Construction Of α-amino Acid Ester Acyltrasferase Producing Recombinant Escherichia Coli And Optimization Of Its Fermentation Conditions

α-Amino acid ester acyltrasferase can synthesize L-alanyl-L-glutamine from L-alanine methyl ester hydrochloride and L-glutamine. L-alanyl-L-glutamine is much more tolerant to high temperature and soluble than each of the constitutive amino acids. Based on this property, it is applied in intravenous infusions as the carrier of L-glutamine. In this study, a strain producing Ala-Gln was constructed. To increase the production of Ala-Gln, the culture condition of this strain and the reaction condition for the enzymatic synthesis of Ala-Gln were optimized. The research content is as follows:α-Amino acid ester acyltrasferase from S. siyangensis has the advantages of higher enzyme activity, and less by-products. However, the difference of codon usage frequency between S. siyangensis and E. coli is significant. To improve the expression level of α-amino acid ester acyltransferase in E. coli, we optimized the sequence and m RNA secondary structure of α-amino acid ester acyltrasferase by using JCat and RNAstructure 5.3. A total of 396 nucleotides were changed, CAI values was increased from 0.26 to 0.82, and the G+C ratio was simultaneously increased from 42.15% to 48.22% after optimization.pho C, which is induced by extracellular phosphate concentration, is a weak promoter. pho C is activated when the extracellular phosphate concentration falls below 4 μM. Tryptophan series promoter(Ptrp2), which is composed of two series of tryptophan promoter, is a strong promoter.Codon-optimized α-amino acid ester acyltransferase gene was cloned into expression vectors with a tryptophan tandem promoter or phosphate promoter. The p ET21a-pho C-SAET and p ET21a-Ptrp2-SAET is significantly higher than other plasmida. It is indicated that p ET21 a plasmid is more suitable forα-amino acid ester acyltransferase expression. This plasmid was introduced into E. coli JM109, BL21(DE3), DH5α, respectively. When the host is E. coli DH5α,the yield was 105 mg/L, higher than others.The whole-cell catalysis reaction conditions of E. coli DH5α/p ET21a-pho C-SAET were optimized. The most appropriate reaction conditions are: Ala-OMe?HCl 100 m M, Gln 50 m M,p H9, at 25℃ for 90 min. The yield is 146 mg/L, showing 1.4 fold improvement over that of the initial condition.The composition of the optimized culture medium for the genetic engineered Escherichia coli to produce L-alanyl-L-glutamine is as follows: glucose 10 g/L, yeast extract 5 g/L, tryptone 5g/L, NH4 AC 2.5 g/L, KH2PO4 30 m M, K2HPO4 15 m M, 2 g/L Mg SO4. The optimal fermentation conditions are: the inoculum age is 10 h, the cultivation temperature is 17℃, the original p H value is 6.5, the inoculation amount of bacteria is 4%. The yield is 498 mg/L, showing 3.4 fold improvement over that of the initial condition.We adopted a thin layer chromatography for analysis of L-alanyl-L-glutamine. An aliquot volume(10 μL) of the sample was spotted on a 10×10 cmplate(Silicagel GF254). Thin layer chromatography analysis was performed with ethanol/Ethyl acetate/water/ammonia/ in a volumeratio of 50/10/15/1 as mobile phase. Spots were observed under UV light at 565 nm after reaction with 1 % ninhydrin ethanol solution at 105℃ for 30 min. 0.4 g/L L-alanyl-L-glutamine was used as the standards. The plate and different plates’ s RSD of precision was 1.44%, 3.65%,respectively.