Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene
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- K Zhang
- Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Japan.
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- M Shiraki
- Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Japan.
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- T Saito
- Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Japan.
書誌事項
- 公開日
- 1997-01
- 権利情報
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- https://journals.asm.org/non-commercial-tdm-license
- DOI
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- 10.1128/jb.179.1.72-77.1997
- 公開者
- American Society for Microbiology
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説明
<jats:p>An extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Pseudomonas sp. strain A1. The purified enzyme hydrolyzed the D-(-)-3-hydroxybutyrate dimer and trimer at similar rates. The enzyme activity was inhibited by a low concentration of diisopropylfluorophosphate. The molecular weight of the hydrolase was estimated to be about 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 10-kbp DNA fragment of A1 was detected by hybridization with the gene (2 kbp) of an extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Subsequent subcloning showed that a SmaI-KpnI fragment (2.8 kbp) was responsible for expression of the hydrolase in Escherichia coli and an in vitro transcription-translation system. The expressed protein detected by immunostaining had the same molecular weight as the purified enzyme from A1. The protein band detected in the in vitro transcription-translation system had a molecular size of 72 kDa. The nucleotide sequence of the SmaI-KpnI fragment was determined, and one open reading frame (2,112 nucleotides) was found. It specifies a protein with a deduced molecular weight of 72,876 (704 amino acids). In this sequence, the consensus sequence of serine-dependent hydrolysis, G-X-S-X-G, did not exist.</jats:p>
収録刊行物
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- Journal of Bacteriology
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Journal of Bacteriology 179 (1), 72-77, 1997-01
American Society for Microbiology
