title: Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells.
abstract: The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme.
authors.affiliation: ASPEX Division, Asahi Glass Co. Ltd., 1150 Hazawa, Kanagawa, Yokohama 221-8755, Japan. aisoai@ag.co.jp
authors.forename: Atsushi
authors.initials: A
authors.lastname: Isoai
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Kimura
authors.forename: Arno
authors.initials: A
authors.lastname: Reichert
authors.forename: Kurt
authors.initials: K
authors.lastname: Schörgendorfer
authors.forename: Kiyokazu
authors.initials: K
authors.lastname: Nikaido
authors.forename: Hideki
authors.initials: H
authors.lastname: Tohda
authors.forename: Yuko
authors.initials: Y
authors.lastname: Giga-Hama
authors.forename: Norihiro
authors.initials: N
authors.lastname: Mutoh
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Kumagai
mesh_terms: D002384:Catalysis; D002478:Cells, Cultured; D002511:Cephalosporins / Q000378:metabolism; D003001:Cloning, Molecular; D003605:D-Amino-Acid Oxidase / Q000096:biosynthesis* / Q000235:genetics*; D004795:Enzyme Stability; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D010865:Pilot Projects; D010957:Plasmids; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics*; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D013045:Species Specificity; D014170:Transformation, Genetic
chemical_list: D002511:Cephalosporins; C025163:cephalosporin C; D003605:D-Amino-Acid Oxidase