Abstract:

【Objective】Isoamylases are debranching enzymes that hydrolyze α-1, 6 linkages in α-1, 4/α-1, 6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. 【Method】The isoamylase-oligomer were fractionated by salting out with ammonium sulphate, DEAE-cellulose column chromatograthy, and gel filtration using sephadex G-200. The constructure and component of ISA complexes were studied by using native polyacrylamide gel electrophoresis (Native-PAGE) and SDS polyacrylamide gel electrophoresis (SDS-PAGE). 【Result】 Results of the study showed that there are two distinct polymeric forms of isoamylase (ISA) in wheat endosperm: Presumably a homo-tetramer of ISA1 and a hetero-oligomer composed of four ISA1 and one ISA2. The molecular sizes of the homo- and hetero-oligomers were approximately 320 and 380 kD, respectively. The ISA1 homo-oligomer optimum temperature was 30℃, after 10 min 40℃ treatment, the activity was completely lost, while the optimum temperature of ISA1/ISA2 heter-oligomer was 35℃, which was active even when incubated at 50℃ for 10 min. Native-PAGE showed that ISA2 not existed separate activity，but when the ISA1 homo-oligomer was incubated with the ISA2, it had the same bands as ISA1/ISA2 hetro-oligomer, indicating that ISA1 and ISA2 combined to form a hetero-oligomer. 【Conclusion】Two multimeric forms of ISA are present in wheat endosperm. The hetero-oligomer was found to be more stable at high temperatures of 40℃ and 50℃. These results indicate that ISA2 plays an important role in amylopectin biosynthesis by enhancing the activity of the ISA1 homo-oligomer and facilitating the binding of the hetero-oligomer to polyglucan substrates.

Key words: wheat, starch debranching-enzyme, isoamylase-oligomer, thermal stability