44C-1 |
Purification and characterization of grape beta-galactosidase |
Y. D. HANG and E. E. Woodams. Dept. of Food Science & Technology, Cornell Univ., W. North St., NYSAES, Geneva, NY 14456 Beta-galactosidase (E.C. 3.2.1.23) are widely distributed in plants, animals and microorganisms. Recent interest in beta-galactosidase as the agent most likely responsible for softening of fruits has increased. Beta-galactosidases from apples and other plant sources have been investigated. However, there is no information in the literature regarding the activity of grape beta-galactosidase. The objective of this study was to purify and characterize beta-galactosidase isolated from Einset Seedless grapes. Einset Seedless grapes grown in Geneva, NY were used in this study. Acetone powder containing beta-galactosidase activity was prepared by homogenizing 50 g of mature berries in 200 ml 0f -10 degree C acetone. The precipitate was collected by centrifugation, washed with 75% ethanol.The crude enzyme was purified by affinity chromatography on a Lactosyl-Sepharose 4B column. The activity was determined by incubating the enzyme with 0.2% PNPG (p-nitrophenyl-B-D-galactopyranoside) in 0.1 M acetate buffer (pH 5.0) at 30 degree C for 2 h. The reaction was stopped by adding 1 M sodium carbonate and the color was measured at 405 nm. One enzyme unit was the amount of enzyme that produced 1 umol p-nitrophenol/h. Beta-galactosidase from Einset Seedless grapes was purified 33-fold by affinity chromatography. The purified enzyme had a specific activity of 1043 units/mg protein. It was most active at pH 4.5 and exhibited the highest activity at 50-55 degree C. The Km and Vmax values of the enzyme for PNPG were 0.5 mmole/L and 0.14 micromol p-nitrophenol/h, respectively. The molecular weight of grape beta-galactosidase was 63 kDa. Beta-galactosidase from Einset Seedless was purified and characterized. Because of its unusual properties, the enzyme could serve as a biocatalyst for food applications.
Session 44C, Fruit & Vegetable Products: Chemistry
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