Cloning And Expression Of Nitrilase From Arthrobacter Nitroguajacolicus

In this study, a nitrilase gene was cloned from Arthrobacter nitroguajacolicus and a recombinant E. coli strain expressing nitrilase, BL21(DE3)-pETNYNitd, was constructed. After culture optimization, the enzyme production of the recombinant strain was 3 times higher than that of the wild strain. Because the fermentation period of the recombinant strain was halved and the highest enzyme production increased by 3 folds, the calculated power comsuption was cut down to 60% while the enzyme productivity was increased up to 300%. The recombinant strain has the potential for industrial production. The main research contents and results are as follow:1. A nitrilase gene was cloned from Arthrobacter nitroguajacolicus, which was named NYNit. through the steps such as purification of the enzyme, screening of genomic library and amplification of flanking fragments. The combinant plasimd pETNYNit was constructed. A recombinant E. coli strain BL21(DE3)-pETNYNit was resulted by transforming the plasmid pETNYNit into E. coli BL21(DE3)2. Two ways were used to improve the enzyme production of E. coli BL21(DE3)-pETNYNit. One is the optimization of the induction conditions, the other is optimization of the fermentation medium by three rounds orthogonal tests and then exploring the proper seed age. After the former optimization, the combinant strain's enzyme production was one-third of the wild-type strain's. The latter optimization makes the combinant strain's enzyme production equal to the wild strain's.3. Deletion of the DNA which encoded tag proteins before the nitrilase gene in the plasmid pETNYNit resulted in a new recombinant plasmid, named pETNYNitd. Construct a new recombinant bacterium by transforming the plasmid pETNYNitd into E. coli BL21(DE3). Compared to the E. coli BL21(DE3)-pETNYNit, the enzyme production of the E. coli BL21(DE3)-pETNYNitd was 2 folds enhanced under the same culture conditions.4. In the process of culturing E. coli BL21(DE3)-pETNYNitd, the inducer IPTG was replaced by lactose, which reached the better induction efficiency. In the end, the highest enzyme production of the E. coli BL21(DE3)-pETNYNitd increased by 3 folds compared to the wild strain.