Study On The Synthesis Of L-alanyl-L-glutamine Using Enzymatic Method

L-alanyl-L-glutamine has the advantages of high solubility,strong water-solubility and thermal stability,and has gradually replaced glutamine as the main parenteral nutrition drug.Chemical synthesis methods have many disadvantages,such as complex reaction steps and byproducts,toxic reagents and environmental pollution.The Japanese patented technology for the production of dipeptide catalyzed by the L-amino acid ligase from Bacillus subtilis ATCC 15245 overcomes the shortcomings of chemical production,but still has the problems of large ATP consumption and low catalytic production efficiency.In this study,Escherichia coli was used as a host,and L-amino acid ligase,polyphosphokinase,and acetic acid kinase were used as recombinant expression vectors to form an enzyme-catalyzed reaction system in which L-amino acid ligase-coupled ATP regeneration was used to produce dipeptide.After optimization of the amount of enzymes and the reaction condition,the yield and conversion rate of L-alanyl-L-glutamine were improved,and the economic and industrial feasibility of the reaction was enhanced.According to the coding sequence of the L-amino acid ligase YwfE in Bacillus subtilis ATCC 15245,the gene was codon optimized,cloned and then ligated into the vector of pET-His and transferred into Escherichia coli BL21(DE)for expression.Next,Ni-NAT affinity purification was used to recover the recombinant protein based on the His-tag.The purified YwfE had the highest activity of 1843 U/mg at 37 ? and pH 9.0.Under the optimal condition,21.8 mM L-alanyl-L-glutamine was finally produced from 30 mM L-alanine,30 mM L-glutamine and 30 mM ATP for 7 h.The polyphosphate kinase encoding gene ppkl and acetate kinase encoding gene ackl were successfully amplified from E.coli MG1655.The ppk2 gene was amplified from Rhodobacter Sphaeroides,and the ackl gene and ack2 gene was amplified from Bulgaricus ATCC 11842 and Clostridium acetobutylicum ATCC 824,respectively.The cloned genes wereligated into the vector of pET-His and transferred into Escherichia coli BL21(DE)for expression.The Ni-NAT affinity purification was used to recover the recombinant protein based on the His-tag,and the enzymatic properties were analyzed.Ppk1 and Ppk2 had the highest enzyme activity of 237 U/mg and 356 U/mg,respectively,at 47 ? and pH 8.0.The conversion rates of ADP to ATP were 15.4%and 20.1%,respectively.Acetate kinases Ackl,Ack2,and Ack3 had the highest enzyme activity of 327 U/mg,453 U/mg and 476 U/mg,respectively,at 37 ? and pH 8.0.The conversion rate of ADP to ATP was 24.5%,24.5%and 25.1%,respectively.The polyphosphate kinase Ppkl and Ppk2 was respectively used as ATP regeneration enzymes for L-alanyl-L-glutamine production.The reaction system contained 30 mM Ala,30 mM Gln,5 mM ADPand 30 mM sodium polyphosphate,at 37 ? and pH 9.0.The highest yield with Ppkl and Ppk2 was 45%and 51.3%,respectively.The acetokinase Ackl,Ack2 and Ack3 was respectively used as as ATP regeneration enzymes for L-alanyl-L-glutamine production.The reaction system contained 30 mM Ala,30 mM Gln,5 mM ADP,30 mM acetyl phosphate,at 37 ? and pH 9.0.The highest yield with Ackl,Ack2 and Ack3 was 64.3%,68.4%,and 71.1%,respectively.The results suggest that Ack3 can be effectively used for ATP regeneration and can couple with YwfE for L-alanyl-L-glutamine production.