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) 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 degrees C and the half-life at 50 degrees 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 (omega-1)-ketones; primary alcohols or aldehydes are not accepted.
Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria.
Birgit
B
Kosjek
Wolfgang
W
Stampfer
Mateja
M
Pogorevc
Walter
W
Goessler
Kurt
K
Faber
Wolfgang
W
Kroutil
D000426:Alcohol Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D000438:Alcohols / Q000737:chemistry*; D004356:Drug Storage; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D012240:Rhodococcus / Q000145:classification / Q000201:enzymology*; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature
D000438:Alcohols; D000426:Alcohol Dehydrogenase