腈水合酶NHaseK在大肠杆菌中的功能表达

Functional Expression of a Nitrile Hydratase from Klebsiella oxytoca KCTC 1686 in E.coli

腈水合酶由α亚基和β亚基组成,活化元件对其功能表达至关重要,研究腈水合酶基因簇中各元件的表达比例对酶重组表达的影响具有重要意义。以来源于Klebsiella oxytoca KCTC 1686的腈水合酶（NHaseK）为研究对象,构建了多种表达策略,以期实现α亚基、β亚基和活化元件17k差异表达。利用pETDuet-1质粒具有双T7启动子的特点,将上述基因以八种不同的组合方式分别插入于两个启动子之后。当将三段基因同时插入于第一个启动子之后时,亚基表达量均衡,比活力为0.78 U/mg蛋白,是亚基表达量比例为5∶3时的124%。在此基础上,在第二个启动子之后插入活化元件基因,活化元件表达水平提升2倍,比活提升5%,为0.82 U/mg蛋白。当将α亚基和β亚基插入于不同启动子之后时,酶活仅为对照组的10%,说明NHaseK的亚基必须同时转录才可形成成熟蛋白。进一步考察质粒拷贝数对大肠杆菌表达NHaseK的影响,确定15~20的质粒拷贝数足够实现NHaseK的功能表达。结果表明,亚基的均衡表达以及活化元件的充分表达对NHaseK的重组表达具有积极作用。

Nitrile hydratase is consisted of α-and β-subunit and regulated by the activator for its functional expression. To investigate the influence of different proportions of subunits and activator（ 17k） on recombinant expression,the nitrile hydratase（ NHaseK） from Klebsiella oxytoca KCTC 1686 was studied and several recombinant strategies were constructed. Taking the advantage of two T7 promoters（ T7 promoter-1 and T7promoter-2） in pETDuet-1,the three gene segments of NHaseK were inserted into this plasmid in eight different arrangements. When all the three gene segments were expressed under T7 promoter-1,NHaseK showed a specific activity of 0. 78 U/mg as α-and β-subunits equivalently expressed,and was 24% higher than at the ratio of α-and β-subunit as 5∶ 3. With this optimal condition,17 k was added following the T7 promoter-2,which enhanced the expression level of 17 k by 2-folds and improved the activity to 0. 82 U/mg. Noted that the activity reduced to10% when subunits were separately in control of different promoters,indicating that the α-and β-subunits needed to be transcribed simultaneously. Further,the influence of plasmid copy number was investigated and the number of 15 ~ 20 was appropriate to express NHaseK in E. coli. In conclusion,the equivalent expression of subunits and the abundant expression of 17 k were in favor of the functional expression of NHaseK.