蔗糖异构酶突变菌株的构建及其应用研究

Engineering of Mutant Sucrose Isomerase Producing Strain and Its Application

蔗糖异构酶是糖苷水解酶13家族的重要成员,可以异构化蔗糖生成异麦芽酮糖和海藻酮糖,同时水解生成少量的葡萄糖和果糖。通过定点突变法对土壤发散菌来源的蔗糖异构酶基因进行定点突变,在大肠杆菌中实现异源表达,获得3例突变菌株R-M1(Q299E),R-M2(Q299D),R-M3(Q299N)。R-M1转化蔗糖的产物当中,异麦芽酮糖的比例从90.28%升至93.16%,海藻酮糖比例从3.09%降低到1.79%。对突变菌株R-M1游离细胞转化蔗糖底物的条件进行了优化,确定了最适转化条件为:30℃条件下,投入浓度为8×109cfu/m L的细胞到蔗糖浓度为50%的10 m L的磷酸柠檬酸缓冲液中,反应90 min,可实现蔗糖最大程度的转化,麦芽酮糖产物浓度达到460 mg/m L。

Sucrose isomerase was a major member in the glucoside hydrolase family 13. In addition to catalyzing the isomerization of sucrose to isomaltulose, it also produced trehaltulose and small amounts of fructose and glucose. Sim derived from Pantoea dispersa had been subject to site-directed mutagenesis; 3 mutants（RM1（Q^299E）,R-M2（Q^299D）,R-m^3（Q^299N）） were obtained. Q^299 E exhibited increased isomaltulose content（from90.28 % to 93.16 %） and reduced percentage of trehalulose（from^3.09 % to 1.79 %）. The optimal conditions for sucrose conversion of mutant R-M1 was determined as follows ： the culture（substrate concentration was 8 × 10^9cfu/mL） was centrifuged and then resuspended in 10 mL of citrate/phosphate-buffered 50 % sucrose solution and incubated for 90 min at 30 ℃. Sucrose was completely converted under the above conditions and the concentrations of isomaltulose reached up to 460 mg/mL.