Expression Purification Of Nitrile Hydratase In Escherichia Coli

The nitrile hydratase from Microbial is the important biological catalysts with which as catalyst acrylonitrile were synthesized into acrylamide. It has the advantages. Such as mild reaction conditions, high yields, fewer by-products, the small loss of the product of Self-Polymerization, regio-and stereoselectivity and can be widely used in synthesis of amino acids, amides, carboxylic acid and its derivatives. In this paper, a fragment of the nitrile hydratase gene from Nocardia was obtained by PCR. The recombinant bacteria of BL21-pGEX-4T-1/NHase were constructed to express the fusion protein of GST-NHase. However, due to the relative molecular weight of the GST is a little large which hampered the correct combination of nitrile hydrataseβ-subunit andαsubunit. So that nitrile hydratase activity can not be displayed. The fusion protein GST-NHase,expressed in BL21(DE3)strain,was purified with Glutathione Sepharose 4B affinity chromatography followed by thrombin cleavage.The digested product was further purified with additional Glutathione Sepharose 4B affinitychromatography and Benzamidine affinity chromatography step.And the recombinant bacteria of BL21-pET-30c/NHase was constructed and wsa induced by IPTG. The results show that the expression of theα-subunit of nitrile hydratase has not been largely affected the activity of nitrile hydratase. Bioinformatic analysis found that nitrile hydratase the start codon of theα-subunit was gtg, may be difficult to be recognized by the RNA polymerase of E. coli. Then by the site-directed mutation the gtg was change into atg. The expression of the positive recombinant bacteria was induced by IPTG. The results show that the expression of theβ-subunit andαsubunit ofnitrile hydratase has been enhanced. Nitrile hydratase activity also increased to 200U/mg which wsa detected by Gas chromatography. The bacterial suspension which was induced by IPTG was deal with by ultrasonication. The crude enzyme solution was obtained by centrifugation. Nitrile hydratase with the 6×His tag was Purified by affinity chromatography column containing EPI-30-ARG-IDA-Co²⁺.