重组水稻α-半乳糖苷酶的分离纯化及酶学性质研究-食品科学.PDF

304 2010, Vol. 31, No. 21 食品科学 ※生物工程 α- 1 1 2 , 李苏红 ，朱旻鹏 ，李拖平 * (1.沈阳师范大学工程技术学院，辽宁 沈阳 110034 ；2.辽宁大学轻型产业学院，辽宁 沈阳 110036) ：对重组水稻α- 半乳糖苷酶的分离纯化及其酶学性质进行研究。结果表明：该重组酶分子质量约为 59kD ； 最适酶反应温度为 45 ℃，最适pH 值为 5.0 ；酶活力在0 ～20 ℃，pH4.0 ～7.0 最为稳定；酶学动力学常数 Km 为 0.78mmol/L ，Vmax 为 10.16mmol/(mg ·min) 。多数的金属离子和有机离子对酶催化作用没有影响，Hg2+ 、Ag+ 及－ SH 基团抑制剂p -chloromercuribenzoic acid(p CMB)对酶活性有强烈抑制作用。 ：重组； α- 半乳糖苷酶；纯化；酶学性质 Isolation, Purification and Characterization of Recombinant α-Galactosidase from Rice 1 1 2, LI Su-hong ，ZHU Min-peng ，LI Tuo-ping * (1. College of Engineering and Technology, Shenyang Normal University, Shenyang 110034, China； 2. College of Light Industry, Liaoning University, Shenyang 110036, China) Abstract ：A crude enzyme solution rich in recombinant rice α-galactosidase from the engineering strain E. coli pET-32a (+)- Gal/ Origami DE obtained from our laboratory was prepared and purified on a Ni-Sepharose affinity column. The purified α- galactosidase showed a single protein band in SDSwith a molecular mass of 59 kD. This enzyme exhibited the highest activity at the conditions of pH 5.0 and 45 ℃. Meanwhile, it showed the best stability at pH 4.0－7.0 and 0 －20 ℃. The kinetic parameters Km and Vmax were 0.78 mmol/L and 10.16 mmol/(mg ·min), respectively. Most of metal and organic ions did not affect the activity of this enzyme. However, Ag+, Hg2+ and p CMB (p -chloromercuribenzoic acid) significantly decreased its activity by 84%, 87% and 92%, respectively.