Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171005, India.
Appl Biochem Biotechnol. 2012 Oct;168(3):465-86. doi: 10.1007/s12010-012-9790-9. Epub 2012 Jul 26.
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-Bertani 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.
通过 PCR 扩增分离出突变体 4D 的 NHase 编码基因。利用 Ek/LIC Duet 克隆试剂盒（Novagen），成功地在大肠杆菌中克隆和表达了突变体 4D 的 NHase 基因。为了使 NHase 基因的活性表达，还与 NHase 基因共同表达了小钴转运蛋白基因（P 蛋白基因）。该 NHase 基因的核苷酸序列与红球菌 J1 的 H-NHase 具有高度同源性。与野生型（4.1 U/mg dcw）相比，重组大肠杆菌细胞表现出更高的 NHase 活性（5.9 U/mg dcw），但低于突变株（8.4 U/mg dcw）。需要在 Luria-Bertani 培养基中添加钴离子，其浓度低至非常小（0.4 mM），才能使 NHase 活性增加。重组大肠杆菌在孵育 6 小时时表现出最大的 NHase 活性，并用 56%的产率进行了纯化，比活性为 37.1 U/mg 蛋白。
