Molecular cloning and expression of a cold-adapted lipase gene from an Antarctic deep sea psychrotrophic bacterium Pseudomonas sp. 7323.
A psychrotrophic bacterium producing a cold-adapted lipase was isolated from the deep-sea sediment of Prydz Bay, Antarctic and identified as a Pseudomonas strain. Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp. 7323 (lipA) revealed that LipA is composed of 617 amino acid residues with a calculated molecular weight of 64,466 Da. LipA has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine. The lipase purified from the Escherichia coli transformant (rLipA) by metal-chelating chromatography exhibited the same electrophoretic mobility as did the wild-type lipase (wLipA) purified from strain 7323, and both enzymes were quite similar in physicochemical properties. The optimal temperature and pH value for the lipases activity were 30 degrees C and 9.0, respectively. They were unstable at temperatures above 25 degrees C and only retained half of their highest activity after incubation at 60 degrees C for 5 min. These results indicated that the enzymes were typical alkaline cold-adapted enzymes. Both enzymes were particularly activated by Ca2+. Additionally, the enzymes hydrolyzed p-nitrophenyl caprate and tributyrin at the highest velocity among the other p-nitrophenyl esters and triglycerides.
Zhang
Jin-Wei
JW
Institute for Biomedical Research, Xiamen University, Xiamen, People's Republic of China. jinweizhang@21cn.com
Zeng
Run-Ying
RY
D000595:Amino Acid Sequence; D000864:Antarctic Regions; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009792:Oceans and Seas; D010802:Phylogeny; D011549:Pseudomonas / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D003902:Detergents; C079999:LipA protein, Bacteria; D012336:RNA, Ribosomal, 16S