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Gene Cloning, Expression and Characterization of a Cold-adapted Lipase from a Psychrophilic Deep-sea Bacterium

Author: ChenRuiPeng
Tutor: GuoLiZhong；DangHongYue
School: Qingdao Agricultural University
Course: Microbiology
Keywords: cold-adapted lipase genomic DNA library recombinant lipase Psychrobacter deep-sea sediment
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 56
Quote: 0
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Abstract


Lipases (triacylglycerol acylhydrolases, E.C.3.1.1.3) can catalyze the hydrolysis of ester bonds of triacylglycerols at the interface between an insoluble substrate and water. This feature distinguishes lipases from esterases, which act only in water-soluble substrates. Cold-adapted lipases from psychrophilic bacteria have received increased attention for the fact that they raise considerable interest in both basic and applied research. These enzymes are characterized by higher kcat and physiological efficiency (kcat/km) and by a lower and rather constant km at temperatures close to 0℃. In addition, cold-adapted lipases display an apparent optimal activity shifted toward lower temperature and manifest pronounced heat lability.A psychrophilic bacterium Psychrobacter sp. C18 previously isolated from the Southern Okinawa Trough deep-sea sediments showed extracellular lipolytic activity towards tributyrin. A genomic DNA library was constructed and screened to obtain the corresponding lipase gene. The sequenced DNA fragment contains an open reading frame of 945 bp, which was denoted as the lipX gene, from which a protein sequence was deduced of 315 amino acid residues with a molecular mass of 35,028 Da. This protein LipX contained the bacterial lipase GNSMG (GxSxG, x represents any amino acid residue) and HG consensus motifs. The recombinant pET-28a(+)/lipXHis gene was overexpressed in heterologous host Escherichia coli BL21 (DE3) cells to overproduce the lipase protein LipXHis with a 6×histidine tag at its C-terminus. Nickel affinity chromatography was used for purification of the expressed recombinant lipase. The maximum lipolytic activity of the purified recombinant lipase was obtained at temperature of 30℃and pH 8.0 withρ-nitrophenyl myristate (C14) as a substrate. Thermostability assay indicated that the recombinant LipXHis is a cold-adapted lipase, which was active in 10% methanol, ethanol, acetone and 30% glycol, and inhibited partially by Zn2+, Co2+, Mn2+, Fe3+ and EDTA. Additionally, most non-ionic detergents, such as’DMSO, Triton X-100, Tween 60 and Tween 80 enhanced the lipase activity but 1% SDS completely inhibited the enzyme activity. Theρ-nitrophenyl myristate was the most suitable substrate for the recombinant LipXHis lipase.

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