Synthesis Of Functional Dipeptides Catalyzed By Amino Acid Ligase Coupled Polyphosphate Kinase Expression System

Because of its special function,dipeptide is widely used in food,medicine,health care products and cosmetics.Chemical synthesis of dipeptides is cumbersome and environmentally unfriendly,so in recent years,the use of enzyme catalytic method to produce dipeptides gradually emerged.L-amino acid ligase（EC 6.3.2）can directly use amino acids as substrates to synthesize dipeptides,however,for its ATP dependence,exogenous ATP supplementation or ATP regeneration system is needed.In this study,using Escherichia coli as the host,co-expressing L-amino acid ligase,polyphosphokinase and dual enzymes were explored.Functional dipeptides were synthesized by L-amino acid ligase and polyphosphokinase without ATP supplementation（ATP-free）,and the yield of dipeptides was improved by optimizing the reaction conditions.The specific research contents are as follows:Firstly,the recombinant strains were constructed by using L-amino acid ligase Ywf E from Bacillus subtilis ATCC 15245 and L-amino acid ligase Tab S from Pseudomonas syringae NBRC 14081 ATCC 27881.Ywf E and Tab S were induced,expressed and purified.Ywf E catalyzed the synthesis of alanine（Ala）and glutamine（Gln）to L-alanyl-L-glutamine（Ala-Gln）with the highest yield at 22-37℃,p H 8-9,Mg²⁺concentration 20-25 mmol/L,ATP concentration 30 mmol/L,and substrate Ala and Gln concentration 30 and 60 mmol/L respectively:the Ala-Gln yield reached 61.0%at 6 h;The synthesis of alanine dipeptide（Ala-Ala）from 2 molecules of Ala catalyzed by Tab S had the highest yield at 37℃,p H 9-9.5,ATP concentration 15 mmol/L,and the Ala-Ala yield reached 47.3%at 11 h.It shows that Ywf E and Tab S have good ability to synthesize different dipeptides,and can complete the synthesis of a variety of functional dipeptides.Secondly,using the genomes of Corynebacterium glutamicum 299 and Sphingobacterium siyangensis CGMCC 1.6855 as templates,the polyphosphate kinase gene was amplified,and the recombinant strain was constructed.PPK2（1.6855）with ATP synthesis activity was induced,expressed,isolated and purified.The yield of ATP synthesis catalyzed by PPK2（1.6855）was the highest at 22-37℃,p H 7-9,Mg²⁺concentration of 30 mmol/L,substrate ADP and sodium hexametaphosphate concentration of 5 and 20 mmol/L:when the reaction lasted for3 hours,the yield of ATP was as high as 71.0%.Finally,coupling L-amino acid ligase Ywf E and polyphosphate kinase PPK2（1.6855）to achieve high-efficiency synthesis of Ala-Gln:the concentrations of Ala and Gln were 30 and 60mmol/L respectively,the concentrations of ADP and sodium hexametaphosphate were 10 and15 mmol/L respectively,the reaction temperature was controlled at 27℃,the p H was 7-8,and the yield of Ala-Gln could be as high as 64.1%after 3 h of PPK2 reaction,which was equivalent to the yield of direct addition of ATP.In order to reduce the purification steps of the enzyme,reduce the reaction cost,and make it more suitable for industrial amplification,a whole cell catalyst with co-expression system of amino acid ligase Ywf E and polyphosphokinase PPK2（1.6855）was established by using single-plasmid and double-plasmid methods.This study laid the foundation for the efficient synthesis of functional dipeptide.