[Screening of strain producing an novel esterase with high enantioselectivity and molecular cloning of the enzyme gene].
A novel strain producing an enantioselective lipolytic enzyme was isolated from soil samples, and identified as Pseudomonas putida NH33. A genomic library of P. putida NH33 was constructed and screened for esterase activity in E. coli. One positive clone was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.7kb DNA fragment carrying esterase gene. The nucleotide sequence of the DNA was found to contain an open reading frame of 1142 nucleotides encoding esterase of 381 amino acid residues and designated PPEst. The primary structure of the esterase exhibited 35%-40% homology to those of related enzymes from various sources and 80%-90% homology to esterases from the genus Pseudomonas. Amino acid sequence deduced from the nucleotide sequence contains of the consensus active site sequence, GXSXG, of serine esterase. The PPEst fragments were cloned into the expression vector pET-22b( + ) and transformed into E. coli BL21 (DE3), and the recombinant protein fused with 6 x His at its C-terminus was purified to homogeneity by a single immobilized metal ion affinity chromatographic step. The molecular mass of the esterase was determined to be approximately 42kDa by SDS-PAGE. The purified enzyme could convert ethyl esters of 2-arylpropanoic acid to S-isomer of 2-arylpropanoic acids with an optical purity of > 99%. The result suggests that this esterase is excellent biocatalyst for synthesis of chiral pharmaceuticals. The enzyme is an novel esterase, and its nucleotide sequence has been submitted to GenBank under accession number AY896293.
Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China. sxzlb@263.net
Shao-xin
SX
Chen
Bing-zhao
BZ
Shi
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007536:Isomerism; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D012988:Soil Microbiology
D001426:Bacterial Proteins; D004950:Esterases