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.

Modeling the chromatographic enantioseparation of aryl- and hetarylcarbinols on ULMO, a brush-type chiral stationary phase, by 3D-QSAR techniques.

The enantiomers of a series of chiral aryl- and hetaryl-carbinols were separated on a chiral stationary phase (ULMO) based on (S,S)-3,5-dinitrobenzoylated 1,2-diphenylethane-1,2-diamine. In all cases, the homochiral analogs of (S)-1-phenylethanol (mostly also S-enantiomers) were retained more. Log alpha values were modeled with the aid of the 3D-QSAR techniques CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) as well as a descriptor based on normal mode eigenvalues (EVA). Partial least-squares analysis with two (CoMSIA) or three (CoMFA, EVA) latent variables on a set of 22 training analytes gives cross-validated correlation coefficients q(2) = 0.85-0.91 and conventional correlation coefficients r(2) = 0.94-0.99. The quantitative structure-enantioselective retention relationships derived thereby were used to predict the separation factors of the test set, containing also hetaryl-carbinols, e.g., furan and thiophene analogs, with good accuracy.

On the organic solvent and thermostability of the biocatalytic redox system of Rhodococcus ruber DSM 44541.

The sec-alcohol dehydrogenase activity of whole cells of Rhodococcus ruber DSM 44541 has been employed as an efficient biocatalytic redox system due to the use of acetone and 2-propanol at elevated concentrations for cofactor regeneration in the oxidation and reduction mode, respectively, and external addition of NADH/NAD(+) can be omitted. The operational half-life time of the redox system is 29 hours in 20% v/v acetone and 37 hours in 30% v/v 2-propanol. The Redox system allows the enantioselective oxidation of sec-alcohols and the asymmetric reduction of ketones to furnish (S)-configurated alcohols in high optical purity. The stability of the cells towards further organic solvents was investigated. In addition, the system displays thermostability of up to 60 degrees C and pH stability of up to pH 11. The system represents a simple to handle tool for environmentally benign redox reactions.

Biocatalytic asymmetric hydrogen transfer employing Rhodococcus ruber DSM 44541.

Nonracemic sec-alcohols of opposite absolute configuration were obtained either by asymmetric reduction of the corresponding ketone using 2-propanol as hydrogen donor or by enantioselective oxidation through kinetic resolution of the rac-alcohol using acetone as hydrogen acceptor employing whole lyophilized cells of Rhodococcus ruber DSM 44541. The microbial oxidation/reduction system exhibits not only excellent stereo- and enantioselectivity but also a broad substrate spectrum. Due to the exceptional tolerance of the biocatalyst toward elevated concentrations of organic materials (solvents, substrates and cosubstrates), the process is highly efficient. The simple preparation of the biocatalyst and its ease of handling turns this system into a versatile tool for organic synthesis.