Enzyme Synthesis Of N-Acetyl-Amino Acid And N-Acetyl-Dipeptide,Appliation And Umami-Enhancing Mechanism Evaluation

In food,the preparation and application of kokumi compounds is an important research topic in the field of food flavor.As a new type of kokumi compound,N-acetyl amino acid not only has a strong effect on improving the taste of food,but also has various physiological activities,such as antioxidant activity,anti-inflammatory activity,etc.which could be condisered as an green,healthy and safe kokumi compounds.In this paper,the chemical and enzymatic preparation of N-acetyl amino acids were optimized respectively,the law of enzymatic synthesis of N-acetylated dipeptide was studied,and the enzymatic preparation of N-acetyl amino acid and N-acetyl dipeptide was applied to soy sauce system.In this paper,the effect of this method on the taste of soy sauce was evaluated,and finally the taste characteristics and freshness-enhancing mechanism of N-acetyl amino acid and N-fatty phenyalanine were discussed.The specific research conclusions are as follows:1.The process of preparing N-acetylamino acids by heating by using acetic acid and amino acid as substrate was optimized.The results show that the heating method can efficiently prepare N-acetylalanine,N-acetylvaline,N-acetylleucine,N-acetylisoleucine,N-acetylmethionine and N-acetylmethionine and N-acetylphenylalanine（high synthetic yield and few by-products）.among 18 amino acids.Using 0.030 mol amino acid and 0.400 mol acetic acid as substrates for 12 h at 130 ^oC,the synthetic yields of the six N-acetyl amino acids were 41.21%,82.52%,53.56%,59.82%,99.61%and 45.13%,respectively.After extraction with ethyl acetate,the purity of the above-mentioned six N-acetyl amino acids was all above 90%.The electronic tongue evaluation found that the above-mentioned N-acetyl amino acids could increase the salty and umami taste of soy sauce and chicken soup.2.The conditions of N-acetylphenylalanine synthesis catalyzed by transglutaminase（TG-1）,Protease A 2SD and Protease AXH were optimized,and their substrate selectivity was analyzed.The results showed that TG-1,Protease A 2SD and Protease AXH catalyzed the synthesis and hydrolysis of N-acetylphenylalanine in aqueous solution.TG-1 and Protease A2SD have high synthetic yields in water（in 10 different solvents）,and Protease AXH has low synthetic efficiency.At 50 ^oC,p H=7.0,100 m M phenylalanine,and 2 M acetic acid,reaction time 24 h,and the synthetic yield of N-acetylphenylalanine catalyzed by TG-1 was 22.75%.Protease A 2SD at 60 ^oC,p H=7.0,100 m M phenylalanine,100 m M phenylalanine,1.5 M acetic acid,reaction time for 24 h,the yield of N-acetylphenylalanine catalyzed by Protease A2SD was 30.78%.TG-1 and Protease A2SD are synthetically specific for N-acetyl-valine,N-acetyl-leucine,N-acetyl-isoleucine,N-acetyl-methionine,N-acetyl-tryptophan and N-acetyl-tyrosine,and for N-formyl-,N-propionyl-,N-butyridine,N-hexanoyl-,and N-octanoyl-phenylalanine,suggesting that TG-1 and Protease A2SD were good enzyme for synthesis of N-acetyl-amino acid.After the reaction product is extracted with ethyl acetate,the purity of the above five N-acetyl amino acids（except N-acetyl tryptophan and N-acetyl tyrosine）are all above 90%.The electronic tongue evaluation found that the above N-acetyl amino acids can increase the salty and umami taste of soy sauce and chicken soup.3.The enzymatic properties of Acylase H were studied,the enzymatic synthesis process of N-acetylphenylalanine was optimized,and its substrate selectivity was clarified.The results showed that the hydrolase activity and synthase activity of Acylase H on N-acetylphenylalanine were 3948 u/g and 70u/g（p H=7.0,37^oC）,respectively.The optimal hydrolysis conditions of Acylase H were:p H=8.0,55 ^oC,0.1 m M Co Cl₂,which had hydrolysis activity to N-acetyl amino acids（20 common types）except N-acetylglycine and N-acetylproline,the degree of hydrolysis was in the range of 2.31%-41.22%;the optimal synthesis conditions were:p H=6.0,37 ^oC,0.1 m M Zn Cl₂,which had synthesis specificity for N-acetylvaline,N-acetylleucine,N-acetylisoleucine,N-acetylmethionine and N-acetyltryptophan,and yields was ranging from 2.55%to 9.42%.Adding ethyl acetate（50%,m/m）or trypsin（1:1）could effectively increase the synthetic yields of the above five N-acetyl amino acids by 2.05 times,6.60 times,3.02 times,1.88 times and 1.24 times,respectively.times and 2.65 times,1.36 times,1.38 times,2.47 times and 1.78 times.Meanwhile,Acylase H could also catalyze the synthesis of N-formyl-,N-propionyl-,N-butyryl-,N-hexanoyl-and N-octanoylphenylalanine.4.The N-acetylation efficiencies of Acylase H,TG-1 and Protease A2SD on dipeptide and tripeptide were compared.The results showed that the N-acetylation yields of common oligopeptides（Gly-Gly,carnosine,glutathione,Gly-Leu,Gly-Phe,Phe-Gly and Phe-Phe）by the three enzymes were all<1.0%（carnosine was not synthesized）.At the same time,these three enzymes have hydrolysis and transpeptide effects on oligopeptides.Using Gly-Phe and Phe-Phe as substrates,the hydrolyzed products of dipeptide and the production of N-acetylphenylalanine gradually increased with the prolongation of reaction time,but the synthetic yield of N-acetyldipeptide remained unchanged.Further analysis showed that the three enzymes had both aminopeptidase and carboxypeptidase activities,which were 867.31u/g,222.10 u/g,5678.45 u/g and 1346.80 u/g,and 495989.01 u/g and 2772.00 u/g,respectively.Electronic tongue analysis showed that the addition of Gly-Phe and Phe-Phe enzymatic products improved the salty and umami taste of soy sauce better than dipeptide.5.The effects of Acylase H,TG-1 and Protease A2SD on the physical and chemical properties of soy sauce,the type and content of N-acetyl amino acids,the type and content of N-acetyl dipeptide and the taste characteristics of soy sauce were evaluated.In soy sauce,adding three enzymes directly or adding acetic acid and three enzymes at the same time could increase the content of amino acid nitrogen and total nitrogen,reduce the average relative molecular weight of peptides,and enhance the salty and umami taste of soy sauce.Compared to control,the contents of 19 kinds of N-acetyl amino acids and 28 kinds of N-acetyl dipeptides in soy sauce were increased after TG-1 and glutaminase compound acetic acid treatment.Sensory evaluation showed that TG-1 treatment could improve the taste of soy sauce.Sensory results showed that 20 kinds of N-acetyl amino acids and 8 kinds of N-fatty acyl phenylalanine were kokumi compounds and their umami-enhancing threshold was 0.3-2.5 g/L.Using the T1R3-MSG complex as the umami taste receptor,the results of molecular docking showed that the interaction between N-acetyl amino acids and receptors was quite different.Among them,the main mode of action of N-acetylalanine and N-acetylglutamic acid with better taste and T1R3-MSG is hydrogen bonding,and the main action sites are His341 and Lys 77,and their umami enhancement effect may be due to enhance the interaction of sodium glutamate and T1R3 receptors.