Use of the enzymes in the microbial and plant "aldoxime-nitrile pathway"for chiral synthesis

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(1) Development of hydroxynitrile lyase (HNL) and its use in optically active cyanohydrin : To construct a library of HNLs, several HNL genes were expressed in heterogonous hosts. We succeeded in cDNA cloning, primary structure elucidation and expression of cDNA of P. mume (Plum) HNL in methanol yeast Pichia pastoris. Full-length cDNA and genomic DNA of a novel S-HNL from a plant, Baliospermum montanum were cloned and sequenced. The genomic DNA contained two introns and one ORF encoding a 263-residue protein (subunit : 29.5 kDa). C-terminal short truncations proved to be successful to access higher productivity in culture medium of E. coli up to 3-fold. The hnl gene was expressed in E. coli and the enzyme was characterized including kinetic studies of 20 substrates for (S)-cyanohydrin synthesis. An R-HNL from Arabidopsis thaliana accepts nitromethane (MeNO2) as a donor in a reaction with aromatic aldehydes to yield (R)-b-nitro alcohols (Henry reaction). This is the first example of the … More R-HNL-catalyzed synthesis of (R)-b-nitro alcohols. (R)-Mandelonitrile was successfully synthesized by an enzymatic transcyanation reaction of benzaldehyde and acetone cyanohydrin catalyzed by a HNL from Eriobotrya japonica (EjHNL) in an aqueous-organic biphasic system. An HNL from leaves of Passiflora edulis (PeHNL) was purified and characterized for the first time. Asymmetric synthesis of (R)-mandelonitrile in a biphasic system employing PeHNL as carried out.(2) Mechanism of functional expression of HNL from Manihot esculenta : Functional expression of HNL from M. esculenta, MeHNL, was achieved in E. coli. The highly soluble mutant MeHNL-His103Leu were expressed in the systems such as Pichia pastoris, Leishmania tarentolae and two cell-free translation systems, such as E. coli PURE system and Wheat germ translation system. The phenomenon of the functional expression was proven to be specific to the E. coli system.(3) Dynamic kinetic resolution of amino acid amide : The first crystal structures of a fold-type I racemase are solved for the native form and e-caprolactam-complexed form of a-amino-e-caprolactam racemase. Mutation was successfully done to affect the substrate specificity of the enzyme. Further screening for new amino acid amide racemase was carried out. Less