L-Alanyl-L-Glutamine Production By Recombinant Escherichia Coli Expressing?-amino Acid Ester Acyl Transferase

L-alanyl-L-glutamine is one of the most mature dipeptide product,which has the advantages of high solubility,good thermal stability and high decomposition rate.It has been widely used in food,health care and other fields.At present,bio-enzymatic method has been widely studied as a new method for production of L-alanyl-L-glutamine.Compared with the traditional chemical method,this method has the advantages of environmental protection,less by-products and rapid reaction,which has become a new method for the industrialization of dipeptide.However,most of the domestic research on bio-enzymatic method is still in the laboratory stage,and there is no relevant research on its industrialization method.In this paper,the recombinant Escherichia coli expressing ?-amino acid ester acyl transferase to produce Lalanyl-L-glutamine was taken as the starting strain.The high density fermentation strategy and immobilization strategy of the recombinant E.coli were studied for the industrial production process,respectively.These methods can realize the high efficiency of dipeptide and lay a foundation for the industrial production with economization.Firstly,the high density fermentation process was optimized.In order to achieve the high density of the strain,the medium types,carbon and nitrogen source were selected and optimized.The optimal fermentation medium was found to be glucose 10 g/L,mixed nitrogen source 24 g/L,potassium dihydrogen phosphate 4.62 g/L,potassium dihydrogen phosphate 25.08 g/L.In 3 L fermenter,the control of p H,feeding medium and feeding mode were investigated.And the optimum conditions were feedback by dissolved oxygen.When the semi-synthetic medium was used,the OD620 value of the bacteria reached up to 67 in 30 h,and that's 14 times better than before.Finally,the high density culture of recombinant E.coli was used to synthesize dipeptide as the whole cell catalyst.The concentration of Ala-Gln reached 34.56 g/L and he substrate conversion rate was about 53%,The recombinant E.coli by high density fermentation still maintain good activity.Secondly,immobilized cells were used to optimize the reaction process after fermentation.It was found that immobilization of cells before induction could enhance the reactivity effectively.Compared with different methods,the best immobilized material was sodium alginate-calcium chloride.The properties of the immobilized cell after immobilization were further explored.It was found that the p H and temperature stability of the immobilized cells were improved significantly compared with those of free cells.Good activity can be maintained at the reaction temperature from 20 ?~35 ?,and at a p H of 8.0~9.0.In addition to that,they were still active after 10 repetitions.In this way,the immobilized cells were put into the reactor to realize continuous production,and the continuous flow packed bed reactor was finally used,which was optimal at the flow rate of 5 m L/min.The production efficiency of 2.79 mg/(min× ml-CV)and the substrate conversion rate of 51% could be achieved in the continuous process.Furthermore,the separation and purification of the product after the reaction were briefly studied.It was found that L-alanyl-L-glutamine could be separated by methanol due to its solubility.The specific process was to add hydrochloric acid into the reaction liquid and then placed it in round bottom flask.Spin steaming equipment was used to obtain dry product.Pure methanol was added,and the supernatant was removed by extraction and filtration.The insoluble solid was mixed with 70% methanol solution and dissolved uniformly.The supernatant was extracted by filtration.At last,the supernatant was placed in round bottom flask with decompress distillation to get L-alanyl-L-glutamine.Finally,based on the above results,a brief economic accounting for high density and immobilization methods was conducted respectively.The cost can be reduced by 80% when the same quantity of strain is produced by high density fermentation.The economic accounting of immobilized cell reaction shows that the production cost is about 40% of the product,which has the profit margins for the makers.