In Semillon wine, the inactivation or insolubilization of invertase by TABLE 3. Amino acids Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine a Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan b
Amino acid composition of invertases tool% Wine
Juice
14.9 7.8 7.8 7.3 5.9 9.4 7.1 6.4 0.1 2.4 3.7 8.7 4.4 4.5 2.1 3.0 2.1 2.5
14. l 7.5 7.4 8.2 5.3 9.4 6.6 5.8 0.2 1.7 4.9 9.1 4.7 4.1 2.1 3.2 2.8 2.8
0.5 mg of the purified enzyme was used for the analysis of amino acid. Not corrected for destruction. b Measured spectrophotometrically (8).
68
NAKANISHI ET AL.
the formation of tannin-invertase complexes seemed to be relatively s m a l l d u r i n g t h e f e r m e n t a t i o n a n d a g i n g p r o c esses. I n a d d i t i o n , j u i c e i n v e r t a s e s e e m e d to b e s t a b l e to p r o t e o l y s i s b y yeast cells o r p r o t e a s e s c o n t a i n e d in w i n e . REFERENCES 1. Nakanishi, K. and Yokotsuka, K.: Characterization of thermostable invertase from wine grapes. J. Ferment. Bioeng., 69, 16-22 (1990). 2. Arnold, W.: fl-Fructofranosidase from grape berries. Biochim. Biophys. Acta, 110, 134-147 (1965).
J. FERMENT. BIOENG., 3. Ishikawa, N., Nakagawa, H., and Ogura, N.: Isoforms of invertase in grape berries. Agric. Biol. Chem., 53, 837-834 (1989). 4. Gabriel, O.: Analytical disc gel electrophoresis. Methods Enzymol., 22, 565-579 (1971). 5. Weber, K. and Osborn, M.: The reliability of molecular weight determination by dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem., 244, 4406-4412 (1969). 6. Dubois, M., GlUes, K., Hamilton, J.K., Rebers, P.A., and Smith, F.: Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-356 (1956). 7. Okada, Y. and Hanabusa, H.: Jikken kagaku koza, vol. 23, p. 146-148. Maruzen, Tokyo (1957).
Purification and Some Properties of Thermostable Invertase from Wine K O T O Y O S H I N A K A N I S H I , * W E N WU, AND KOKI Y O K O T S U K A
Institute of Enology and Viticulture, Yamanashi University, Kofu 400, Japan Received 2 July 1990/Accepted 90clober 1990 Invertase from a white table wine made from Semillon grapes was purified to homogeneity on polyacrylamide gel electrophoresis. The enzymatic and physicochemical properties of the enzyme were compared with those of invertase purified from Semillon grape juice. The invertases from the two sources showed similar properties, suggesting that the wine invertase originated from the juice and was stable during the vinification and aging processes.
In a previous paper (1), we examined the characteristics of invertases collected from 14 varieties o f wine grapes. Although the activities o f the invertases showed variation among the grape varieties, all the enzyme preparations exhibited a high degree of thermostability, and were stable toward acidic p H and high concentrations of alcohol or SO2. Significant levels of invertase activity were also detected in several white table wines. The enzymatic properties of these wine invertases suggested they originated from the grapes rather than from the yeast cells. Usually, vinification and aging processes involve many biological, chemical, and physical reactions, which influence the activity level of various enzymes originating from grape juice. However, any enzyme which originates from grape juice has not been purified from fermenting must or the resultant wine. It is, therefore, o f interest to elucidate whether wine invertases maintain their original properties even after vinification and aging. As far as we know, there have been no reports on the purification of invertase from wines until now, although a few studies (1-3) have been published on the purification and properties of invertase from grapes. The present paper deals with the purification and some properties of invertase from Semillon wine. The enzymatic and physicochemical properties of the enzyme were compared with those of the purified invertase from Semillon grape juice. Semillon grapes were harvested from our lnstitute's vineyard in 1986, and wine was produced by conventional vinification procedures at our winery. The wine was stored at 15°C for two years before use. The activity o f invertase was measured by following the release of reducing sugar from sucrose, as described previously (1). One unit of enzyme activity was defined as the a m o u n t o f enzyme which was capable of liberating 1 p m o l o f glucose in 1 min. Specific activity (units/rag) was represented as the enzyme activity to 1 mg of protein. Protein concentration was measured by a Bio-Rad protein assay kit (Bio-Rad Laboratories, New York, USA) with bovine serum albumin as the standard. Polyacrylamide disc gel electrophoresis was done on a 12.5% acrylamide gel with 0.05-M Tris0.38 M glycine buffer (pH 8.3) (4). Isoelectric focusing was performed using polyacrylamide gel (5.3%) containing 2.59/00 p h a r m a l a t e (pH 2.5-5). The p h a r m a l a t e and a pI m a r k e r protein kit were purchased from P h a r m a c i a Fine Chemical Corp. (Uppsala, Sweden). A b o u t 20/Jg of the
