Quasi-irreversible oxidation of sec-alcohols was achieved via biocatalytic hydrogen transfer reactions using alcohol dehydrogenases employing selected ketones as hydrogen acceptors, which can only be reduced but not oxidized. Thus, only 1 equiv of oxidant was required instead of a large excess. For the oxidation of both isomers of methylcarbinols a single nonstereoselective short-chain dehydrogenase/reductase from Sphingobium yanoikuyae was identified and overexpressed in E. coli.

Ketones with two bulky substituents, named bulky-bulky ketones, as well as less sterically demanding ketones were successfully reduced to the corresponding optically highly enriched alcohols using a novel identified recombinant short-chain alcohol dehydrogenase RasADH from Ralstonia sp. DSM 6428 overexpressed in E. coli.

No abstract

Alternative oxidases (Aox or Aod) are present in the mitochondria of plants, fungi and many types of yeast. These enzymes transfer electrons from the ubiquinol pool directly to oxygen without contributing to the proton transfer across the mitochondrial membrane. Alternative oxidases are involved in stress responses, programmed cell death and maintenance of the cellular redox balance. The alternative oxidase gene of the methylotrophic yeast Pichia pastoris was isolated and cloned to study its regulation and the effects of deregulation of the alternative respiration by overexpression or disruption of the gene. Both disruption and overexpression had negative effects on the biomass yield; however, the growth rate and substrate uptake rate of the strain overexpressing the alternative oxidase were slightly increased. These effects were even more pronounced when higher glucose concentrations were used. The occurrence of free intracellular radicals and cell death phenomena was investigated using dihydrorhodamine 123 and the TUNEL test. The results suggest a major contribution of the alternative oxidase to P. pastoris cell viability. The negative effects of deregulated alternative respiration clearly indicated the importance of precise regulation of the alternative oxidase in this yeast.

A novel short-chain alcohol dehydrogenase from Paracoccus pantotrophus DSM 11072, which is applicable for hydrogen transfer, has been identified, cloned, and overexpressed in E. coli. The enzyme stereoselectively reduces several ketones in a sustainable substrate-coupled approach using 2-propanol (5% v/v) as hydrogen donor. The enzyme maintained its activity in organic co-solvents in biphasic as well as monophasic systems and was even active in micro-aqueous media (1% v/v aqueous buffer). In general, a higher conversion was observed at higher log P values of the solvent, however, DMSO, which exhibits the lowest log P value of all solvents investigated, was not only tolerated but led to a higher conversion and relative activity (110-210%). For example, the conversion after 24 h in 15% v/v DMSO was double that for the reaction performed in buffer. This tolerance to DMSO may be attributed to the ability of the wild-type strain to adapt and grow in media with high sulfur content.