A resting-cell assay for cholesterol reductase activity inEubacterium coprostanoligenes ATCC 51222 |
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Authors: | L Li T A Freier P A Hartman J W Young D C Beitz |
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Institution: | (1) Department of Animal Science, Nutritional Physiology Group, 313 Kildee Hall, Ames, Iowa 50011-3150, USA. Fax: 515-294-6445, US;(2) Department of Microbiology, Immunology and Preventive Medicine, Iowa State University, Ames, Iowa 50011, USA, US |
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Abstract: | A resting-cell assay was established to evaluate the cholesterol reductase activity of Eubacterium 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 mixture 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 of E. coprostanoligenes.
Received: 2 August 1994/Received revision: 15 November 1994/Accepted: 8 December 1994 |
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