Protein Engineering Of Prolyl Aminopeptidase Using Random Mutagenesis Combined With Semi-rational Design And Preliminary Study Of Protein Crystallization

Prolyl aminopeptidase(PAP;E.C.3.4.11.5)was used as an exopeptidase which can specifically cleave the N-terminal proline residue of proteins.PAP is used in the debittering of hydrolysates and the preparation of bioactive peptides,and can also be used in biomedical detection and diagnosis.In this study,PAP derived from Aspergillus oryzae JN-412(AoPAP)was researched by random mutation,semi-rational design,hot spot of mutation,analysis of enzyme characteristics of B.subtilis expression and protein crystallization.The main research content is as follows:In order to construct a random mutation library with appropriate mutation frequency,error-prone PCR was carried out by adding different concentrations of Mn2+.It was found that 1-3 amino acid mutations in AoPAP gene sequence could be produced when the concentration of Mn2+was 0.05 mmol·L-1.The best mutant 3D9(V147A/K415R)was screened from 3000 variants by high-throughput screening method.Its specific enzyme activity increased from 100 U·mg-1to 127 U·mg-1,which was 27%higher than that of WT.According to the analysis of enzymatic characteristics,the optimum temperature increased from 50? to 55?,and the half-life at 50? was 4.5 h longer than that of WT.Through the analysis of the simulated structure of AoPAP,the non-conservative amino acids Cys-185,Tyr-393,Val-399 and Leu-427,near the substrate binding pocket,were mutated by site-directed saturation,respectively.The screening results showed that Cys-185 was the key site for the hydrolysis of AoPAP.The specific enzyme activities of the optimal mutants C185V,C185A,C185I and Y393W were 156,128,116 and 120 U·mg-1,respectively.The specific enzyme activity of C185V increased by 56%compared with WT,and the kinetic analysis showed that the value of kcat/Km increased from 154.9 s-1·mM-1 to 293.5 s-1·mM-1.According to the analysis of enzymatic characteristics,the optimum temperature of C185V was increased to 55?,the half-life of C185V was prolonged by 1.0 h,and the optimum pH of C185V decreased from 7.5 to 6.5.When pH was 6.5,the half-life of C185V was 2.0 h longer than that of WT.Based on AoPAP molecular modification and enzymatic characteristics,mutation C185V was carried into B.subtilis for expression and enzymatic characterization.The mutant B.subtilis WB600-pMA5-AoPAPC185V was successfully constructed.The total enzyme activity of the mutant was 253 U·mL-1,which was 2.25 times than that of the original strain B.subtilis WB600-pMA5-AoPAPWT in shake flask,and that of the 5 L fermenter was 338 U·mL-1,which was 1.5 times than that of the original strain.According to the analysis of enzymatic properties,the optimum temperature and pH of the mutant were 50? and 6,respectively.The half-life of the mutant at 50? was 8 h,which was 1 hour longer than that of the original strain,and the half-life of the mutant was 8 h,6 h longer than that of the original strain when pH was 6.5.In order to further study the mechanism of substrate catalytic hydrolysis of AoPAP,this study attempted to explore its crystallization conditions.The crude enzyme produced by fermentation was purified by Ni2+affinity chromatography,Superdex G-75 gel column and 30 kDa ultrafiltration.The final protein concentration was 20 mg·mL-1,and the enzyme reached electrophoretic purity,which met the requirements of crystallization.After the initial screening and optimization of the protein crystal,the high quality crystal was obtained under the condition of 0.2 mol·L-1 magnesium chloride,0.1 mol·L-1 HEPES,pH 7.5 and 35%PEG 400.