purified enzyme was charged and the focusing was done at 200V for 12h, and then at 400V for 1 h. The molecular weight of the purified enzyme was estimated by sodium dodecyl sulfate slab gel electrophoresis (5). M W - M a r k e r containing five kinds of proteins (Oriental Yeast Co., Ltd., Tokyo) was used as the m a r k e r proteins. The amino acid composition was analyzed with an amino acid analyzer (Kyowa KTC-104, Tokyo) after hydrolysis of the enzyme with 6 N HCI for 24 h at 110°C in a sealed evacuated tube. The c a r b o h y d r a t e content o f the enzyme was measured by the phenol-sulfuric acid method (6). Results of the enzyme purification are summarized in Table 1. Two liters of wine were sufficiently dialyzed against distilled water at 4°C. The dialyzed wine (2.53/) was concentrated to 490 ml by a flash evaporator at 40°C, and filtered by filter paper (Toyo Roshi no. 5C) to remove insoluble materials. A m m o n i u m sulfate was added to the filtrate to 80% saturation and the mixture was stored overnight at 4°C. The precipitate obtained by centrifugation was dissolved in 0.1 M acetate buffer (pH 5.0) and the solution was dialyzed against the same buffer. The dialyzed solution was applied on a DEAE-Sephadex A-25 column (2.1 ~ 55 cm) equilibrated with the same buffer. Gradient elution was performed with increasing NaCI concentration from 0 to 0.35 M. The elution profile is shown in Fig. 1. The active fractions (fraction nos. 130-170) were collected and concentrated by a rotary evaporator. Ammonium sulfate was added to the concentrated enzyme
lOO
E iO
e-
1.0 , D
>
50~ ~0.5 ..Q <
70
100
150
200
Fraction no.(Sml/tube) FIG. 1.
DEAE Sephadex A-25 column chromatograph}'. Ex-
perimental conditions are described in the text. Symbols: @, 280 nm;, :, enzyme activity. Line: --, NaCI concentration.
Corresponding author. 66
VOL. 71, 1991
NOTES
67
TABLE 1. Summary of purification of invertase from wine Procedure
Volume (ml)
Total activity (units)
Total protein (rag)
Specific activity (units/mg)
Yield (%)
2530 490 102 200 40
19234 18875 15452 8116 5275
185 152 72 12 2.2
104 124 215 676 2398
100 98 80 42 27
Dialyzed wine" Concentration Ammonium sulfate precipitate (80O/oo) DEAE-Sephadex A-25 Sephadex G-100 a Two liters of wine were dialyzed.
(50 ml) to a saturation of 80P/o, and the resultant precipitate was dissolved in a small a m o u n t of 0.1 M acetate buffer (pH 5.0) and dialyzed against the same buffer. The dialyzed enzyme (10 ml) was applied on a Sephadex G-100 column (2.6 × 90 cm) equilibrated with the same buffer and eluted with the same buffer at a flow rate o f 15 ml per hour. After gel filtration on Sephadex G-100, the enzyme was purified to about 25-fold. The purified enzyme gave a single protein band on polyacrylamide disc gel electrophoresis ( p h o t o g r a p h not shown). In the previous report (1), the invertase activity from Semillon grape juice was recovered in two fractions which were eluted with about 0.2 M and 0.25 M o f NaC1, on a D E A E - S e p h a d e x A-25 column. The m a j o r fraction eluted with about 0.2 M o f NaCI (the ratio of activity between the m a j o r and minor fractions was about 4 : 1) was further purified by Sephadex G-100 c h r o m a t o g r a p h y . The elution profiles o f the wine invertase by D E A E - S e p h a d e x A-25 and Sephadex G-100 c h r o m a t o g r a p h i e s were somewhat different from those of the juice invertase. The specific activity of the purified enzymes was slightly higher than that o f the purified invertase from the juice. The enzymatic and physicochemical properties of the purified wine invertase were c o m p a r e d with those o f juice invertase purified as described previously (1), and stored at 20°C until use. The optimal p H and temperature, p H and thermal stabilities, molecular weights, apparent Km values for sucrose, isoelectric points, and carbohydrate contents o f these two enzymes are summarized in Table 2. In addition, the amino acid composition o f both enzymes is given in Table 3. The two tables show that there are no significant differences between the two enzymes. They both exhibited r e m a r k a b l e thermostability and high stability toward acidic p H , as well as high concentrations of alcohol or SO2. The effects o f various metal ions and other reagents on
TABLE 2.
