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

Wilailak Klomklang, Akiwo Tani, Kazuhide Kimbara, Rie Mamoto, Takashi Ueda, Masayuki Shimao, Fusako Kawai

Research output: Contribution to journalArticle

22 Citations (Scopus)

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, 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. Km and Vmax 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.

Original languageEnglish
Pages (from-to)1255-1262
Number of pages8
JournalMicrobiology
Volume151
Issue number4
DOIs
Publication statusPublished - Apr 2005

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Sphingomonas
Polyvinyl Alcohol
Hydrolases
Serine
Enzymes
Mesorhizobium
Sinorhizobium meliloti
Amino Acids
Periplasm
Rhizobium
Proteins
Protein Sorting Signals
Pseudomonas
Lipase
Recombinant Proteins
Open Reading Frames
Genes
Amino Acid Sequence
Pseudomonas sp. oxidized polyvinyl alcohol hydrolase
Escherichia coli

ASJC Scopus subject areas

  • Microbiology

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Biochemical and molecular characterization of a periplasmic hydrolase for oxidized polyvinyl alcohol from Sphingomonas sp. strain 113P3. / Klomklang, Wilailak; Tani, Akiwo; Kimbara, Kazuhide; Mamoto, Rie; Ueda, Takashi; Shimao, Masayuki; Kawai, Fusako.

In: Microbiology, Vol. 151, No. 4, 04.2005, p. 1255-1262.

Research output: Contribution to journalArticle

Klomklang, Wilailak ; Tani, Akiwo ; Kimbara, Kazuhide ; Mamoto, Rie ; Ueda, Takashi ; Shimao, Masayuki ; Kawai, Fusako. / Biochemical and molecular characterization of a periplasmic hydrolase for oxidized polyvinyl alcohol from Sphingomonas sp. strain 113P3. In: Microbiology. 2005 ; Vol. 151, No. 4. pp. 1255-1262.
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