Abstract:Isoamylase is able to cleave α-1, 6-linkages in amylopectin, which is essential for starch saccharification. Research on mechanism of Thermobifida fusca isoamylase(IAM) by instrumental analysis methods has not been reported. In this study, IAM was purified by affinity chromatography and used to hydrolyze amylopectin from maize(AP). Results of size exclusion chromatography (SEC) indicated that IAM hydrolyzed both inner and outer branching linkages of amylopectin. The polymer structure was completely destroyed by IAM and the mean molecular weight(Mw) decreased from 2 812 000 to 760 000 and 7.2 000 after 2 h, the ratio of large glucans to the small ones was 1∶4.4. The population of starch fragments released was characterized by high performance anion exchange chromatography with pulsed amperometric detector(HPAEC-PAD), the degree of polymerization(DP) of final hydrolysates distributed mainly in 15~35, released maltose and maltotriose demonstrated that the smallest units hydrolysed by IAM were side chains with 2~3 glucose residues. 1H nuclear magnetic resonance(1H-NMR) spectrum showed that IAM specifically and completely cleaved α-1, 6 glycosidic linkages and produced amylose chains or maltoligosaccharides with α- or β-anomeric reducing ends. The mechanism analysis of IAM could lay the foundation for its applications.
