Purification, molecular cloning, and expression of lipase from Pseudomonas aeruginosa.
An extracellular lipase secreted by Pseudomonas aeruginosa TE3285 was purified. A genomic library of this strain was constructed in lambda EMBL3, and a DNA fragment 2.7 kb long containing the lipase gene, lipA, was isolated with an oligonucleotide probe synthesized on the basis of the partial amino acid sequence of a purified preparation of the enzyme. Nucleotide sequence analysis showed an open reading frame of 933 bases, and the deduced amino acid sequence agreed well with the molecular mass and partial amino acid sequences of mature lipase. The results of alignment of the amino acid sequences of five lipases from Pseudomonas species considered together with the published crystal structure studied with human pancreatic lipase showed that Ser82, His251, and Asp209 were catalytic residues and that a surface loop from residues 172 to 204 was responsible for the substrate specificity. About 50 bases downstream of lipA, there was another gene, lipB. The sequence of lipB was highly homologous to that of putative modulators of the production of active lipases in other Pseudomonas species. Expression plasmids encoding lipA followed by the complete or incomplete lipB gene downstream of the lac promoter of pUC18 were constructed. lipA was expressed in Escherichia coli 1100 only in the presence of the complete lipB gene.
Chihara-Siomi
M
M
Institute for Chemical Research, Kyoto University, Japan.
Yoshikawa
K
K
Oshima-Hirayama
N
N
Yamamoto
K
K
Sogabe
Y
Y
Nakatani
T
T
Nishioka
T
T
Oda
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D004269:DNA, Bacterial / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010179:Pancreas / Q000201:enzymology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D003062:Codon; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D008049:Lipase