D-甘露糖异构酶在大肠杆菌中的表达及催化条件的研究

根据已知的放射性土壤杆菌体内的D-甘露糖异构酶（Fmi）氨基酸序列，设计适合在大肠杆菌内表达的Fmi基因序列（1245bp），采用体外基因拼接合成。构建了表达Fmi的重组大肠杆菌BL21(DE3)/pET30-Fmi，诱导表达的Fmi蛋白经SDS-PAGE验证为可溶性蛋白，分子量44000。通过实验优化了Fmi的催化条件，结果表明Fmi催化的最适pH值为7.5，最适温度为45℃，催化1h酶活达5.29U/mL。以25%的果糖溶液为底物时，催化2h果糖转化率达29.4%。

Expression of D-mannose isomerase in E.coli and a study of its catalytic activity

The Fmi gene sequence has been designed based on the amino acid sequence of Fmi in agrobacterium radiobactor in order to express Fmi efficiently in E.coli.The designed sequence was synthesized using the gene splicing method,and the recombinant plasmid pET30-Fmi was obtained and then transformed into the expression host BL21(DE3).After induction and culture,the expressed protein molecular mass was estimated to be 44000 and shown to be soluble by SDS-PAGE.The enzyme showed a maximum activity at pH 7.5 and a temperature of 45℃,under which conditions the enzymatic activity reached 5.29U/mL in 1h;under these optimum conditions,the conversion of 25% fructose as the initial substrate was 29.4% after 2h.