通过PCR克隆得到了不包括信号肽序列的109～1803 bp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10 μmol/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46.8 mmol/L,Vmax为152.2 U/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 isom erase (SI) gene (109-1803 bp , excluded its signal peptide sequence ) from Erwiniarhapontici NCPPB 1578 was cloned and expressed in Escherrhia coliw ith pQ E30 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 condilions for expression , the cell was induced with lO μm ol/L IPTG at 28℃ , the solubje expression was realized in the cell . The optimum pH and temperature for the purified recom binant SI activity was 6.0 and 30℃ respectively , and its Km was 46.8 mm ol/L and its Vm ax was 152 .2 U/mg . SI was not reactive to maltose , trehalose , raffiose , sucralose and α-m ethylgjucoside . It only reacted with sucrose and ρNPG lc.Inadditon , isomalulose as the substrate might be transformed into trehalulose .
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