真杆菌属GLH中甘草次酸单葡萄糖醛酸β-D-葡萄糖醛酸酶的纯化与特性分析

Purification and characterization of glycyrrhetic acid mono-glucuronide beta-D-glucuronidase in Eubacterium sp. GLH.

作者信息

Akao T

机构信息

Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.

出版信息

Biol Pharm Bull. 1999 Jan;22(1):80-2. doi: 10.1248/bpb.22.80.

DOI:10.1248/bpb.22.80

PMID:9989667

Abstract

Glycyrrhetic acid mono-glucuronide (GAMG), 1-(18beta-glycyrrhet-3-yl)-beta-D-glucopyranuroic acid, was hydrolyzed to glycyrrhetic acid (GA) by GAMG beta-D-glucuronidase in Eubacterium sp. GLH from human intestinal bacteria. The enzyme had an optimum pH of 5.0 and was purified from a crude extract by Butyl Toyopearl 650 S, Toyopearl HW-55 S, Hydroxyapatite and DEAE-Toyopearl 650 M column chromatography. The purified enzyme showed a specific activity of 495 nmol/min/mg protein and a single band on Coomassie brilliant blue staining and a molecular weight of about 43 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The apparent molecular weight was 49.5 kDa, as estimated by Toyopearl HW-55 S column chromatography. Also, the enzyme seemed to have a sulfhydryl group(s) in its active site with a Km value of 77 x 10(-3) M.

摘要

甘草次酸单葡萄糖醛酸苷（GAMG），即1 -（18β - 甘草次酸 - 3 - 基）-β - D - 吡喃葡萄糖醛酸，在人肠道细菌的真杆菌属GLH中被GAMGβ - D - 葡萄糖醛酸酶水解为甘草次酸（GA）。该酶的最适pH为5.0，通过丁基Toyopearl 650 S、Toyopearl HW - 55 S、羟基磷灰石和DEAE - Toyopearl 650 M柱色谱从粗提物中纯化得到。纯化后的酶在考马斯亮蓝染色下显示出495 nmol/分钟/毫克蛋白质的比活性和一条单一谱带，在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳上的分子量约为43 kDa。通过Toyopearl HW - 55 S柱色谱估计其表观分子量为49.5 kDa。此外，该酶在其活性位点似乎含有一个巯基，Km值为77×10⁻³ M。