Properties of an intracellular poly(3-hydroxybutyrate) depolymerase (PhaZ1) from Rhodobacter spheroides.
The gene of an intracellular poly(3-hydroxybutyrate) (iPHB) depolymerase from Rhodobacter sphaeroides was cloned and sequenced. The nucleotide sequence of the cloned gene was homologous to that of the iPHB depolymerase gene from Ralstonia eutropha H16 (phaZ1(Reu)) and the gene was designated phaZ1(Rsh). PhaZ1(Rsh) was purified from E. coli harboring an expression vector containing phaZ1(Rsh) and its properties were examined. PhaZ1(Rsh) degraded amorphous PHB granules, and the 3-hydroxybutyrate tetramer and pentamer, but not crystalline PHB granules. The enzyme activity was inhibited by p-chloromercuribenzoate and Triton X-100. Diisopropylfluorophosphate, phenylmethylsulfonylfluoride, and dithiothreitol had no effect on the activity. A mutant having alanine instead of cysteine at 178 lost the activity. These results show that PhaZ1(Rsh) is a quite similar enzyme to PhaZ1(Reu).
Kobayashi
Teruyuki
T
Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan.
Nishikori
Kenji
K
Saito
Terumi
T
D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase; D003545:Cysteine