Improving The Activity And Thermal Stability Of Thermomyces Lanuginosus Lipase By Rational Design

Thermomyces lanuginosus lipase has been widely used in feed,oil and fat processing,biodiesel and other fields due to its good temperature resistance.On the other hand,the expansion of the application field puts forward higher requirements on the characteristics and economy of the lipase.In order to make lipase can be better applied to the corresponding field,through rational design of T.lanuginosus lipase,we obtained the varieties of lipase with high enzyme activity and high temperature resistance,which laid a foundation for the application of lipase in.In order to obtain lipase strains with high enzyme activity and high heat resistance,we analyzed the phylogenetic relationships between the lid and loop regions of the typical lipase domain of T.lanuginosus and found and selected the appropriate mutation sites.By mutating Gly at position 91 to Ala and Leu at position 227 to Gly,the structures of the lid region and loop region of lipase were optimized,respectively,to improve enzyme activity and temperature stability.The Pichia pastoris expression system of Pichia pastoris was constructed and a lipase recombinant PAO815-tll2 a with significantly improved lipase activity and high temperature resistance was obtained.Compared with the enzyme activity of wild-type T.lanuginosus lipase,the enzyme activity was increased by 251.8% after induction expression.At the same time,its high temperature stability was significantly improved,and 78.94% of the enzyme activity was still retained after 12 h at 80 ?.The designed lipase gene was used to construct a multi-copy vector,and the copy number of P.pastoris lipase gene was determined by fluorescent quantitative PCR.The evaluation of enzyme production ability in a 50-L fermentor was optimized and completed.The lipase recombinant was fermented and induced in the 50-L fermentor for 168 h,and the enzyme activity of the supernatant reached 29,000 U/m L.Through rational design of lipase TLL,a novel high activity and high temperature resistant lipase variety TLL2 A was successfully designed and obtained,and high-efficiency expression was achieved,which laid a foundation for its industrialization and industrial application.