Expression And Characterization Of Streptomyces Canus Aminopeptidase And Its Application In Preparation Of Rice Peptides

Rice protein is a by-product of rice processing,and its low solubility and high denaturation limit the development of rice protein resources.In addition to retaining the original excellent properties of rice protein,rice peptide also has the functions of regulating human immunity,alleviating aging,improving the homeostasis of intestinal flora,and is not easy to cause overnutrition,so it is recognized as a high-quality food peptide.At present,high-quality rice peptides are mainly prepared by the synergistic hydrolysis of rice protein by protease and aminopeptidase,that is,protease hydrolyzes rice protein to release peptide,and then aminopeptidase removes the hydrophobic amino acids that cause bitterness at the N-terminal of the peptide.At present,the research on rice peptidase preparation method is mainly focusing on proteases,but there are few reports on debittering aminopeptidase.So,the debittering effect of existing aminopeptidase on rice peptides is not significant.Therefore,it is urgent to obtain aminopeptidase with significant debittering effect of rice peptides,and to investigate its role compounding with protease in the preparation of rice peptides.In this study,aminopeptidase ScAP,was obtained from natural strain T20,with assayed high aminopeptidase activity during fermentation.It was heterogeneously expressed in food-grade host strain Bacillus subtilis for characterization and structure analysis of enzymatic property,and the debittering effect of ScAP on rice peptides was investigated.Then ScAP and trypsin were compounded to hydrolyze rice protein,and the preparation conditions were optimized to obtain high quality rice peptides.The main research results are as follows:(1)Strain T20,which belongs to Streptomyces canus,was induced by taking different proteins/peptides as the only nutrient source to produce aminopeptidase,among different proteins/peptides,rice protein has the best induction effect.The extracellular aminopeptidase activity reached the highest as S.canus T20 induced by 2%rice protein for 4 d,which was up to310.90 U·m L^-1.Peptides fingerprint analysis showed that the potential aminopeptidase isolating from fermentation broth had the highest similarity with S.fulvoviolaceus-derived aminopeptidase(WP?043454179.1),and was named ScAP.(2)The target gene scap,was amplified by PCR from the genome of S.canus T20 and recombinant expressed in B.subtilis.The intracellular ScAP activity reached 271.03 U·m L^-1 after fermentation of recombinant strain for 24 h.The specific activity of purified ScAP was up to 6216.67 U·mg^-1,which was the highest level of aminopeptidase from Streptomyces reported in the literature.The optimum temperature of ScAP is 60?,the optimum p H is 8.0.the half-life of ScAP is 285 h at 50?and 48 h at p H 9.0.0.1 mmol·L^-1 Ca²⁺and Co²⁺could increase ScAP activity to115.44%and 104.03%,respectively,while 0.1 mmol·L^-1 EDTA could completely inactivate ScAP.After removing EDTA-Zn²⁺chelates,re-adding Co²⁺,Zn²⁺and Mn²could partially restore ScAP activity.ScAP showed a high hydrolytic preference for N-terminal hydrophobic amino acids such as Leu,Met and Phe,and the structural analysis of ScAP indicated its substrate binding region showed higher hydrophobicity.(3)The debittering effect of ScAP was evaluated by taking rice peptide as substrate.When the rice peptide was treated with ScAP(final concentration 0.48 mg·m L^-1)for 2 h,the bitterness decreased by 50.68%,and the content of free hydrophobic amino acids increased by 80 times,indicating that ScAP can effectively remove the N-terminal hydrophobic amino acids of rice peptides,and results in a decrease in bitterness of the peptides.Then,ScAP and trypsin were compounded to prepare rice peptides.After optimization,the optimum preparation condition are as follows:Enzymolysis of rice protein by trypsin and ScAP at 1:5 and 3.33%(w·w^-1)at 50?for 4 h through one-step hydrolysis.Compared with trypsin hydrolysis alone,in regards to product of compounding of two enzymes,the content of small molecular peptide(180-1000 Da)increased by145.00%,reached 4.41 mg·m L^-1,and the bitterness decreased by 15.76%.Then,the biological activity of the prepared rice peptides was evaluated,and it was found that its free radical scavenging ability,reducing ability and ACE inhibitory activity were overall better than commercial rice peptide or commercial aminopeptidase participating in rice peptide preparation.