PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and kcat/Km. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze β-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.
Keywords: ACE inhibitory activity; X-prolyl-dipeptidyl aminopeptidase; catalytic mechanism; pH robustness; semirational design.