Cloning, Expression and Characterization of The Cold Active Lipase (Lip3) From Metagenomic DNA of an Antarctic Deep Sea Sediment / 生物化学与生物物理进展

ABSTRACT

The metagenomic DNA was extracted from the deep sea sediment with the depth of 900m of Prydz Bay, Antarctic. A lipase gene (lip3) with the size of 948bp was cloned from the metagenomic DNA by PCR with the primers designed. The deduced Lip3 protein was composed of 315 amino acids (AA) with a molecular mass of 34.577 ku. The motifs GFGNS(GXGXS)and G-N-S-M-G(GXSXG)in the AA sequences of Lip3 were found to be conserved in other lipase. They were most conserved sequence among the serine hydrolase and were necessary for the activity. A 35 ku of Lip3 was purified by Ni-NTA chelating sepharose column from the extract of recombinant E.coli Top 10F′ cell harboring a pLLP-OmpA plasmid inserted with lip3. The purified Lip3 was most active at 25℃ and kept 22% of activity at 0℃. Only 10% of activity was retained after it was incubated at 35℃ for 60 min. The optimal pH value for the Lip3 activity was 8.0. The Km value of the enzyme towards p-nitrophenyl palmitate increased with the increasing of assayed temperature. These results indicated that Lip3 was a typical alkaline cold active enzyme.