Expression Optimization And Application Of Carboxypeptidase M32

Plant proteins are a high quality protein resource comparable to animal proteins,with high nutritional value and many physiological functions.However,they tend to produce a bitter taste during hydrolysis,which to some extent limits their application in the food industry.Carboxypeptidase is an exonuclease that acts at the C-terminus of peptide chains or proteins and gradually hydrolyses them to release free amino acids,and is of interest for its ability to reduce the bitterness of plant protein hydrolysates.In this study,we aimed to obtain a new carboxypeptidase and applied it to the preparation of plant oligopeptides,in order to reduce bitterness and enhance its functional properties,and provide a promising carboxypeptidase for food processing.（1）Based on the genomes of Bacillus subtilis、Bacillus amyloliquefaciens、Bacillus megatherium、Bacillus pumilus and Bacillus cereus in the NCBI database,several suspected carboxypeptidase genes were mined and 20 gene fragments were obtained by PCR cloning.The E.coli and B.subtilis expression systems were used for expression screening.The B.subtilis WB600/p MA5-cpm32 expressed carboxypeptidase M32 with the highest carboxypeptidase activity of 1520 U·m L^-1,as determined by SDS-PAGE electrophoresis and enzyme activity.The recombinant carboxypeptidase M32 had a distinctive sequence of the peptidase M32 family,"His-Glu-X-X-His",and the active centre contained a Zn binding site linking His and Glu.The sequence similarity between this enzyme and Priestia veravalensis was the highest,at 79.3%.（2）To improve the production of extracellular carboxypeptidase M32,three methods were used:fusion signal peptide,overexpression of transfer kinetic protein Sec A and optimization of medium and fermentation conditions,respectively.The results showed that the fusion signal peptide Amy Q increased the extracellular enzyme activity from 1520 U·m L^-1 to 4233 U·m L^-1.While after the optimization of medium and fermentation conditions,the final enzyme activity of carboxypeptidase M32 reached 5320 U·m L^-1,which was 25.3%higher than that before the optimized expression.The metal ions Co²⁺and Zn²⁺could increase the enzymatic activity by600.9%and 334.6%respectively.The substrate specificity study showed that carboxypeptidase M32 could cleave most of the amino acid residued at the C-terminus except Pro and was a broad-spectrum carboxypeptidase.The kinetic study showed that Z-Phe-Tyr was the best substrate,and the K_m value for carboxypeptidase M32 was 0.6 mmol·L^-1 and the k_cat/K_m value was 82.2 L·mmol^-1·s^-1.（3）Low bitterness soybean oligopeptide and pea oligopeptide were prepared and functionally characterized by using carboxypeptidase M32 in combination with alkaline protease.Compared with the oligopeptides prepared by alkaline protease,the solubility and hydrolysis of soybean oligopeptides increased by 18.6%and 22.4%,respectively,and that of pea oligopeptides increased by 37.4%and 33.4%,respectively.The content of short peptides with molecular weight less than 1 k Da in both soybean and pea oligopeptides could reach more than 80%.The free amino acid analysis showed that carboxypeptidase M32 had a preference for bitter amino acids Met,Leu,Val,His and Arg,indicating the removal of these C-terminal bitter amino acids was beneficial to reduce the average hydrophobicity of bitter peptides and bitterness.Electron tongue assay showed that the bitterness values of soybean and pea oligopeptides were reduced by 47.1%and 22.8%,respectively,which achieved a good effect of bitterness removal.The antioxidant activity test showed that the DPPH radical scavenging rate of soybean oligopeptide and pea oligopeptide increased by 65.1%and 23.9%respectively,and the hydroxyl radical scavenging rate increased by 32.4%and 25.1%,respectively.Therefore,the dual enzymatic method combined with carboxypeptidase M32 can significantly reduce the bitterness of plant oligopeptides and improve their functional properties,indicating the promising application of carboxypeptidase M32 in the preparation of plant bioactive peptides.