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Purification and characterization of glycyrrhetic acid mono-glucuronide beta-D-glucuronidase in Eubacterium sp. GLH
1Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
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.