Molecular Modification Of Prolyl Endopeptidase

The specific proteases are highly valuable for the processing of protein food products.Prolyl endopeptidases?PEPs?are important proteases which hydrolyze protein to C-terminal bioactive peptides and for the treatment of celiac disease.However,PEPs still have the potential to improve the catalytic efficiency.At present,there are few studies to modify the catalytic efficiency of prolyl endopeptidase through theoretical calculations,modifying this enzyme could further strengthen their industrial and therapeutic applications.In this work,Sphaerobacter thermophiles PEP with enzymatic property was heterologous expression and purification.And the enzymatic properties were investigated.The optimum pH of prolyl endopeptidase was 6.6,and the optimum temperature was 63°C.The specific enzyme activity for SEKTTMPLW was 11.99 U/mg.The K_M value of the kinetic parameters was 7.48 mM,the k_catat value was 164.47 min^-1,and the k_cat/K_M value was22 min^-1·mM^-1.A novel rational design method based on perfect conformation was adopted.Based on the catalytic mechanism of PEPs,the distance between O of Ser and C of Pro,was recognized as the criterion of near-attack conformation to increase catalytic activity of PEP.The stable structure of the enzyme-substrate complex was obtained through methods such as homology modeling,molecular docking,and MD simulation.The near-attack conformation was promptly obtained by constrained molecular dynamics?MD?simulation.The total binding free energy of 575 single-point mutants was obtained by energy minimization.Then,nine site-directed mutations were virtually screened,and the mutants were constructed and expressed.Finally,the experimental results showed that compared with the WT enzyme,the k_cat/K_M values of both mutants?I462W and Q560Y?were increased by 32.7%and 6.3%,respectively.That proved our rational design method effectively improves the catalytic activity of prolyl endopeptidase.