为研究Ⅱ型Xylose/Glucose的N端蛋白序列对I型Xylose/Glucose的作用，实验将源自超嗜热菌Thermoanaerobacter thermohydrosulfuricus的Ⅱ型葡萄糖异构酶（TTGIase）N端31个氨基酸序列融合到嗜热菌Thermobifida fusca的Ⅰ型葡萄糖异构酶（TFGIase）的N端，构建了融合蛋白N-TFGIase。实验结果显示：在相同培养和诱导条件下，N-TFGIase，菌体单位浓度产酶量比TFGIase高出约40%，比酶活比TFGIase高出26%，最适温度比TFGIase高出5 ℃，75 ℃下的半衰期较TFGIase延长30%，最适pH较TFGIase降低1.0。序列分析表明：TTGIase N端序列的mRNA二级结构不形成有阻碍的颈环结构，提高了融合蛋白的表达效率；其含有的31个氨基酸残基的疏水性指数均小于0，利于融合蛋白的初始折叠和包装；其含有的酸性氨基酸残基比例约为碱性氨基酸残基比例的两倍，减小了酸性介质环境对融合蛋白分子表面的影响。

To study the effect of the N-terminal protein sequence of type II xylose/glucose on type I xylose/glucose, the N-terminal 31 amino acid sequence of type II glucose isomerase (GIase) from the hyperthermophilic bacterium, Thermoanaerobacter thermohydrosulfuricus (TTGIase) was fused to the N-terminus of the type I glucose isomerase from Thermobifida fusca (TFGIase), to construct the fusion protein N-TFGIase. The experimental results showed that under the same culture and induction conditions, total yield of N-TFGIase produced by the bacteria was approximately 40% higher than that of TFGIase. The specific enzyme activity of N-TFGIase was 26% higher than that of TFGIase and the optimum temperature of N-TFGIase was 5 ℃ higher than that of TFGIase. The half-life of N-TFGIase at 75 ℃ was extended by 30% compared to that of TFGIase, and the optimum pH of N-TFGIase was reduced by 1.0 compared to that of TFGIase. Sequence analysis showed that the mRNA secondary structure of the N-terminal sequence of TTGIase did not form a blocked cervical-loop structure, thereby improving the efficiency of expression of the fusion protein. The hydrophobicity indices of 31 amino acid residues of the N-terminal sequence were all less than zero, favoring the initial folding and packaging of the fusion protein. The proportion of acidic amino acid residues was approximately twice that of the basic amino acid residues of the N-terminal sequence, thereby reducing the impact of the acidic medium environment on the surface of the fusion protein molecules.

国家863计划项目(2012AA021500)；国家自然科学基金项目（81274021）；国家自然科学基金项目（H2801）；皖西学院科研基金项目（WXZR201616）