Purification and properties of a nicotinamide adenine dinucleotide-linked cyclohexanol dehydrogenase from aNocardia species |
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Authors: | Lynne A. Stirling J. J. Perry |
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Affiliation: | (1) Papanicolaou Cancer Research Institute, 1155 N. W. 14th Street, P. O. Box 016188, 33101 Miami, Florida, USA;(2) Department of Microbiology, North Carolina State University, P. O. Box 5476, 27650 Raleigh, North Carolina, USA |
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Abstract: | An inducible pyridine nucleotide-linked cyclohexanol dehydrogenase activity was present in crude extracts from aNocardia species following growth on cyclohexane. The enzyme was purified 126-fold by affinity chromatography and has an oligomeric molecular weight of 145,000 ±5,000. There was an absolute requirement for NAD for activity and the products of the dehydrogenase reaction were stoichiometric amounts of NADH and cyclohexanone. The enzyme had a broad specificity for secondary alcohols including straight-chain secondary alcohols, cyclic and substituted cyclic alcohols, and cyclohexane diols. The apparentK m values for cyclohexanol and NAD were 3.7×10−5 M and 2.4×10−5 M, respectively, and the optimal pH for cyclohexanol oxidation was 10.5. The enzyme was heat sensitive, losing about 50% activity after a 1-min incubation at 45°C. Enzyme activity was completely inhibited by the thiol agent,p-chloromercuribenzoate but not by metal chelating agents. |
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