The Cloning And Efficient Expression Of Aspartase Transaminase In E.coli

Among the eight essential amino acids, L-phenylalanine(L-Phe) is known for its commercial value in both pharmaceutical and chemical industries. In the past ,the need for L-Phe is met mainly by imports. However, the critical demand for L-Phe has called for investigation of high-efficient bioprocessing. Among the methods of producing L-Phe, the enzymatic preparation of L-Phe remains a major interest because the production process is relatively simple and efficient. In this processing, hydantoin used as a starting material is transformed to benzilidene glycolycurea , which is then hydrolyzed to phenylpyruvic acid(PPA). L-Phe can be synthesized through the reaction mediated by aspartase using PPA and L-Asp as substrates. After intensive studies, Life Sciecnces and Pharmacy College of Nanjing University of Technology had already solved the problems in the processing from hydantoin to PPA, so screening high efficient aspartase-producing strains is in urgent need. In this study, three strains overexpressing transaminase were constructed by gene engineering, and the expression of the aspartate aminotransferase (encoding by aspC gene) in E.coli was studied. In order to acquire gene engineering strains with high activity of aminotransferase, the primer of AspC was designed to obtained the ORF of gene aspC. Then it was connected to the promoter p-121 in our lab, the fragment was subcloned into three vectors: pUC19; pSE380; pet22b, to give three constitutive plasmids:pUC-aspC; pSE-aspC; Pet-aspC. They were transformed into different host strains. As the host strains, BL21 had showed the well adaptability the faster growth rate and the higer enzymatic activity than others Growth curve, and expressions of enzyme of the recombinant strains all had been studied, the results indicated indicated that when promoters were uninducing in constitutive plasmids, expression of the clone genes is affected by copy number of plasmids. In the strains with high copy number plasmid, the overexpression of the allogenic genes may take a metabolic burden on host strains, which in turn may cause growth impairment of the constitutive cells. In the strains having low copy number plasmid, the expressing of enzymes was low in unitary cell, but total growth of strains was very high because it depressed the poison effect of the allogenic genes. The optimum composition of the recombinant strain BL21/Pet-aspC culture medium were obtained by orthogonal design method and Fermentation condition is also study in this study. Furthermore, the cultured recombinant strain was used to produce L-Phe L-Asp and PPA(30g/L) as substrates,. As a result, the conversion yields obtained for L-Phe was 85.1%, showing a well perspective of industrialization,...