Improvement Of Recombinant Escherichia Coli Producing L-alanyl-L-glutamine By Whole-cell Catalysis And Optimation Of Systhesis Conditions

L-alanyl-L- glutamine（Aln- Gln） has been well recognized as the carrier of L-glutamine（Gln） home and abroad because of its high water solubility, good stability and rapid in vivo degradation. Therefore, Aln-Gln plays an important role in clinical medicine and nutrition. α-amino acid ester acyltrasferase（SAET） from Sphingobacterium siyangensis is capable of catalyzing the synthesis of Aln-Gln from L-alanine methyl ester hydrochloride（Ala-OMe·HC l） and L-glutamine. Previously, gene Saet was optimized and the recombinant Escherichia coli producing SAET was successfully constructed. In order to reduce the degradation of Aln-Gln and improve the production efficiency, the researcher adopts several strategies, as follows: transforming the host E. coli, optimizing the fermentation conditions together with the reaction condition for the enzymatic synthesis of Ala-Gln, expanding culture was conducted in a fermenter.In order to determine the suitable recombinant plasmid express ing SAET in the host E. coli JM109, we firstly optimized the type of the plasmids which were constructed. Secondly, pepD and pepN in E. coli JM109 encoding dipeptides and aminopeptidases were knocked out by Red-mediated recombination to reduce the degradation of Ala-Gln. Recombinant E. coli strains JM109Δpep D/p AMP-phoC-SAET and JM109ΔpepDΔpepN/pAMP-phoC-SAET were constructed. Meanwhile, the whole cell activity of the double knockout strain reached 93.33 U·mg^-1, which was 1.29 times as high as the original strain JM109/pAMP-phoC-SAET.By single factor analysis and orthogonal design on the medium compositions and fermentation conditions of E. coli strains JM109ΔpepDΔpepN/pAMP-phoC-SAET, the optimal medium was determined as follows(g·L^-1): glucose 12, yeast extract 10, tryptone 10,（NH₄）₂SO₄ 1, KH₂PO₄ 3, K₂HPO₄, MgSO₄ 0.2. The most suitable cultivation temperature was 27 °C and the inoculation amount of bacteria was 6%. Recombinant E. coli was cultivated for 36 h as crude enzyme source under the optimal fermentation conditions, the yield of Aln- Gln reached 5.08 g·L^-1, which was 1.66 times more than before(3.06 g·L^-1).The whole-cell catalysis reaction conditions of E. coli JM109ΔpepDΔpepN/pAMP-phoC-SAET were optimized under the optimal fermentation conditions. The most appropriate reaction conditions were as following: reaction temperature 27 °C, reaction pH 8.5, substrate concentration Gln 200 mmol·L^-1 and Ala-OMe·HC l 200 mmol·L^-1. The recombinant E. coli was cultivated under the optimal fermentation conditions for 36 h, the yield of Aln-Gln reached 11.51 g·L^-1, which was 3.76 times more than before.To further increase the amount of bacteria and improve the yield of Aln- Gln, recombinant E. coli was cultivated in a 5 L fermenter. After 32 h fermentation, OD600 value reached 35.7. After 1.5 h reaction in the 20 mL substrate system, the yield of Aln- Gln reached 17.6 g·L^-1, which provides an important technical support for the industrialization of the Aln-Gln production.