Screening, Cloning And Expression Of The Cold Active Lipase From Microorganism Of An Antarctic Deep Sea Sediment

A strain Lip001,which produces a lipase,was isolated from the Antarctic Ocean sediment samples.The cells of the strain Lip001 were neutrophilic,Gram-negative,separate motile rods,with a size of 0.5-0.8μmx1-1.5μm。Growth was observed between 5 and 30℃(optimum temperature:10-20℃),between pH 4 and 10(optimum:pH6-8),and between NaC1 concentrations of 0 and 8%(w/v)(optimum NaC1 concentration:1-4%).The strain Lip001 could utilize various single carbon sources and produce lipase,the optimum condition is 24 hours under 20℃and pH6-8.The purification was performed by ammonium sulfate fractionation,column chromatography with Q Sepharose XL and Sephadex G-75,a purified enzyme,with a molecular mass of 55kDa was obtained.The optimum condition for the lipase activity was 35℃and pH 9-10,which suggested the enzyme was a typical alkaline cold-active lipase,with sensibility to high temperature.This enzyme sensitivity to Urea and SDS.From the result that EDTA showed obvious inhibiting effect on the enzyme,it can be suggested that metal ions played an important role in the conservation of enzyme conformation for the catalytic activity.The lipase activity was stimulated by Ca²⁺,Mg²⁺,Cu²⁺, and was inhibited by Cd²⁺,Co²⁺ and Zn²⁺.Using the Fosmid vector,we have constructed the labrary of Lip001 DNA,picked up a positive clone to sequence and obtain a section of the gene encoding carboxylesterase.It's a menber of the lipase superfamily by amino acid sequence analysis.A strain Lip004,which produces a lipase,was isolated from the Antarctic Ocean sediment samples.We also obtained the lipase gene(lipA)through PCR cloning with metagenome DNA and the method of genome walking.Then the gene was expressed in E. coli and purification of-lipase has been done.The results and technques will contribute to the futher research in the abundant cold-adaptive enzymes from the Antarctic.The gene diversities of dissimilatory sulfite reductase(DSR)and methyl-coenzyme M reductase MCR in metagenomic DNA of deep sediment of the west Pacific "warm pool" were studied by PCR-RFLP using primers specific for dsrAB and mcrA,respectively.The results indicated that the dsrAB were come from 6 genus ofδ-Proteobacteria,mainly Desulfovibrio and Desulfobacter.All of the mcrA were come from archaeal methanogens,mainly Methanomicrobia.These genes were tend to clustered within independent clades on phylogenetic tree respectively.Furthermore,many dsrAB and mcrA genes showed high similarity to those from unknown or new species.These results indicated that the microbial metabolisms,which may include some new type of metabolic pathway,were active in the S and CH₄ cycles in deep sediment of "warm pool".