Effects Of Substitution And Mutation Of C-terminal Loop PE Domain Of Lipase On Its Fatty Acid Site Selectivity And Activity

Penicillium expansum lipase(PEL)has little position selectivity in catalyzing triglycerides.The structure of PEL(PDB:3g7n)was analyzed by Yasara View.It was found that there was a flexible ring protruding(located at the C-terminal of the whole structure)above the substrate binding pocket,which had a hydrophobic amino acid 237V closely related to the enzyme catalytic activity.Through the comparison of the tertiary structure,we found that the C-terminal ring structure of PEL is quite different from the corresponding structure of homologous lipases.Therefore,this domain is likely to cause the difference of properties(such as positional selectivity)between PEL and other lipases.In order to explore the relationship between this domain and the catalytic properties of Penicillium expansum lipase,we systematically explored the C-terminal cyclic domain of PEL by homologous alignment,homologous substitution,saturation mutation,recombinant protein expression,thin layer chromatography,high performance liquid chromatography and molecular docking.The main research contents are as follows:(1)The C-terminal domain of PEL-Loop PE(GMYA),was replaced by homologous peptides of different lengths.The obtained homologous substitution mutants PEL-Loop TL,PEL-Loop AO,PEL-Loop RN,and PEL-Loop RM decreased the catalytic activity of ticaprylin The results of TLC and HPLC analysis showed that mutants PEL-loop TL,PEL-loop RN and PEL-loop RM have no position selectivity;only mutant PEL-loop AO has obvious positional selectivity.(2)The G233NNK/M234NNK and Y235NNK/A236NNK mutant libraries were constructed,and the mutants with different types of hydrolysis loops were screened from the trioctyl glyceride emulsion plate.Three mutants were selected from these mutants:M234Q(semi transparent hydrolysis circle),M234F(fully transparent circle is similar to WT)and Y235T/A236G(fully transparent circle is larger than WT).The k_cat/K_mvalues of mutants M234Q and M234F were lower than WT;the k_cat/K_m values of Y235T/A236G were higher than WT.The results of TLC and HPLC analysis showed that the mutant Y235T/A236G has no position selectivity;M234F and M234Q have different degrees of sn-1,3 position selectivity(the specificity ratio are about 8.5 and 4.4 times that of WT,respectively.).(3)Study on the catalytic activity of mutant lipase for benzyl butyrate.The k_cat/K_mvalues of mutants M234F and Y235T/A236G were higher than WT,respectively.Yasara view analysis showed that maintaining or improving the hydrophobicity of the C-terminal"protruding"flexible domain could improve the affinity of lipase for benzyl butyrate.(4)The results of homologous substitution and saturation mutation showed that the change of the C-terminal cyclic domain of PEL could significantly affect the regioselectivity of the lipase.The analysis results of bioinformatics software such as autodock showed that there may be two main reasons for the position selectivity change:One is because the chemical bonds C1-C2,C2-C3,and C2-O14 form a rigid structure,and C2 is the center of this rigid structure.The sn-2 ester bond is closest to the rigid center of tricaprylin,so the"flexibility"of twisting the single bond on the sn-2 ester bond is lower than sn-1(3).When the sn-1(3)catalytic conformation is formed,since the sn-1(3)ester bond can easily adapt to changes in the structure of the substrate binding pocket,it can continue to form correct carbonyl oxygen orientation and reasonable attack distance.;when the sn-2 catalytic conformation is formed,the low"flexibility"of the sn-2ester bond makes it difficult to adapt to changes in the structure of the substrate binding pocket and adjust to form the correct carbonyl oxygen orientation and reasonable attack distance.In short,due to the difference in"flexibility",the sn-1(3)catalytic conformation is easier to form than the sn-2 catalytic conformation.Second,the space under the"bridge"-like structure in the mutant is significantly smaller than that of WT(the bridge-like structure between 237V and 71F),and the increase in steric hindrance makes the catalytic conformation of sn-2,which is susceptible to the binding space,more difficult formed,so the mutant showed a more obvious sn-1,3 selectivity.