Characteristics of an alpha-galactosidase associated with grape flesh

alpha-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 alpha-galactosidase. alpha-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 alpha-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-alpha-D-Gal (PNP-alpha-D-Gal) was 4.2 times higher than that of PNP-beta-D-Gal, implying an apparent alpha-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-alpha-D-Gal at pH 6.0 and 7.0, respectively. EDTA or 1,10-phenanthroline did not substantially affect enzyme activity.