大黄欧文氏菌蔗糖异构酶基因克隆表达及其酶学特性研究

Expression and characterization of sucrose isomeras from Erwinia rhapontici

通过PCR克隆得到了不包括信号肽序列的109～1803bp大黄欧文氏菌蔗糖异构酶基因,以pQE30为表达载体可以在大肠杆菌中高效表达SI基因,但容易形成包涵体。通过降低诱导剂IPTG至10μmol/L,28℃低温诱导表达可以改进表达产物的可溶性。重组SI的最适pH为6.0,最适反应温度为30℃。测定的Km为46.8mmol/L,Vmax为152.2U/mg。重组SI对麦芽糖、海藻糖、棉子糖、三氯蔗糖及α-甲基葡萄糖苷等均不起作用,只利用蔗糖为底物转糖苷,也可以水解对硝基苯酚-α-葡萄糖苷。同时发现重组SI具有转化异麦芽酮糖生成海藻酮糖的活性。

Sucrose isomerase （SI） gene （109-1803 bp, excluded its signal peptide sequence） from Erwinia rhapontici NCPPB 1578 was cloned and expressed in Escherichia coli with pQE30 as expression vector, which muld realize high-quality expression of SI in E. coli, but inclusion body was likely to appear in the expression product. Then, by optimizing conditions for expression, the cell was induced with 10 μmol/L IPTG at 28℃, the soluble expression was realized in the cell. The optimum pH and temperature for the purified recombimnt SI activity was 6.0 and 30℃ respectively, and its Km was 46.8 mmol/L and its Vmax was 152.2 U/ mg. SI was not reactive to maltose, trehalose, raffiose, sucralose and a-methyl gluoaside. It only reacted with sucrose and ρNPGlc. In addition, isomalmlose as the substrate might be transformed into trehalulose.