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KMID : 0545120070170040604
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 4 p.604 ~ p.610
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Cloning, Expression, and Characterization of a Cold-Adapted Lipase Gene from an Antarctic Deep-Sea Psychrotrophic Bacterium, Psychrobacter sp.7195
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Zhang JinWei
Lin Shu
Zeng Runying
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Abstract
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A psychrotrophic strain 7195 showing extracellular lipolytic activity towards tributyrin was isolated from deepsea 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 30oC, and was unstable at temperatures higher than 30oC, 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 4oC. 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).
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KEYWORD
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Deep-sea sediment, cold-adapted lipase, Psychrobacter, recombinant protein
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