徐冲左军廖军杨永辉陶丽梅
朱国萍伍传金滕脉坤王玉珍
(中国科学技术大学生命科学学院，合肥230027)
Prelin nary Study on Reformation of Engheered Bacter s
E. coli of Glucose Isomerase
XU Chong, ZUO Jun,L IAO Jun, YAN G Yonghui, TAO L in ei,
ZHU Guop ing,W U Chuanjin, T EN G M aikun,W AN G Yuzhen
}Schoolof LifeScience,University of Science and Technology of China,Hefei 230027)
Abstract In order to realize stableoverexpressionofmutantglucoseisomerase(GI)genefrom Streptanyces
dlastatlcusNo. 7M 1033 in E. toll, a L 2 kb fragment containing the intact coding sequence of the protein
w as amplified sp ecifically from p lain id p T KI〕-G Il by PCR. A t the sam a tin e, 45 by unnecessary sequence
at GIgene upstream was deleted The amplified fragmentwas inserted into the expression vectorpBV 220
to obtain the recombinant plagnid pBZGIl,which was introduced into E. toll DHSIX Data gathered from
passage of the generations of the strains showed thatpBZGIl inDH50(wasmuchmore stable thanpTKD-
GIl in K38}pGP1-2 Induced at 420C，pBZGIl overexpressed the mutant GI,which accounted for about
55% of total soluble proteins andwaspurified through heat treatment,DEAE-Sepharose FF column and
Sephacrcyl S-300 column. It also showed that the thennostability of the purified GIdidn't decline though
the undesired 15 am ino acids p resent in N一teen final w as deleted
Key words Glucose isom erase, Gene expression, Stability of plagn ids
葡萄糖异构酶(gluco se isom erase, G I)是使用量最大的工业酶之一，可用于高果糖浆的生产，也可以用含木聚糖物质及废料为底物发酵生产乙醇，具有重要的经济价值
本文选择了表达载体pBV 220「"，利用PCR方法删除了原表达质粒pTKD-GI1中GI结构基因5}端多余的核普酸，并添加了合适的酶切位点，重新构建了能在大肠杆菌DHSa中高效表达G IG 138P的表达质粒pB ZG Il.传代实验表明，新表达体系的稳定性明显优于原表达体系粗酶液经热处理、DEAE-Sepharose FF和分子筛Sephacryl S-300柱层析分离纯化后，得到电泳纯的葡萄糖异构酶酶学实验表明，其性质与pTKD-GIl所产GIG138P蛋白完全一致1L,发酵液可得纯酶60mg.
