源于类芽胞杆菌的低聚糖脱支酶底物特异性及其应用研究

Studies on Substrate Specificity and Application of an Oligo-1,6-glucosidase from Paenibacillus sp.STB16

脱支酶是淀粉深加工产业的常用酶制剂,然而传统脱支酶难以完全满足淀粉糖绿色生产及副产物综合利用的需求,因此有必要挖掘适合的新型脱支酶。本研究筛选并构建来源于类芽胞杆菌STB16的低聚糖脱支酶基因(oga)的枯草芽孢杆菌表达系统,实现低聚糖脱支酶的异源表达,并探究重组酶的酶学性质和底物特异性。结果表明,枯草芽孢杆菌发酵上清液的酶活力可达162.33 U/mL,重组酶的最适反应温度50℃、最适反应pH值6.0,且专一地作用于底物中聚合度为1的分支的α-1,6-糖苷键。相比于其它类型的脱支酶(底物转化率低于检出限),重组低聚糖脱支酶对异麦芽糖、异麦芽三糖、潘糖等异麦芽低聚糖具有更彻底的水解能力,底物转化率达97.4%~100%,且产物为葡萄糖,因此更适合用于处理葡萄糖母液,可使一次母液、二次母液和色谱分离尾液中葡萄糖含量分别提升3.6%,12.7%和34.4%。相关研究结果可为新型脱支酶的开发和利用奠定理论基础,也为淀粉糖加工副产物的综合利用提供重要参考。

Debranching enzymes are commonly used enzymes in the starch deep processing industry.However,traditional debranching enzymes are unable to fully meet the requirements of green production of starch sugars and comprehensive utilization of by-products.Therefore,it is necessary to explore suitable new types of debranching enzymes.This study screened an oligo-1,6-glucosidase gene(oga)from Paenibacillus sp.STB16 and constructed its Bacillus subtilis expression system.This system achieved heterologous expression of the oligo-1,6-glucosidase.The enzymatic properties and substrate specificity of the recombinant oligo-1,6-glucosidase were explored.The results showed that the enzyme activity of the fermentation supernatant of B.subtilis could reach 162.33 U/mL.The recombinant oligo-1,6-glucosidase had the optimum reaction temperature at 50℃and optimum reaction pH at 6.0.It specifically acted on theα-1,6-glycosidic bonds of branches with degree of polymerization of 1.Compared to other types of debranching enzymes(substrate conversion rate under the detection limit),the recombinant oligo-1,6-glucosidase exhibited stronger hydrolytic activity towards isomaltose,isomaltotriose,and panose.The substrate conversion rate reach 97.4%-100%,with glucose as the product.Therefore,it is more suitable for treating glucose mother liquor.The glucose content in the first mother liquor,second mother liquor and tail liquor of chromatographic separation was significantly increased by 3.6%,12.7%and 34.4%,respectively.The research results provide a theoretical basis for the development and utilization of new types of debranching enzymes,and also serve as an important reference for the comprehensive utilization of by-products in starch sugar processing.