Development of New Bioreactor Systems and Their Applications in the Chiral Industry

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The D-p-hydroxyphenylglycine (D-HPG) production process from DL-5-p-hydroxyphenylhydantoin by a combination of two immobilized enzymes, hydantoinase and N-carbamoyl-D-amino acid amidohydrolase (DCase), was established. A hydantoinase obtained from a thermophilic bacterium and a DCase from a mesophilic bacterium improved the thermostability by a protein engineering technique. The latter enzyme could be used over 700 times as a bioreactor. An enzymatic reduction system was developed to produce optically active alcohols from the corresponding carbonyl compounds. Effective production of ethyl (S) -4-chloro-3-hydroxybutyrate was achieved by the use of the recombinant Escherichia coli that was co-harbored in the genes of a carbonyl reductase (S1) from Candida magnoliae and glucose dehydrogenase from Bacillus megaterium, in high productivity (350 g/l, over 99% e.e.). Successful change in the cofactor dependency of S1, from NADPH to NADH, was accomplished highly efficiently by the use of in silico screening.