本文以质粒pET-22b(+)为载体，Thermomonospora curvata淀粉分支酶基因(TcSBE)为客体，采用构建重组大肠杆菌BL21(pET-22b(+)-TcSBE)的方法,实现TcSBE过量表达。目标分支酶经分离纯化，酶活达90.28 U/mg，并分别以长直链淀粉和短直链糊精为底物，研究了TcSBE作用淀粉机理。结果表明：TcSBE以链间反应模式催化长直链淀粉生成大分子的支链淀粉和小分子的低聚糖；以短直链糊精为底物时，TcSBE通过水解作用和转糖基作用同时作用于底物，反应初期，水解作用较强，将底物随机水解成聚合度不等的小分子直链糊精，所需供体链的最低聚合度（DP）为12，随着反应的进行，水解作用不断减弱，转糖基作用增强，将DP 3~8的短链糊精通过α-1,6糖苷键与产物相连接，使产物中分支侧链含量增加，反应12 h后，TcSBE作用产物的α-1,6糖苷键含量达到0.9 mM。

A gene (TcSBE) encoding starch branching enzyme from Thermomonospora curvata was cloned into the plasmid of pET-22b(+) and overexpressed in Escherichia coli BL21. The recombinant enzyme was purified and showed a high specific activity to amylose with 90.28 U/mg. The reaction mechanism of TcSBE was examined relative to its reaction model and transglycosylation for amylose and linear dextrin. The results indicated that TcSBE could catalyze the inter-chain branching of amylose into macromolecular amylopectin and micromolecular oligosaccharides. The enzyme simultaneously catalyzed the hydrolysis and transfer reaction as it was incubated with linear dextrin. The recombinant enzyme cleaved linear dextrin into short chains with different degree of polymerization (DP) at a random mechanism, and the minimal donor chain length was DP 12. As the reaction proceeded, the enzyme exhibited a high transglycosylation activity, and predominantly transferred short chains of DP 3-8 throughout the branching process. After 12 h of incubation, the highly branched product contained 0.9 mM α-1,6 linkages from the enzyme reaction with linear dextrin as the substrate.

国家自然科学基金项目（31571792）