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

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Abstract
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.

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