绿色乳杆菌α－羟基戊二酸脱氢酶的纯化和特性研究

PURIFICATION AND CHARACTERIZATION OF AN α-HYDROXYGLUTARATE-DEHYDROGENASE FROM LACTOBACILLUS VIRIDESCENS SSP.HALOTOLERANS B175

本文对绿色乳杆菌（Ｌ．Ｖｉｒｉｄｅｓｃｅｎｓ）Ｂ１７５产生的α－羟基戊二酸脱氢酶用硫酸铵盐析；ＤＥＡＥ—纤维素柱层折；超滤浓缩；苯基琼脂糖凝胶（ＰｈｅｎｙｌＳｅｐｈａｒｏｓｅ）疏水层析等方法纯化，使酶的纯度提高１１７倍。通过ＨＰＬＣ和等电聚焦测得该酶的分子量约为６６，０００，等电点为４．２。酶催化作用的最适ｐＨ５．５，最适温度为６０℃，对α—酮戊二酸和ＮＡＤＨ的Ｋｍ值分别为０．１４×１０（－３）Ｍ和０．０３×１０（－３）Ｍ。

An a- Hydroxyglutarate - Dehydrogenase was purified from a crude extract of Lactobacilles Viridescens ssp. halotolerans B175. The enzyme was purified 117 - fold by ammonium Sulfate fractionation, Ion - exchange Chromatography on DEAE - Cellulose and Hydrophobic Chromatography on phenylsepharose. The purified enzyme had a molecular Weight of 66, 000 and isoelectric point of 4. 2. The enyme activity was optimal at pH 5. 5 and 60℃.Kinetic Studies indicated that the enzyme’s Km is 0. 14×10-3 M for a ketoglutarat and 0. 03× 10-3 M for NADH.