1887

Abstract

Oxidized polyvinyl alcohol hydrolase (OPH) and polyvinyl alcohol dehydrogenase were found to be constitutively present in the periplasm of sp. strain 113P3 (formerly 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 and Zn. The enzyme hydrolysed oxidized polyvinyl alcohol (oxidized PVA) and -nitrophenyl acetate (PNPA), but did not hydrolyse any of the mono- or diketones tested. and values for oxidized PVA and PNPA were 0·2 and 0·3 mM, and 0·1 and 3·4 μmol min mg, 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 sp. strain VM15C (63 % identity) and the polyhydroxybutyrate depolymerases from , sp. and (29–32 % identity). The gene was expressed in under the control of the promoter. The recombinant protein had the same molecular mass and N-terminal amino acid sequence as the purified OPH from strain 113P3.

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2005-04-01
2019-11-21
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