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Biochemical and molecular characterization of a periplasmic hydrolase for oxidized polyvinyl alcohol from Sphingomonas sp. strain 113P3

¹ Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan
² Faculty of Nutrition, Kobegakuin University, Kobe, Hyogo 651-2180, Japan
³ Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-8552, Japan

Correspondence
Fusako Kawai
fkawai{at}rib.okayama-u.ac.jp

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, Hg²⁺ and Zn²⁺. 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 µmol 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.

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AB190288.

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				R. Hirota-Mamoto, R. Nagai, S. Tachibana, M. Yasuda, A. Tani, K. Kimbara, and F. Kawai Cloning and expression of the gene for periplasmic poly(vinyl alcohol) dehydrogenase from Sphingomonas sp. strain 113P3, a novel-type quinohaemoprotein alcohol dehydrogenase Microbiology, July 1, 2006; 152(7): 1941 - 1949. [Abstract] [Full Text] [PDF]