Some properties of invertases from wine and grape juice
Optimum pH Optimum temperature pH stability" Thermal stability b Km value for sucrose Molecular weight Isoelectric point Carbohydrate content (/~g as glucose/rag protein)
Wine
Juice
4.0 75°C 2-8 70°C 10.3 mM 65000 3.8 290
4.0 75°C 2-8 70°C 9.1 mM ~ 65000 3.9 330 ~
a The enzyme solution was maintained at various pHs at 30°C for 30 rain and the residual activity was measured at the optimum pH. b The enzyme solution was maintained at various temperatures for 30 min, followed by rapid cooling in ice-cold water, after which the residual activity was measured. See reference 1.
the activities of the wine and juice invertases were examined. The enzymes were preincubated with each ion or reagent in 0.05 M acetate buffer (pH 4.0) at 30°C for 30 min and then the residual activities were assayed. The activities o f both enzymes were almost completely inactivated or inhibited by 10 2 M o f HgCI2, sodium dodecyl sulfate, and N-bromosuccinimide, as well as 10 4 M of sodium p-chloromercuric-benzoate. Furthermore, both enzyme activities were inhibited by 10 2 M o f CdCI2, CuCI2, and FeCI2 to 50-80O/oo. The effects o f 10 2 M KC1, NaC1, MnC12, MgCI2, CaC12, and ZnC12 on the activities o f both enzymes were negligible. No significant differences between the two enzymes were observed. The substrate specificity of the purified wine invertase was examined. One ml of each 0.1 M substrate solutions was incubated with l ml o f the enzyme (1.4units for sucrose and raffinose, or 7 units for melezitose, treharose, maltose, and cellobiose) and 3 ml of 0.1 M acetate buffer (pH 4.0) for 3 0 m i n at 45°C. The enzyme activity toward the substrates was determined by measuring the increase in reducing sugars in the reaction mixture. The relative activities of the enzyme toward sucrose and raffinose were 100 and 28, respectively. The enzyme was inactive toward all other disaccharides tested. These results were also similar to those obtained with juice invertase (1). Several results obtained in this study suggested that the wine invertase was identical with the juice invertase, and that the former originated from the latter. In Semillon wine, the inactivation or insolubilization of invertase by TABLE 3. Amino acids Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine a Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan b
Amino acid composition of invertases tool% Wine
Juice
14.9 7.8 7.8 7.3 5.9 9.4 7.1 6.4 0.1 2.4 3.7 8.7 4.4 4.5 2.1 3.0 2.1 2.5
14. l 7.5 7.4 8.2 5.3 9.4 6.6 5.8 0.2 1.7 4.9 9.1 4.7 4.1 2.1 3.2 2.8 2.8
0.5 mg of the purified enzyme was used for the analysis of amino acid. Not corrected for destruction. b Measured spectrophotometrically (8).
68
NAKANISHI ET AL.
the formation of tannin-invertase complexes seemed to be relatively s m a l l d u r i n g t h e f e r m e n t a t i o n a n d a g i n g p r o c esses. I n a d d i t i o n , j u i c e i n v e r t a s e s e e m e d to b e s t a b l e to p r o t e o l y s i s b y yeast cells o r p r o t e a s e s c o n t a i n e d in w i n e . REFERENCES 1. Nakanishi, K. and Yokotsuka, K.: Characterization of thermostable invertase from wine grapes. J. Ferment. Bioeng., 69, 16-22 (1990). 2. Arnold, W.: fl-Fructofranosidase from grape berries. Biochim. Biophys. Acta, 110, 134-147 (1965).
J. FERMENT. BIOENG., 3. Ishikawa, N., Nakagawa, H., and Ogura, N.: Isoforms of invertase in grape berries. Agric. Biol. Chem., 53, 837-834 (1989). 4. Gabriel, O.: Analytical disc gel electrophoresis. Methods Enzymol., 22, 565-579 (1971). 5. Weber, K. and Osborn, M.: The reliability of molecular weight determination by dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem., 244, 4406-4412 (1969). 6. Dubois, M., GlUes, K., Hamilton, J.K., Rebers, P.A., and Smith, F.: Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-356 (1956). 7. Okada, Y. and Hanabusa, H.: Jikken kagaku koza, vol. 23, p. 146-148. Maruzen, Tokyo (1957).