Cloning, expression and characterization of a lipase gene (lip3) from Pseudomonas aeruginosa LST-03.
A lipase gene (lip3) was cloned from the Pseudomonas aeruginosa strain LST-03 (which tolerates organic solvents) and expressed in Escherichia coli. The cloned sequence includes an ORF consisting of 945 nucleotides, encoding a protein of 315 amino acids (Lip3 lipase, 34.8 kDa). The predicted Lip3 lipase belongs to the class of serine hydrolases; the catalytic triad consists of the residues Ser-137, Asp-258, and His-286. The gene cloned in the present study does not encode the LST-03 lipase, a previously isolated solvent-stable lipase secreted by P. aeruginosa LST-03, because the N-terminal amino acid sequence of the Lip3 lipase differs from that of the LST-03 lipase. Although the effects of pH on the activity and stability of the Lip3 lipase, and the temperature optimum of the enzyme, were similar to those of the LST-03 lipase, the relative activity of the Lip3 lipase at lower temperatures (0-35 degrees C) was higher than that of the LST-03 lipase. In the absence of organic solvents, the half-life of the Lip3 lipase was similar to that of the LST-03 lipase. However, in the presence of most of the organic solvents tested in this study (the exceptions were ethylene glycol and glycerol), the stability of the Lip3 lipase was lower than that of the LST-03 lipase.
Ogino
H
H
Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, 599-8531 Sakai, Osaka, Japan. ogino@chemeng.osakafu-u.ac.jp
Hiroshima
S
S
Hirose
S
S
Yasuda
M
M
Ishimi
K
K
Ishikawa
H
H
D000595:Amino Acid Sequence; D002847:Chromatography, Agarose; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics* / Q000302:isolation & purification*; D008066:Lipolysis / Q000235:genetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature
D017931:DNA Primers; D008049:Lipase