甲基营养菌MP688甲醛脱氢酶的表达、纯化及酶活性质  

Expression,purification and enzymatic characteristics of aldehyde dehydrogenase from MP688

作　　者：景爱霞[1] 毕波[2] 李彤[2] 熊向华[1] 汪建华[1] 张惟材[1] 

机构地区：[1]北京生物工程研究所,北京100071 [2]沈阳药科大学,沈阳110016

出　　处：《军事医学》2017年第5期381-384,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31300083)

摘　　要：目的克隆甲基营养菌MP688甲醛脱氢酶(aldehyde dehydrogenase)基因adhA,在大肠杆菌中表达、纯化,并研究其酶学性质。方法 PCR扩增甲醛脱氢酶基因adhA,构建表达载体p TIG-AdhA,在BL21(DE3)中诱导表达,经Ni+亲和层析纯化,利用AHMT法进行体外酶学性质分析。结果成功构建甲醛脱氢酶表达载体,在大肠杆菌中AdhA表达量达总蛋白的50%以上,其中大部分表达产物可溶,纯化得到纯度为95%的甲醛脱氢酶。甲醛脱氢酶能以甲醛为底物,最适pH为7.0,最适反应温度为30℃,室温保存6 d后酶活约保留60%。结论在大肠杆菌中实现了甲基营养菌甲醛脱氢酶的可溶表达,在所考察的底物中,甲醛为该酶的最适底物。Objective To clone the aldehyde dehydrogenase （adhA） gene from Methylovorus glucosotrophus and study its expression, purification and enzymatic characteristics. Methods The adhA gene was amplified and cloned to the expression vector pTIG. The AdhA was successfully expressed with induction in Escherichia coli BL21 （ DE3 ）. The enzymatic characteristics were investigated by AHMT, and AdhA was purified by Ni ＋ exchange chromatography. Results AdhA accounted for more than 50% of the total cell proteins, and the purity was about 95%. With methanol as the substrate, the optimal pH of AdhA was 7.0, while the optimal temperature was 30℃. The enzymatic activity of purified AdhA remained about 60% when stored at room temperature for 6 days. Conclusion AdhA from MP688 is expressed in vitro, and methanol is the optimal substrate among all the substrates investigated.

关 键 词：甲基营养菌属 醛脱氢酶 克隆 纯化 酶学性质 

分 类 号：Q936[生物学—微生物学]