Substitution and mutation of the C-terminal loop domain of Penicillium expansum lipase significantly changed its regioselectivity and activity

In order to explore the effect of the C-terminal loop domain of Penicillium expansum lipase (PEL) on its regioselectivity and activity, homologous substitution and site-directed saturation mutagenesis were used. Two mutants PEL-loopAO and M234F were selected to catalyze the hydrolysis of tricaprylin and the molar ratios of 1,2dicaprylin to 1,3-dicaprylin in the products were 25.57 and 8.57 respectively, showing obvious sn-1,3 regioselectivity of the mutants. Bioinformatics analysis showed that the structures of the substrate binding pocket of the mutants changed significantly. Moreover, due to the different positions of sn-1(3) and sn-2 ester bonds in the substrate molecule, it is easier for the substrate molecule to adapt to the significant changes in the substrate binding pocket and form the correct sn-1(3) catalytic conformation instead of sn-2 catalytic conformation, which makes the mutants exhibit apparent sn-1,3 position selectivity. In addition, the decrease of hydrophobicity of Cterminal loop domain will cause the decrease of lipase activity, whereas maintaining or improving the hydrophobicity can maintain or improve the activity. These findings are of great value in elucidating the relationship between the C-terminal loop domain of the lipase and its regioselectivity and activity, and also provide some reliable clues for obtaining lipases with better regioselectivity.

Automated summary

This study investigates the effect of the C-terminal loop domain of Penicillium expansum lipase (PEL) on its regioselectivity and activity using homologous substitution and site-directed saturation mutagenesis. The mutants exhibited obvious sn-1,3 regioselectivity and significant changes in the substrate binding pocket. Additionally, changes in hydrophobicity affected the lipase activity.