Cloning of an intracellular Poly[D(-)-3-Hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product.
An intracellular poly[D(-)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(-)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.
Saegusa
H
H
Research Institute of Innovative Technology for the Earth Branch in Kanagawa University, Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan.
Shiraki
M
M
Kanai
C
C
Saito
T
T
D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006885:Hydroxybutyrates / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D006885:Hydroxybutyrates; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase