A RESTING CELL ASSAY FOR CHOLESTEROL REDUCTASE-ACTIVITY IN EUBACTERIUM COPROSTANOLIGENES ATCC-51222

A resting-cell assay was established to evaluate the cholesterol reduc tase activity of Eubacterium coprostanoligenes ATCC 51222. Cell suspen sions from cholesterol-free media rapidly reduced cholesterol to copro stanol. Optimal assay conditions in a 1-ml reaction mixture were deter mined to be up to 1 h of incubation and up to 0.25 mg bacterial protei n/assay with at least 1 mM cholesterol as substrate. The cholesterol r eductase activity in cells decreased as a function of storage time at 22 degrees C, 4 degrees C and - 20 degrees C. Filling the headspace of the reaction mixture with H-2 increased the activity about 20%. Optim al cholesterol reductase activity occurred at pH 7.5 in sodium phospha te buffer. Pyruvate and reducing agents in the buffer increased the ac tivity. This study has validated assay conditions for determination of cholesterol reductase activity in resting cells of E. coprostanoligen es.