Overexpression of Starch Branching Enzyme from Thermomonospora curvata and Its Catalytic Mechanism Research
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Abstract:
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.
