A resting-cell assay for cholesterol reductase activity inEubacterium coprostanoligenes ATCC 51222

Abstract

A resting-cell assay was established to evaluate the cholesterol reductase activity ofEubacterium coprostanoligenes ATCC 51222. Cell suspensions from cholesterol-free media rapidly reduced cholesterol to coprostanol. Optimal assay conditions in a 1-ml reaction mixture were determined to be up to 1 h of incubation and up to 0.25 mg bacterial protein/assay with at least 1 mM cholesterol as substrate. The cholesterol reductase activity in cells decreased as a function of storage time at 22°C, 4°C and −20°C. Filling the headspace of the reaction micture with H2 increased the activity about 20%. Optimal cholesterol reductase activity occurred at pH 7.5 in sodium phosphate buffer. Pyruvate and reducing agents in the buffer increased the activity. This study has validated assay conditions for determination of cholesterol reductase activity in resting cells ofE. coprostanoligenes.