Isolation Of Halophilic Lipase-producing Strain, Enzyme Property And Construction Of Genomic Library

Because of their special physiological functions, extremophiles can survive in and adapt to some extreme environments, such as high or low temperature, extremely salty, acidic or alkaline conditions, as well as high pressure etc. They can also, with their unique metabolic pathway formed in special environments, produce many special bioactive substances, such as extremozymes.These extremozymes showed considerable usefulness and great potential in many areas, such as medicine, food, agriculture, chemical engineering and environmental protection.A halophilic archaeon producing halophilic lipase was isolated from Hypersaline lakes of Inner Mongolia. The organism, designated strain Z4, was rod-shaped, Gram-negative.The strain was highly thermotolerant: its temperature optimum was 42℃and growth was possible up to 60℃. The optimal NaCl concentration for the growth was 3.5 mol/L. The results indicated that it was a thermotolerant halophilic archaeon. Through the traditional physiological and biochemical method, together with the 16S rDNA sequence homology and phylogenetic analysis, the strain Z4 was identified as Haloterrigena thermotolerans.The results of fermentation showed that the enzyme production was to follow the growth. The optimal conditions for enzyme production was 50℃,pH 8.0,3.5 mol/L NaCl. Z4 could produce lipase by using various inducers and 1% Olive oil as inducer was the best.This crude lipase was partially characterized using p-NPB. The activity of crude lipase was markedly increased 20~30 % by some metal ions (Ba2+,Fe2+,Cu2+), but was decreased 20~30% obviously by EDTA,SDS and DMSO. Otherwise, the activity of crude enzyme could be completely inhibited by PMSF, indicating the presence of a serine residue in the active site. The activity and heat resistance of enzyme was increased by NaCl, suggesting that the enzyme has strong dependence and specificity on NaCl. The thermostability of the crude enzyme was increased by alcohol and the glycerin is the best one. The optimum conditions for the crude lipase hydrolyzing p-NPB were followed:3.5 mol/L NaCl, 70℃and pH 8.0, and the optimum conditions for the crude lipase hydrolyzing p-NPP were followed: 2.5 mol/L NaCl, 80℃and pH 8.0.These results strongly suggested the presence of a true lipase and therefore, this archaeon deserves further attention, since true lipases produced by Haloterrigena thermotolerans have not previously been reported in the archaeon.we also construced the genomic DNA library for further study.