【摘要】 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。更多还原

【Abstract】 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 could 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 μmoI/L IPTG at 28 ℃,the soluble expression was realized in the cell.The optimum pH and temperature for the purified recombinant 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 glucoside.It only reacted with sucrose and ρNPQc. In addition,isornaltulose as the substrate might be transformed into trehalulose.更多还原