Rebuilding The Oxyanion Hole Of Penicillium Expansum Lipase

In order to explore the effect of the oxyanion hole of Penicillium expansum lipase(PEL)on the catalytic activity,homologous replacement and site saturation mutagenesis technology were used to modify the oxyanion hole of PEL,then the catalytic activity of the mutant lipase was analyzed.The main contents include the following two aspects:1.homologous replacement of the oxyanion hole and researching the properties of mutant lipaseBy means of sequence alignment of more than ten kinds of homologous microbial lipase,we determine to homologously replace 131 and 133 sites,which includes a total of 5 kinds of plan.Escherichia coli expression system was used to express the lipase mutants and the catalytic activity of the mutants was measured.The results indicated that the catalytic activity of the mutants to the glyceryl tributyrate was decreased,which is consistent with wild-type.However,the L133 A and H131 E/L133 A exhibited higher catalytic activity to the R-mandelic acid ethyl ester,and showed an increased enantioselectivity compared with the wild-type lipase,.2.saturation mutagenesis of the oxyanion hole sites and the enzymatic property analysis.The 131 and 133 sites of PEL was mutated by saturation mutagenesis technology and two libraries were constructed,which were expressed in Escherichia coli expression system.According to the result of the last step,we selected mandelic acid methyl ester as the substrate for screening of mutant library.Finally,we got six mutants whose catalytic activity is higher than that of the wild type.They are H 131 Y?H 131 N?L 133 M?L 133 A?H 131 L/L 133 V and H 131 I/L 133 N.The result shows that the change of oxyanion hole sites,can change the substrate specificity of the lipase.