Cloning and expression of the polyhydroxyalkanote depolymerase gene from Pseudomonas putida, and characterization of the gene product.
A polyhydroxyalkanote (PHA) depolymerase gene ( pha Z) was cloned by PCR from Pseudomonas putida and over-expressed in Escherichia coli as inclusion bodies. Nucleotide sequence analysis predicted an 852 bp open reading frame encoding a protein of 283 amino acids with a predicted molecular weight of 31283 Da. The deduced amino acid sequence had at least 80% homology to the PHA depolymerase from other Pseudomonas strains and consisted a conserved lipase box-like sequence (G-X-S(102)-X-G). The inclusion bodies were refolded and biochemically characterized. The depolymerase activity was optimal at 40 degrees C and pH 8.
Jiang
Yaqin
Y
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, P.R. China.
Ye
Jiang
J
Wu
Haizhen
H
Zhang
Huizhan
H
D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015202:Protein Engineering / Q000379:methods*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C077226:poly(3-hydroxyalkanoic acid) depolymerase