重组毕赤酵母产匍枝根霉内切葡聚糖酶Ⅱ的分离纯化及其酶学性质研究  

Separation purification and enzymatic properties of endoglucanaseⅡin pichia pastoris from rhizopus stolonifer

作　　者：程伟[1] 汤斌[1] 李松[1] 

机构地区：[1]安徽工程大学微生物发酵安徽省工程技术研究中心,安徽芜湖241000

出　　处：《安徽工程大学学报》2015年第2期1-5,共5页Journal of Anhui Polytechnic University

基　　金：国家自然科学基金资助项目(31270135)

摘　　要：将来源于匍枝根霉的endoglucanaseⅡ(egⅡ)在毕赤酵母中实现异源表达,对构建完成的重组毕赤酵母进行高密度发酵,制备目标酶蛋白.发酵液经Ni柱分离纯化,得到分子量大小约为38kDa,比酶活为37.8IU/mg的蛋白.酶学性质研究表明:该酶最适反应温度为55℃,最适反应pH为4.8;Mn2+、Zn2+、Fe2+对egⅡ的酶活力有一定的激活作用,Cu2+对egⅡ酶活有抑制作用,Mg2+、Co2+、Na+和K+等离子对该酶的催化活力没有明显影响;该酶以CMC-Na为底物时其反应米氏常数为2.04mg/ml.The endoglucanase Ⅱ （ eg Ⅱ） of Rhizopus stolonifer has been expressed in Pichia pastoris.The high-level expression of recombinant endoglucanase Ⅱ was achieved through high-density fermentation of recombinant Pichia pastoris.Ni column was used to purify the expressed product, the specific activitiy of purified eg Ⅱ has been determined,and its molecular mass was 38 kDa.The results of enzyme character- ization showed that the purified eg Ⅱ has a optimum catalytic activity at 55 ℃ and pH 5.0. The catalytic activity of eg Ⅱ could be enhanced by metal ion Mn2＋ , Zn2＋ and Fe2＋ , but strongly inhibited by Cu2＋ at a high concentration （5 raM）, while it has no effects by Mg2＋ , Co2＋ , Na＋ and K＋. The determined Kr. value of eg Ⅱ was 2.04 mg/ml by using sodium carboxymethyl cellulose （CMC-Na） as the substrate.

关 键 词：匍枝根霉 内切葡聚糖酶 分离纯化 酶学性质 

分 类 号：Q93[生物学—微生物学]