Isolation, characterization, and molecular cloning of a thermostable xylitol oxidase from Streptomyces sp. IKD472

Mitsuo Yamashita, Hironori Omura, Eri Okamoto, Yuji Furuya, Masahiko Yabuuchi, Kimi Fukahi, Yoshikatsu Murooka

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

A thermophilic bacterium, Streptomyces sp. IKD472, that can oxidize xylitol was isolated from a hot spring and was found to produce xylitol oxidase. The purified enzyme was a monomeric protein with an apparent molecular weight of 43 k as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. This novel enzyme is capable of catalyzing the oxidation of one mole of xylitol to form one mole each of xylose and hydrogen peroxide. Since the V(max)/K(m) value for xylitol was two and four times higher than those for galactitol and D-sorbitol, respectively, the enzyme was designated as xylitol oxidase. The enzyme was stable in the pH range from 5.5 to 10.5 and at temperatures up to 65°C. The optimal temperature and pH were 55°C and pH 7.5, respectively. Xylitol oxidase bound one mole of FAD as a coenzyme per mole of protein. The amino acid sequence of the NH2 terminus and the fragments obtained by lysylendpeptidase digestion of xylitol oxidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 2.8-kb chromosomal fragment hybridizing to the probes was cloned into pUC18 in Escherichia coli. The gene consists of an open reading frame of 1245 bp that encodes a protein containing 415 amino acids with a molecular weight of 44,730 but without the conserved nucleotide-binding sequence, Gly-X-Gly-X-X- Gly. The amino acid sequence has 70% identity to putative oxidoreductase from Streptomyces coelicolar, 51% to sorbitol oxidase from Streptomyces sp., and 26% to L-gulonolactone oxidase from rat in terms of the overall amino acid sequence. DNA manipulation of the cloned gene in E. coli, by alteration of a strong promoter and a synthesized ribosome-binding sequence at an appropriate position, resulted in overproduction of xylitol oxidase 100 times more than that produced in the original Streptomyces sp. IKD472. The enzyme properties of recombinant xylitol oxidase were the same as those of the authentic enzyme. Stable xylitol oxidases, which allow easier quantitative analysis of xylitol, are useful for clinical applications.

Original languageEnglish
Pages (from-to)350-360
Number of pages11
JournalJournal of Bioscience and Bioengineering
Volume89
Issue number4
DOIs
Publication statusPublished - 2000
Externally publishedYes

Fingerprint

Xylitol
Cloning
Oxidoreductases
Enzymes
Amino acids
Amino Acids
Proteins
Escherichia coli
L-Gulonolactone Oxidase
Gels
Genes
Molecular weight
Hot springs
Galactitol
Coenzymes
Xylose
Oligonucleotides
Flavin-Adenine Dinucleotide
Sodium dodecyl sulfate
Sorbitol

Keywords

  • FAD
  • Heterologous expression
  • Streptomyces
  • Thermostable
  • Xylitol oxidase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering

Cite this

Isolation, characterization, and molecular cloning of a thermostable xylitol oxidase from Streptomyces sp. IKD472. / Yamashita, Mitsuo; Omura, Hironori; Okamoto, Eri; Furuya, Yuji; Yabuuchi, Masahiko; Fukahi, Kimi; Murooka, Yoshikatsu.

In: Journal of Bioscience and Bioengineering, Vol. 89, No. 4, 2000, p. 350-360.

Research output: Contribution to journalArticle

Yamashita, Mitsuo ; Omura, Hironori ; Okamoto, Eri ; Furuya, Yuji ; Yabuuchi, Masahiko ; Fukahi, Kimi ; Murooka, Yoshikatsu. / Isolation, characterization, and molecular cloning of a thermostable xylitol oxidase from Streptomyces sp. IKD472. In: Journal of Bioscience and Bioengineering. 2000 ; Vol. 89, No. 4. pp. 350-360.
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AU - Furuya, Yuji

AU - Yabuuchi, Masahiko

AU - Fukahi, Kimi

AU - Murooka, Yoshikatsu

PY - 2000

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N2 - A thermophilic bacterium, Streptomyces sp. IKD472, that can oxidize xylitol was isolated from a hot spring and was found to produce xylitol oxidase. The purified enzyme was a monomeric protein with an apparent molecular weight of 43 k as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration. This novel enzyme is capable of catalyzing the oxidation of one mole of xylitol to form one mole each of xylose and hydrogen peroxide. Since the V(max)/K(m) value for xylitol was two and four times higher than those for galactitol and D-sorbitol, respectively, the enzyme was designated as xylitol oxidase. The enzyme was stable in the pH range from 5.5 to 10.5 and at temperatures up to 65°C. The optimal temperature and pH were 55°C and pH 7.5, respectively. Xylitol oxidase bound one mole of FAD as a coenzyme per mole of protein. The amino acid sequence of the NH2 terminus and the fragments obtained by lysylendpeptidase digestion of xylitol oxidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 2.8-kb chromosomal fragment hybridizing to the probes was cloned into pUC18 in Escherichia coli. The gene consists of an open reading frame of 1245 bp that encodes a protein containing 415 amino acids with a molecular weight of 44,730 but without the conserved nucleotide-binding sequence, Gly-X-Gly-X-X- Gly. The amino acid sequence has 70% identity to putative oxidoreductase from Streptomyces coelicolar, 51% to sorbitol oxidase from Streptomyces sp., and 26% to L-gulonolactone oxidase from rat in terms of the overall amino acid sequence. DNA manipulation of the cloned gene in E. coli, by alteration of a strong promoter and a synthesized ribosome-binding sequence at an appropriate position, resulted in overproduction of xylitol oxidase 100 times more than that produced in the original Streptomyces sp. IKD472. The enzyme properties of recombinant xylitol oxidase were the same as those of the authentic enzyme. Stable xylitol oxidases, which allow easier quantitative analysis of xylitol, are useful for clinical applications.

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