| The microorganiams living in the extreme environment have their own special characters, compared with those of common microorganisms, to survive and adapt in harsh environment. Since 1970s, they become the focus of scientific interests and being the one of the hot research fields. Their habitat, the Antarctic region, has prolific biodiversity, provides unidentified new microbe species which might be the source of producing many bioactive substances such as low-temperature enzyme, antibiotic, polysaccharide, polyunsaturated fatty acid (PUFA), etc. Cold-adapted microorganisms inhabiting in Antarctica can tolerant the drastic temperature fluctuation, this property is possibly maintained by the activity of cold adapted enzymes which keeps the normal metabolism of these microorganism in extreme environment.Cold-adapted enzymes are characteried by higher Kcat and physiological efficiency (Kcat/Km) and by a lower and rather constant Km at temperatures from 0 to 30C. In addition, cold-adapted enzymes display an apparent optimal activity at lower temperatures and manifest pronounced heat lability. These unique properties potentially make them a great economical value as being utilized in a temperature range from 0 to 30C at which homologous mesophilic enzymes are essentially inactive.Our study started from the screening of bacteria demonstrating lipase activity. More than 500 strains were selected from samples collected by CTD during Chinese 18th Antarctic Scientific Expedition in the Southern Ocean, of which 120 strains showed lipolytic activity. Through comparison, 5 strains were selected for their higher lipase activity. Theycan produce lipase only if there existed lipase inducer. Through 16S rDNA sequences analysis of 5 strains, the strain 1-1-10-2 and 9-2 were classified as genus Pseudoalteromonas sp. and genus Psychrobacter sp. respectively. The strain 2-5-10-1, 2-2-2-1 and 1-2-8-1 were determined as genus Moritella sp. Phylogenetic analysis indicated that all of these five strains belong to Bacteria, r-Proteobacteria.To our knowledge, there are little published researches on Antarctica Moritella sp., thus Moritella sp.2-5-10-1 was chosen as the model for the following studies. The highest lipase secretion of this strain is observed at temperature 5C and pH 7-8, which is also for their optimal growth. Tween 80 and olive oil can induce lipase secretion. The optimal temperature and pH for lipase activity are 35C and 7.5C, respectively. The lipase keeps active in the temperature range of 0-20C and still has 37% relative lipase activity at 0C. The enzymatic kinetic parameter Km of the lipase is 3.13X10-5M and activation energy Ea is 20kJ/mol. The lipase shows high thermolability, more than 50% activity lost after incubation at 60 C for 15 min. EDTA has no effect on lipase activity, indicating the lipase activity is independent of divalent cation. In contrast, the lipase activity is inhibited drastically by Cu2+ and Zn2+All these characteristics proved that the lipase secreted by strain 2-5-10-1 is cold-adapted lipase and this enzyme will have great industrial prospect if manipulated by genetic engineering. The chromosomal DNA was extracted from strain 2-5-10-1, partially digested with Sau3AI. The DNA fragments of 2-9kb were isolated and then ligated with pBluescript vector that had been previously digested with BamHI and dephosphorylated with bacterial alkaline phosphatase. The resultant plasmids were introduced into E.coli DH5 a producing a gene library containing 18700 recombinant clones. The clones producing a lipase were detected due to the formation of halos around the colonies on LB agarplates supplemented with ampicillin, IPTG and tributyrin. After incubation at 25 C until clones became visible, the plates were incubated further at 4 C for 4 days. 1 clone showed a clearest halos was selected for DNA sequence analysis. We determined its nucleotide sequence and found an open reading frame comprising 837bp, which encodes a putative protein of 279 amino acids. The nucleotide sequence have a unique c...
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