Molecular cloning of the carboxylesterase gene and biochemical characterization of the encoded protein from Pseudomonas citronellolis ATCC 13674.
A genomic library of Pseudomonas citronellolis ATCC 13674 was constructed and screened for esterase activity in Escherichia coli using tributyrin-containing medium. One positive transformant was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.1-kb DNA fragment potentially carrying an esterase gene. The deduced nucleotide sequence of the DNA was found to contain an open reading frame encoding carboxylesterase and designated estA. Amino acid sequence analysis of estA showed the serine conservative motif, GDSAG, located between residues 208 and 212. Together with Ser, residues 310 and 334 corresponding to aspartic acid and histidine, respectively, comprised the catalytic triad. With the aid of immobilized metal ion affinity chromatography, the carboxylesterase fused with poly His at its C-terminus was purified and shown to be strongly inhibited by the tryptophan modifier and mercuric ion, indicating the important role of conservative Trp (189) and cysteine (152 and/or 183) residues in maintaining the structural integrity of the protein. Further analyses showed that the carboxylesterase functioned optimally at 37-40 degrees C with pH ranging between 8 and 9 and displayed a broad substrate spectrum. The protein exhibited greater preference toward short-chain (C2-C4) than medium- and long-chain fatty acids. Higher substrate specificity on para-nitrophenol butyrate was observed in comparison with para-nitrophenol acetate as indicated by the higher kcat/Km value of the former.
D000595:Amino Acid Sequence; D001483:Base Sequence; D043182:Carboxylesterase / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015698:Genomic Library; D008969:Molecular Sequence Data; D017510:Protein Folding; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature
D043182:Carboxylesterase