Cloning, expression, and characterization of a cold-adapted lipase gene from an antarctic deep-sea psychrotrophic bacterium, Psychrobacter sp 7195.
A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deep-sea sediment of Prydz Bay and identified as a Psychrobacter species. By screening a genomic DNA library of Psychrobacter sp. 7195, an open reading frame of 954 bp coding for a lipase gene, lipA1, was identified, cloned, and sequenced. The deduced LipA1 consisted of 317 amino acids with a molecular mass of 35,210 kDa. It had one consensus motif, G-N-S-M-G (GXSXG), containing the putative active-site serine, which was conserved in other cold-adapted lipolytic enzymes. The recombinant LipA1 was purified by column chromatography with DEAE Sepharose CL-4B, and Sephadex G-75, and preparative polyacrylamide gel electrophoresis, in sequence. The purified enzyme showed highest activity at 30 degrees C, and was unstable at temperatures higher than 30 degrees C, indicating that it was a typical cold-adapted enzyme. The optimal pH for activity was 9.0, and the enzyme was stable between pH 7.0-10.0 after 24 h incubation at 4 degrees C. The addition of Ca2+ and Mg2+ enhanced the enzyme activity of LipA1, whereas the Cd2, Zn2+, Co2+, Fe3+, Hg2+, Fe2+, Rb2+, and EDTA strongly inhibited the activity. The LipA1 was activated by various detergents, such as Triton X-100, Tween 80, Tween 40, Span 60, Span 40, CHAPS, and SDS, and showed better resistance towards them. Substrate specificity analysis showed that there was a preference for trimyristin and p-nitrophenyl myristate (C14 acyl groups).
Key Laboratory of Marine Biogenetic Resources, State Oceanic Administration, Xiamen, P R China.
Jinwei
J
Zhang
Shu
S
Lin
Runying
R
Zeng
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000864:Antarctic Regions; D003001:Cloning, Molecular; D003080:Cold Temperature; D003300:Copper / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D044096:Psychrobacter / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014871:Water Microbiology
D003902:Detergents; D011994:Recombinant Proteins; D003300:Copper; D008049:Lipase