Kiso T, Nakano H, Nakajima H, Terai T, Okamoto K, Kitahata S
Osaka Municipal Technical Research Institute, Japan.
Biosci Biotechnol Biochem. 2000 Aug;64(8):1702-6. doi: 10.1271/bbb.64.1702.
p-Hydroxybenzoyl beta-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of beta-galactosyl ester linkage by beta-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl beta-galactoside (pNP-Gal), a usual substrate with a beta-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that beta-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.
化学合成了对羟基苯甲酰β-半乳糖（pHB-Gal），以检测β-半乳糖苷酶对β-半乳糖基酯键的水解活性。多色青霉的酶水解底物的速度与对硝基苯基β-半乳糖苷（pNP-Gal，一种具有β-半乳糖苷键的常用底物）一样快。大肠杆菌和米曲霉的酶水解pHB-Gal的速度与pNP-Gal几乎相同。环状芽孢杆菌、脆壁酵母和牛肝中的酶活性则低得多。对pHB-Gal进行酶促反应的pH-活性曲线、抑制分析和动力学特性表明，β-半乳糖苷酶只有一个水解半乳糖基酯和半乳糖苷的活性位点。多色青霉的酶在H218O存在下能水解pHB-Gal，释放出含18O的半乳糖。这一结果表明，降解发生在pHB-Gal酯键的异头碳和相邻的O原子之间。
