Purification and characterization of a chemotolerant alcohol dehydrogenase applicable to coupled redox reactions

The purification and characterization of an organic solvent tolerant, NADH‐dependent medium‐chain secondary alcohol dehydrogenase (denoted sec‐ADH “A”) from Rhodococcus ruber DSM 44541 is reported. The enzyme can withstand elevated concentrations of organic solvents, such as acetone (up to 50% v/v)...

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Published in:Biotechnology and bioengineering 2004-04, Vol.86 (1), p.55-62
Main Authors: Kosjek, Birgit, Stampfer, Wolfgang, Pogorevc, Mateja, Goessler, Walter, Faber, Kurt, Kroutil, Wolfgang
Format: Article
Language:English
Subjects:
alcohol dehydrogenase
Alcohol Dehydrogenase - biosynthesis
Alcohol Dehydrogenase - chemistry
Alcohol Dehydrogenase - isolation & purification
Alcohols - chemistry
Biological and medical sciences
Biology of microorganisms of confirmed or potential industrial interest
Biotechnology
Drug Storage
Enzyme Activation
Enzyme Stability
Fundamental and applied biological sciences. Psychology
Hydrogen-Ion Concentration
Kinetics
Miscellaneous
Mission oriented research
Molecular Weight
organic solvent tolerance
purification
Rhodococcus - classification
Rhodococcus - enzymology
Rhodococcus ruber
Species Specificity
Substrate Specificity
Temperature
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Summary:The purification and characterization of an organic solvent tolerant, NADH‐dependent medium‐chain secondary alcohol dehydrogenase (denoted sec‐ADH “A”) from Rhodococcus ruber DSM 44541 is reported. The enzyme can withstand elevated concentrations of organic solvents, such as acetone (up to 50% v/v) and 2‐propanol (up to 80% v/v). Thus, it is ideally suited for the preparative‐scale enantioselective oxidation of sec‐alcohol and the asymmetric reduction of ketones, using acetone and 2‐propanol, respectively, as cosubstrates for cofactor‐regeneration via a coupled‐substrate approach. The homodimeric protein was found to bear tightly bound zinc and displayed a molecular mass of 38 kDa per subunit as determined by SDS gel electrophoresis. The optimal temperature ranged from 30–50°C and the half‐life at 50°C was 35 h. In addition, excellent storage stability was found. The pH optimum for reduction is pH 6.5; pH 9.0 is preferred for oxidation. The enzyme followed a sequential reaction mechanism. The substrates are medium chain sec‐alcohols or (ω‐1)‐ketones; primary alcohols or aldehydes are not accepted. © 2004 Wiley Periodicals, Inc.
ISSN:0006-3592
1097-0290
DOI:10.1002/bit.20004