We have developed a practical methodology for bio-catalyst discovery by screening of micro-organisms, which consists of a) preselection of micro-organisms possibly containing the necessary enzymes based on their degradation ability on specific compounds and b) high-throughput screening with a miniaturized system (Figure 1) coupled with fast and sensitive analysis of activity/ enantio-selectivity by liquid chromatography-mass spectroscopy, high performance liquid chromatography, or gas chromatography. By use of this method, we have discovered and developed several novel monooxygenases and ketoreductases with high activity as well as high and complementary enantio-selectivity for the following reactions: 1) regio- and stereo-selective hydroxylation of non-activated carbon atoms; 2) enantio-selective epoxidation of C-C double bond; 3) enantio-selective trans-dihydroxylation of C-C double bound; 4) enantio-selective reduction of ketone. These catalysts have been used for the preparation of several useful and valuable pharmaceutical intermediates.

We also developed efficient methods for recycling the expensive cofactor by the use of permeabilized microbial cells that are easily available, stable, and can be repeatedly used. Enantio-selective reduction of ketone with the recycling of nicotinamide adenine dinucleotide phosphate (NADPH) for 4200 times was achieved by the use of a permeabilized bacterium containing both enantioselective NADPH-dependent ketoreductase for the reduction and glucose-6-phosphate-dehydrogenase for the NADPH regeneration. Moreover, bio-reduction with cofactor recycling was also realized with a new concept that, as schematically shown in Figure 2, involves the use of “coupled permeabilized micro-organisms” – one containing ketoreductase for the reduction and the other containing glucose dehydrogenase for the regeneration of NADPH.