Heterogeneous Expression And Directed Evolution Of Lipase Gene From Geotrichum Candidum 42

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are hydrolytic enzymes that catalyze the hydrolysis, synthesis and transesterification of a variety of acylglycerols at the interface of lipid and water. There are so many advantages of lipases, such as mild reaction conditions, less by-products, and less environmental pollution. Therefore, lipases have been widely applied to food processing, oil chemicals, organic synthesis and separation, bio-energy, environmental management, medicine, paper, and washing industry, etc. In this study, Cloning of promoter, secretory expression and display expression of the G. candidum lipase gene (lip42), and directed evolution of the G. candidum lipase (GGL) were all investigated. The main results are as follows:1 The 5' flanking sequence of lip42 with 910bp was isolated using adaptor PCR. Analysis of result shows this sequence has the typical structural features of promoter of eukaryotic genes.2 Prokaryotic expression vector pET26-lip42 harboring lip42 with mature peptide sequence was constructed. However, there is no lipase activity was detected in recombinant Escherichia coli BL21. Some short peptides were observed by SDS-PAGE. This phenomenon may be explained as the early termination of translation due to the existing of rare codon in lip42.3 The selective medium for yeast to produce lipase was improved. The recombinant yeast could not only produce a clear and transparent hydrolysis cycle, but also grow normally on this medium. It provides an ideal functional screening medium for the study of recombinant yeast producing lipase, in particular, for screening of target strains in directed evolution of lipase.4 The recombinant expression vector pHBM354-lip42 was constructed, and realized the correct expression in S. cerevisiae INVScâ… . Lipase activity was measured by alkali titration method, and the lipase activity of the initial fermentation liquid was 0.3U/mL, the specific activity of the lipase solution was 130.58U/mg.5 We constructed both the display expression vector pAMB121-lip42 harboring the leader peptide sequence of lip42 and the display vector pPIC9K-Flo1-lip42 harboring the mature peptide sequence of lip42 respectively. Both vectors were transformed into P. pastoris GS115 and the display expressions of the genes were achieved.6 A set of technique for directed evolution of lipase were established, which are composed of a construction method of mutant lipase library by in vivo recombination technology and screen method by a functional selective medium. Fortunately, 13 mutants with higher lipase activity were selected. One of them, ep3 with the specific activity of 514.98U/mg, is nearly four times to the activity of wild-type lipase.In conclusion, three host strains, E. coli BL21, S. cerevisiae INVScâ… , P. pastoris GS115 were used for expression of lipase gene of G. candidum. The GGL was secreted in INVScâ… and was display on the cell surface of GS115. In addition, a directed evolution technology for lipase was established in this study. 13 mutants with higher lipase activity were obtained, and the specific activity of the mutant ep3 was increased nearly three times.