Over-expression Of Prolyl Aminopeptidase In Pichia Pastoris And Its Characterization

Prolyl aminopeptidase(PAP;EC 3.4.11.5),utilized to hydrolyze proteins and debitterize cheese often,is an exopeptidase which is able to catalyze the hydrolysis of the N-terminus proline residue of peptides.Besides,it is used to prepare active peptides and detect the pathogen.In this study,the prolyl aminopeptidase gene(pap)from Aspergillus oryzae was inserted into the plasmid pPIC9 K and introduced into P.pastoris.Expression of PAP was achieved succesessfully.Further,the research included the following aspects: the optimization of fermentation conditions,purification of PAP by one-step,analysis of N-glycosylation,enzymatic properties and the application of recombinant PAP on hydrolyzed glutenin.In this study,the pap gene of A.oryzae JN-814 was used as the research object.To improve the PAP expression level,the pap gene was codon optimized according to the codon usage in P.pastoris.The Codon Adaptation Index(CAI)was increased from 0.65 to 0.85.The GC content and unfavorable peaks were optimized to prolong the half-life of the mRNA.The optimized gene(opt-pap)shared 76.93% nucleotide sequence identity with the native gene.Mut+ and Muts strains were constructed using the optimized proline aminopeptidase gene(opt-pap).Muts and Mut+ clones were screened,and the activity of the intracellular enzyme and extracellular enzyme,expressed by the Mut+ strain containing recombinant optimized pap gene,is 2.1-fold and 1.67-fold higher than that of the native gene.The optimization experiments showed that the highest recombinant PAP production was achieved at pH 6.0 and 1.8% methanol with an induction time of 168 h after an initial cultivation for 18 h,and the intracellular activity of the recombinant PAP reached nearly 61.26 U·m L-1 after codon optimization.The His-tag was added to N-terminal with the PCR technique.The analysis revealed that the N-terminal His-tag showed no effect on PAP expression.The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography.The specific activity of the purified PAP from cell lysate was 166.52 U·mg-1.The final recovery of the enzyme was 86.49% and the purification factor was 13.38.The SDS-PAGE analysis of the purified PAP showed one band.Studies on enzymatic properties of purified PAP showed that the optimal temperature of the purified PAP were 60 °C and 7.5,respectively.Half of the activity remained after incubation at 50 °C for 50 h.The PAP presence of two residues in the active site with pK values of approximately 7.5 and 11.Additionally,the recombinant PAP was recovered at a yield greater than 65% at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h.Furthermore,PAP could be activated by the appropriate salt concentration and the activity of PAP in the presence of 5 mol·L-1 NaCl was 1.07-fold times higher values than that detected in the presence of 0 mol·L-1 NaCl.Using the Pro-pNA as the substrate,the Km and Vmax were estimated to be 0.16 mmol·L-1and 6.25 ?mol·L-1·min-1,respectively.PAP amino acid sequence had three potential N-glycosylation sites.According to the results of MALDI-TOF mass spectrometry analysis and endoglycosidase Endo Hf treatment,it was indicated that PAP was modified by N-glycosylation when expressed in P.pastoris.And the intracellular and extracellular levels of glycosylation were consistented.Circular dichroism spectroscopy indicated that the percentage of ?-sheet in the recombinant aminopeptidase,a protein secondary structure,was increased by 7.8% than that observed in E.coli.Glycosylated PAP showed better thermostability compared with other reported proline aminopeptidases.The remaining activity of glucosylated PAP expressed in P.pastoris after 60 d at 30 °C was 83.73%.Using the wheat glutenin as the substrate,they were hydrolyzed with aminopeptidase and alkaline protease enzyme by single,binary-enzymes or double enzyme hydrolysis methods.Compared with the control group,in the three enzymatic substrate solution,the free amino acids increased significantly,especially the amount of hydrophobic amino acid content of Pro,Leu,Val and Ala in the hydrolyzates of complex enzymes respectively.After double enzyme hydrolysis,the content of small peptides,oligopeptides and polypeptides were significantly increased.Among small peptides,oligopeptides and polypeptides,oligopeptides substance(with molecular weight with 500-180 Da)and small peptide(with molecular weight under 180 Da)content accounted for 60.58% and 19.78%,respectively.The results showed that the recombinant prolyl aminopeptidase had a good compound application characteristics.