Fermentation Optimization Of Recombinant E.Coli Expressing Nitrilase From Acidovorax Facilis 72W

A nitrilase from Acidovorax facilis 72W strains was over-expressed in E. coli BL21(DE3) by using two different kind of plasmids, and it can efficiently convert aliphatic dinitriles to cyanocarboxylic acids. This enzyme exhibited great potential for industrial applications due to its mild reaction conditions and excellent selectivity.In this study, the fermentation condition of a recombinant E. coli harboring a nitrilase gene (E.coli pET21a-NIT) was optimized by the combination of the single factor optimization and response surface method optimization to achieve the maximum nitrilase enzyme production. Secondly, using pRSFDuet plasmid to construct a recombinant E. coli pRSF-NIT2, realizes the double expression of target enzyme. Finally, the culture conditions of the recombinant E.coli pRSF-NIT2, such as carbon source, nitrogen source, and metal ions were studied systematically. The main conclusions of this study are as follwing:(1) We obtained the optimal culture conditions of.E.coli pET21a-NIT for the production of nitrilase by single factor experiment. Results are as follows (g/L):corn starch 10, yeast extract 10, NaCl 5, starch 20, K2HPO44, ZnSO4-7H2O 1, cultivated under 30℃, the initial pH of 7.0, added 0.2 mM IPTG after 4 h cultivation.(2) Basised on the single factor experiment, three main factors that have the significant influence on enzyme production were chosen according the experimental design using PB, Using the steepest climbing experimental, center point of the combination experimental design was found. In the end, using the response surface analysis to determine the optimal concentration of the main influence factors and the best formula for the cultivation of recombinant strains (g/L):23.55 starch, corn starch and yeast extract (1:1) 20.15,0.18 mM IPTG. Verified, the experiment value (202.04 U/L) is close to the theoretical value (201.93 U/L).(3) About the structure and double-expression of engineering bacterium E.coli pRSF-NIT2, first, we insert the target gene between Nde 1 and Xno 1 on MCS2, then we design a pair of primers, to PCR and insert the second same purpose gene between Pst 1 and Nco 1 on MCS1, to construct recombinant plasmid, then double-expressed in E. coli BL21.(4) The comparison of the protein expression of recombinant E.coli pET21a-NTT and E. coli pRSF-NIT2 by SDS-page revealed that the expression level of E. coli pRSF-NIT2 is obviously higher than that of E.coli pE721a-NIT. With nickel column for protein purification, we get a single strip, about 40 KDa, consistent with which was reported.(5) The culture conditions of E.coli pRSF-NIT2 were optimized to obtain the better result of cell growth and enzyme production. The results are as follows (g/L):yeast powder 15, NaCl 5, starch 20, K2HPO44, MgSO4-7H2O 1, cultivated under 30℃, the initial pH of 7.5, adding 0.4 mM IPTG after 4 h induction. At the later stage of logarithmic growth, around 12 h of fermentation, the enzyme activity also reached the maximum value (83.93 U/g), total activity is about 260.22 U/L.