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. 1997 Jan;179(1):72–77. doi: 10.1128/jb.179.1.72-77.1997

Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene.

K Zhang 1, M Shiraki 1, T Saito 1
PMCID: PMC178663  PMID: 8981982

Abstract

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.

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Selected References

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