Characteristics of an α-galactosidase associated with grape flesh

• Published in: Phytochemistry (Oxford) 2001-09, Vol.58 (2), p.213-219
• Main Authors: Kang, Han-Chul, Lee, Seon-Hwa
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; alpha-Galactosidase - metabolism; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Gel; Economic plant physiology; Enzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Grape; Hydrolases; Metabolism; Molecular Weight; Nutrition. Photosynthesis. Respiration. Metabolism; Plant physiology and development; Substrate Specificity; Vitaceae; Vitis - enzymology; Vitis veniferae; α-Galactosidase
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/S0031-9422(01)00207-2

α-Galactosidase activity in grape flesh ( Vitis venifera L. Muscat of Alexandria) was characterized by a marked increase in its activity 4 weeks after fruit bearing. After 12 weeks the specific activity of the enzyme had increased 15-fold. Several other glycosidases were measured at different stages of fruit development but none showed the increased levels of activity displayed by this α-galactosidase. α-Galactosidase activity (unit/g·fresh wt) increased by 52% during postharvest storage, whereas the unripe grape showed a “stagnancy” for 10–15 days prior to the increase. An α-galactosidase was partially purified ca. 103-fold from grape flesh of Vitis labruscana Honey black, by a procedure involving ammonium sulfate fractionation, Biogel P-60, melibiose-agarose, and Sephacryl S-200 chromatographic separations. The enzyme was effectively separated by affinity chromatography on melibiose-agarose, and was a monomer of 40–45 kDa as determined by SDS-PAGE and Sephacryl S-200 chromatographic analysis. The hydrolysis rate of p-nitrophenyl-α- d-Gal (PNP-α- d-Gal) was 4.2 times higher than that of PNP-β- d-Gal, implying an apparent α-anomer specificity, and natural oligosaccharides such as melibiose, stachyose, and raffinose were also considerably hydrolyzed. The enzyme was active over a narrow pH range with an optimal hydrolysis of stachyose and PNP-α- d-Gal at pH 6.0 and 7.0, respectively. EDTA or 1,10-phenanthroline did not substantially affect enzyme activity. α-Galactosidase from grape flesh was purified and characterized.