Nocardia sp.腈水合酶的纯化过程研究

对Nocardiasp.高活力腈水合酶进行了纯化研究。在细胞破碎中,超声时间对腈水合酶比酶活存在一个最优值,超声时间为20.00min时得到的比酶活最高。在离子交换层析过程中,采用DEAE-Sepharose作为层析介质,分别对平衡缓冲液pH、离子强度和线性梯度洗脱体积进行了优化。结果表明,采用pH7.20、50mmolL-1的Na2HPO4-NaH2PO4溶液作为平衡缓冲液,0.00~1.00molL-1的NaCl线性梯度洗脱,洗脱体积为20~25倍柱体积,此条件下腈水合酶的纯化倍数和酶活收率较佳。以Phenyl-SepharoseFF为层析介质研究了疏水层析精制腈水合酶的工艺过程,采用两步层析优化方法纯化出的Nocardiasp.腈水合酶比酶活达到2648.0U穖g-1,酶活收率为40.84%,用SDS-PAGE检测其纯度为99.00%以上。Nocardiasp.腈水合酶的两个亚基分子量分别为22.90kDa和27.38kDa。

Purification of Nitrile Hydratase from Nocardia sp.

Nitrile hydratase (NHase), which catalyzes the hydration of nitriles to amides, has been used in industrial production of acrylamide. To obtain the enzymatic properties of NHase from Nocardia sp., the enzyme was purified from cell free extract by ion exchange chromatography and hydrophobic interaction chromatography. The cell ultrasonication time was optimized aiming at the high specific activity of NHase in cell free extract. The procedures of ion exchange chromatography were optimized in terms of pH, ionic strength of buffer and elution volume. The results show that 50mmolL-1 sodium phosphate buffer at pH 7.20 is optimal for equilibration and sampling, and it is most suitable that the sample is eluted with 0.00 to 1.00mmolL-1 NaCl for 20 or 25 times of column volume in a gradient elution. The sample gathered under the optimized condition of ion exchange chromatography was purified by hydrophobic interaction chromatography. The specific activity of purified nitrile hydratase reaches 2648.0 Umg-1 and the overall purification fold is 9.42 with an enzymatic activity recovery of 40.84%. According to the results of SDS-PAGE, the purity of the Nocardia sp. nitrile hydratase purified by HIC is more than 99.00% and the enzyme comprises two subunits, whose molecular weights are 27.38 kDa and 22.90 kDa respectively.