Identification and Characterization of Reaction Steps and Enzyme Catalysts Involved in the Conversion of Acetate and Methyl Groups to Methane. Annual Report October 1984-October 1985

摘要

A study has been conducted on carbon monoxide dehydrogenase (CODH) from acetate-grown cells of Methanosarcina barkeri. Under conditions of high ionic strength the enzyme was found to exist in an aggregated state along with a discrete set of other proteins (total mol. wt. approx. 3,000,000). Dissociation of the aggregate was accomplished when the ionic strength was decreased by dialysis. The disaggregated form of CODH retained full activity and exhibited a molecular weight of approximately 161,000. An efficient purification procedure was developed which produced high yields of pure CODH. The method consisted of the following steps which were carried out under strictly anaerobic conditions in the NIH Anaerobic Laboratory: (1) Isolation of the CODH aggregate by gel-filtration and subsequent dissociation; (2) Chromatography on Phenyl Sepharose; and (3) Hydroxylapatite chromatography. CODH appeared to be less hydrophobic than any of the other protein components of the aggregate. Among various compounds tested, oxygen and cyanide were potent inactivators. Glyoxalydehyde inacn enzyme intermediate in catalysis.