Expression And Identification Of A Recombiant Carboxypeptidase O From Aspergillus Oryzae Expressed In Pichia Pastoris And Application In Debittering

Aspergillus oryzae is an important fungus in the production of traditional fermentedfoods, such as fermented soybeans, soybean paste and soy sauce. It can secret abundantproteases and amylases. Hydrolysis is an efficient way to improve protein utility efficiencyand produce bioactive peptides, while with the exposure of hydrophobic amino acids duringthe protein hydrolysis, the hydrolysates are usually bitter tasted, which inhibits their utility infood industry. Carboxypeptidases(CPs) can remove hydrophobic amino acids from C terminalof peptides and have debitterring effects in protein hydrolysates. In this paper, we cloned acarboxypeptidase O (OcpO) from Aspergillus oryzae and expressed it in Pichia pastoris.Results showed that the expressed recombinant OcpO(rOcpO) was a glycosylated protein inPichia pastoris with a molecular weight of74KDa. The enzyme yield was20.4nKat/ml.rOcpO was purified by Sephadex G-75and anion-exchanging chromatography. The molecularweight of deglycosylated rOcpO was about56KDa. rOcpO was an acidic protease with theoptimal pH4and was stable in the pH range of3-6; its optimal temperature was about40℃,it retained63%enzyme activity after1h incubation at60℃; PSMF can efficiently inhibit itsactivity, which confirmed it t be a serine-type carboxypeptidase. rOcpO can significantlyimprove the hydrolysis degree of Alcalase hydrolyzed soybean protein and casein, and theirbitterness was greatly reduced.