title: Biochemical and molecular characterization of a periplasmic hydrolase for oxidized polyvinyl alcohol from Sphingomonas sp. strain 113P3.
abstract: Oxidized polyvinyl alcohol hydrolase (OPH) and polyvinyl alcohol dehydrogenase were found to be constitutively present in the periplasm of Sphingomonas sp. strain 113P3 (formerly Pseudomonas sp. 113P3). The OPH was purified to homogeneity with a yield of 40 % and a 5.9-fold increase in specific activity. The enzyme was a homodimer consisting of 35 kDa subunits. Its activity was inhibited by PMSF, Hg(2+) and Zn(2+). The enzyme hydrolysed oxidized polyvinyl alcohol (oxidized PVA) and p-nitrophenyl acetate (PNPA), but did not hydrolyse any of the mono- or diketones tested. K(m) and V(max) values for oxidized PVA and PNPA were 0.2 and 0.3 mM, and 0.1 and 3.4 micromol min(-1) mg(-1), respectively. The gene for OPH was cloned and sequenced. Sequencing analysis revealed that the open reading frame consisted of 1095 bp, corresponding to a protein of 364 amino acids residues, encoding a signal peptide and a mature protein of 34 and 330 amino acids residues, respectively. The presence of a serine-hydrolase motif (a lipase box; Gly-X-Ser-X-Gly) strongly suggested that the enzyme belongs to the serine-hydrolase family. The protein exhibited homology with OPH of the Pseudomonas sp. strain VM15C (63 % identity) and the polyhydroxybutyrate depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti (29-32 % identity). The oph gene was expressed in Escherichia coli under the control of the lac promoter. The recombinant protein had the same molecular mass and N-terminal amino acid sequence as the purified OPH from strain 113P3.
authors.affiliation: Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan.
authors.forename: Wilailak
authors.initials: W
authors.lastname: Klomklang
authors.affiliation: Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Tani
authors.affiliation: Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan.
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Kimbara
authors.affiliation: Faculty of Nutrition, Kobegakuin University, Kobe, Hyogo 651-2180, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Mamoto
authors.affiliation: Faculty of Nutrition, Kobegakuin University, Kobe, Hyogo 651-2180, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueda
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-8552, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Shimao
authors.affiliation: Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan.
authors.forename: Fusako
authors.initials: F
authors.lastname: Kawai
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019897:Periplasm / Q000201:enzymology; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D021122:Protein Subunits; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C048312:polyvinyl-alcohol dehydrogenase (acceptor); D002265:Carboxylic Ester Hydrolases; C409492:oxidized polyvinyl alcohol hydrolase, Pseudomonas sp.