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

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 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.

Biocatalytic Oxidation of Primary and Secondary Alcohols

“…[20,21] Most importantly, the enzyme is remarkably stable towards elevated co-substrate concentrations allowing us to use acetone for cofactor regeneration at 20% v/v with a whole-cell preparation and up to 50% v/v with the partially purified enzyme. [22,23] Elevated acetone concentrations not only shift the equilibrium entirely towards oxidation, but also increase the solubility of lipophilic substrates, thus ensuring enhanced reaction rates.…”

Stereoselective Synthesis of Three Isomers of tert‐Butyl 5‐Hydroxy‐4‐methyl‐3‐oxohexanoate through Alcohol Dehydrogenase‐Catalyzed Dynamic Kinetic Resolution

“…[11] Actually a systematic screening of carbonyl reductases from bakers yeast in most cases resulted in Prelog-type reduction of b-keto esters. [12] Similar to most reductases from bakers yeast, alcohol dehydrogenases from different Rhodococcus species also typically exhibit Prelog-type stereospecificity, as observed for substrate screenings with whole cell preparations of Rhodococcus ruber [13] and for isolated enzymes from Rhodococcus ruber [14] and Rhodococcus erythropolis. [15] In order to obtain syn-(4R,5S)-2 through reduction of 1 we used commercially available NADH-dependent alcohol dehydrogenase 1 from Rhodococcus sp.…”

Stereo‐Complementary Two‐Step Cascades Using a Two‐Enzyme System Leading to Enantiopure Epoxides

“…[25] Excess of 2-propanol is not only used as hydride donor for cofactor recycling but also to solubilise the substrate in the aqueous phase. [22] Among several types of functionalised ketones, ADH-A reduces a-chloro ketones with excellent stereoselectivity affording the Prelog alcohols.…”