Cloning, Expression And Characterization Of The Prolyl Endopeptidase From Aspergillus Fumigatus

Prolyl endopeptidase （PEP） is a serine protease that specifically cleaves peptide bonds atthe carboxyl side of proline residues. In this study, PEP cDNA from the A. fumigatus wasobtained by reverse transcription of a total RNA template. The heterologous expression andsecretion of PEP cDNA gene in Pichia pastoris GS115was further achieved throughmolecular biology techniques. The enzymatic properties and proteolytic characteristics of theresulting recombinant enzyme were likewise studied. This report provides the foundation forthe industrial application of recombinant PEP. The following main conclusions were made:The PEP cDNA was successfully cloned from A. fumigatus and expressly secreted in P.pastoris. The highest yield of the recombinant enzyme in shake flasks was0.65U/mL, whichwas106.1times higher than that of the original strain. The highest enzyme activity of therecombinant enzyme reached5.38U/mL in a25L fermentation tank.The step-by-step separation and purification of the recombinant PEP was achieved byammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtrationchromatography. The specific activity of the purified recombinant enzyme was25.17U/mg.The molecular weight of the purified enzyme was approximately63kDa, as verified bySDS-PAGE.The enzymatic properties of the recombinant PEP were analyzed. The recombinant PEPhad an optimal reaction pH of5.5, and its activity was highly stable from pH3.0to9.0. Theenzyme activity did not decrease at pH values ranging from6.0to8.0, even after10days ofexposure at37°C. The optimal reaction temperature of the recombinant PEP was65°C. Theenzyme was highly thermostable and retained more than90%of its enzyme activity, evenafter8h of incubation at55°C. The metal ions tested in this study had no significant effect onthe recombinant enzyme. The Kmvalue of the recombinant PEP with an Ala-Ala-Pro-pNAsubstrate was1.56mmol/L. Its maximum reaction rate (V_max) was7.19μmol/（L·min）. Theisoelectric focusing result showed the pI of the PEP was4.25.The hydrolytic performance of recombinant PEP on two sets of soybean peptides wasstudied. The recombinant enzyme could hydrolyze soybean peptides with a relative molecularweight lower than6500Da. There was a big change in the distribution map after recombinantPEP hydrolysis with soybean peptide substrates prepared using the1398neutral protease and2709alkaline protease respectively. After soybean peptides were hydrolyzed by therecombinant PEP, the total free amino acids in the neutral protease hydrolyzate were increasedby14.30%, whereas an increase of8.93%was observed in that of the alkaline proteasehydrolyzate. Hydrolysis was likewise investigated using pairs of enzymes. The experimentalresults showed that the double hydrolyzed soybean protein completely achieved identicalresults, as compared with hydrolysis using individual enzymes in sequential order.The recombinant enzyme in this study could significantly reduce, or even remove thebitterness of the above-mentioned polypeptide substrates. Almostly no bitter taste was existedin the protein hydrolysates after the recombinant PEP hydrolysis with2709alkaline proteasehydrolyzate as substrate.The PEP was successfully displayed in cell surface of Saccharomyces cerevisiae using yeast surface display technology. There was a significantly decrease of the bitterness in thehydrolysates after the surface displayed PEP with1398neutral protease hydrolyzate assubstrate. Almostly no bitterness was existed in the hydrolysates after the surface displayedPEP hydrolysis with2709alkaline protease hydrolyzate as substrate.