L-alanyl-L-glutamine (Ala-Gln) is an important functional dipeptide, which has important value in food, medicine and health care products. Using recombinant Escherichia coli to express α-amino acid ester acyltransferase as whole-cell catalyst, which linked alanine methyl ester hydrochloride with glutamine to synthesize Ala-Gln, was an ideal method for the production of Ala-Gln. The high-cell-density cultivation could increase the proliferation concentration of microorganisms and reduce the cost. Firstly, the medium of E. coli BL21-pet29a-SsAET strain which expressing α-amino acid ester acyltransferase was optimized using response surface methodology based on the basic culture medium composition, such as carbon source, nitrogen source, and inorganic salt in flask-shaking experiment. Then, the important factors for fed-batch cultivation, such as pH, feeding medium and feeding mode, were investigated in fermenter. Finally, the recombinant E. coli by the high-density cultivation as whole-cell catalyst were used for the synthesis of dipeptide. The results showed that the optimum concentration of the semi-synthetic medium was: glucose 10 g/L, mixed nitrogen source 24 g/L, KH2PO4 4.62 g/L and K2HPO4 25.08 g/L. And the optimum feeding method was controlled using the automatic feedback by dissolved oxygen against pH-controlled. When the semi-synthetic medium was used with automatic feedback by dissolved oxygen, the OD620 value of the bacteria reached up to 67 at 30 h, which was 14 times better than that of flask culture. When the high density-culture of recombinant E.coli was used to synthesize dipeptide, the concentration of Ala-Gln reached 34.56g /L and the production efficiency was 7 g/(L·min), which could further enhance its potential for industrial applications.
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