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Enzymes from extremophiles have always been of great interest for biotechnology because of their ruggedness against various stress factors. We have isolated, cloned, heterologously expressed and characterized a thermostable old yellow enzyme (OYE) from Geobacillus kaustophilus. In addition to the expected enone reduction, GkOYE also catalyzes the reverse reaction, i.e., the desaturation of C À C bonds adjacent to a carbonyl to give the corresponding a,b-unsaturated ketone. The reaction proceeds at the expense of molecular oxygen without the need for a nicotinamide cofactor and represents an environmentally benign alternative to known chemical dehydrogenation methods.

[reaction: see text] The biocatalytic reduction of alpha-alkyl-1,3-diketones and alpha-alkyl-beta-keto esters employing 1 of 20 different isolated NADPH-dependent ketoreductases proved to be a highly efficient method for the preparation of optically pure keto alcohols or hydroxy esters.

Regio-and stereoselective reductions of asubstituted 1,3-diketones to the corresponding bketo alcohols or 1,3-diols by using commercially available ketoreductases (KREDs) are described. A number of a-monoalkyl-or dialkyl-substituted symmetrical as well as non-symmetrical diketones were reduced in high optical purities and chemical yields, in one or two enzymatic reduction steps. In most cases, two or even three out of the four possible diastereomers of a-alkyl-b-keto alcohols were synthesized by using different enzymes, and in two examples both ketones were reduced to the 1,3-diol. By replacing the a-alkyl substituent with the OAc group, 1-keto-2,3-diols, as well as 1,2,3-triols were synthesized in high optical purities. These enzymatic reactions provide a simple, highly stereoselective and quantitative method for the synthesis of different diastereomers of valuable chiral synthons from nonchiral, easily accessible 1,3-diketones.

Innovative biohydroxylation catalysts for the preparation of drug metabolites were developed from scratch. A set of bacterial and fungal sequences of putative and already known bifunctional P450 enzymes was identified by protein sequence alignments, expressed in Escherichia coli and characterised. Notably, a fungal self-sufficient cytochrome P450 (CYP) from Aspergillus fumigatus turned out to be especially stable during catalyst preparation and application and also in presence of organic co-solvents. To enhance the catalytic activity and broaden the substrate specificity of those variants with high expression levels prominent single mutations were introduced. Selected improved variants were then used as lyophilised bacterial lysates for the synthesis of 4'-hydroxydiclofenac and 6-hydroxychlorzoxazone, the two metabolites of active pharmaceutical compounds diclofenac and chlorzoxazone representing the same metabolites as generated by human P450s.

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