Purification, gene cloning, amino acid sequence analysis, and expression of an extracellular lipase from an Aeromonas hydrophila human isolate.
A structural gene which codes for an extracellular lipase (EC 3.1.1.3) in Aeromonas hydrophila H3, which was isolated from a female hospitalized patient, was cloned in Escherichia coli by using pBR322 as a vector. Lipase purified from both A. hydrophila culture supernatant and the periplasmic fluids of E. coli containing the lip determinant in the original clone (plasmid pLA2) showed an M(r) of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the M(r) determined by Sephacryl S-200 chromatography. Regarding substrate specificity, the optimum chain lengths for the acyl moiety were C6 for ester hydrolysis and C6 and C8 for triacylglycerol hydrolysis. Sequence analysis showed a major open reading frame of 2,052 bp, which predicts a polypeptide with an M(r) of 71,804. The polypeptide was found to contain an amino acid sequence (V-H-F-L-G-H-S-L-G-A) which is highly preserved among lipases.
Anguita
J
J
Departamento de Patología Animal (Sanidad Animal), Facultad de Veterinaria, Universidad de León, Spain.
Rodríguez Aparicio
L B
LB
Naharro
G
G
D016980:Aeromonas hydrophila / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006801:Humans; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D008049:Lipase