Screening And Identification Of Two Lipase Producing Strain And Cloning And Expression Of Lipase Gene

Lipase (Triacylgycerol acylhydrolase, Lipase, EC3.1.1.3) is one of hydrolytic enzymes. It widely lives in microbes, plant seeds, and animal tissues. Furthermore, it can catalyze and hydrolyze the long chain polyglyceryl fatty esters to long chain fatty acids and glycerol. Lipase from microbes is a kind of important industrial enzymes. Because its abundant resource, various types, the wide range of temperature and pH, short production cycle, substrate specificity, high catalytic activity, high transesterification activity and high position selectivity. Therefore, lipase is the hotspot about theoretical and applicable research. Almost all the microbes in the nature have the ability of synthesizing the lipase. But the ability is different. In order to achieve the goal of producing lipase for industry. We must screen the high yield strain that meet the demands of enzymatic characterization. However, in the real situation, the scarcity of good strains has limited the industrial application and theoretical research. Based on it, it is an important and basic job of screening the high activity of producing lipase strain. This paper reported that screening and isolating the producing lipase strains. And the main jobs conclude three points as follows.1. The soil samples that collected from castor-oil plant soil in Wuhan suburb and the bottom soil in Jingsha River,and the screening and isolation of strains were following the steps which are enrichment, initial screening, second screening, and the colorimetric method. This paper has screened ten producing lipase strains, named as HS-L1～HS-L10. The highest enzymatic activity producing lipase strain is HS-L6with the lipase activity5.177U/mL.2. According to the sequence analysis of16S rDNA, the homology between strain HS-L5and Serratia marcescens is up to99%. And the phylogenetic tree was constructed. In addition to morphological characteristics and physiological biochemical test, strain HS-L5is preliminarily identified as Serratia sp., named Serratia sp.HS-L5. Meanwhile, the homology between strain HS-L6and Pseudomonas aeruqionsa is up to100%, the strain HS-L6is preliminarily identified as Pseudomonas sp.3. The lipase gene of HS-L5is cloned and sequenced. It is1842bp long, encoding614amino acids with the molecular weight64.8kDa. The homology compared with lipA of Serratia marcescens SM6is close to98%. The amino acids sequences of Serratia sp.HS-L5contain a lipase consensus sequence-G-X-S-X-G-. The gene of HS-Lip5was cloned into expression plasmid pET-28a and expressed in E.coli. Furthermore, the whole gene sequence of HS-L6is2008bp long, encoding669amino acids with the molecular weight70.8kDa. The gene of HS-Lip6was cloned into expression plasmid pET-28a and expressed in E. coli.