Cloning, Sequencing, and Expression of Nitrile Hydratase Gene of Mutant 4D Strain of Rhodococcus rhodochrous PA 34 in E. coli |
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Authors: | Amit Pratush Amit Seth T C Bhalla |
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Institution: | 1. Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, 171005, India
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Abstract: | The NHase encoding gene of mutant 4D was isolated by PCR amplification. The NHase gene of mutant 4D was successfully cloned and expressed in Escherichia coli by using Ek/LIC Duet cloning kits (Novagen). For the active expression of the NHase gene, the co-expression of small cobalt transporter gene (P-protein gene) has also been co-expressed with NHase gene E. coli. The nucleotide sequence of this NHase gene revealed high homology with the H-NHase of Rhodococcus rhodochrous J1. The recombinant E. coli cells showed higher NHase activity (5.9?U/mg?dcw) as compared to the wild (4.1?U/mg?dcw) whereas it is less than the mutant strain (8.4?U/mg?dcw). Addition of cobalt ion in Luria?CBertani medium is needed up to a very small concentration (0.4?mM) for NHase activity. The recombinant E. coli exhibited maximum NHase activity at 6?h of incubation and was purified with a yield of 56?% with specific activity of 37.1?U/mg protein. |
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