Purification And Enzymatic Characterization Of Isoamylase-oligomer From Wheat Endosperm

Wheat starch is not only the decision of the output, but also have an important impact on the quality of processing. 70%~80% of starch is amylopectin, it is an important factor of physiochemical properties of starch. In the past, the metabolic function of debranching enzymes was thought to be the degradation of amylopectin in storage starch during germination, or in tranditory starch. Several recent observations, however, suggest that starch-debranching enzymes also play essential roles in amylopectin synthesis. However, DBE in starch synthesis in the process of the mechanism has not yet been clearly. In plants, there are two types of starch-debranching enzyme, which hydrolyze theα-1,6-glulcodidic linkages of amylopectin. Both types of debranching enzyme greatly differ in their substrate specificity: isoamylase can debranch glycogen and phytoglycogen, but can scarcely attack pullulan, while the reverse is true for pullulanase.In order to elucidate the structure and function of the three isoamylase isozymes, and the relationship between different oligomer, six wheat cultivars which amylopectin content of the existence of significant differences were used in the present study. We compared the changes of DBE activity during different stages among different wheat cultivars. Furthermore, Isoamylase-oligomerwas purified and its characterization was studied for the first time. This will establish theoretical foundation for further research on the mechanism of amylopectin formation in endosperms of wheat in the future. The main results are as follows:1 Changes of DBE activity during different stages among different cultivarsThe changes of DBE activity among six wheat cultivars, named Nuomai 2, Jinan 16, Jining 13, Lumai 1, Yanyou361 and Lumai 21 were investigated in the paper. As a result, during different stages of filling, the changes of DBE activity were similar among the six cultivars. They all reached the highest activity at 15th day after anthesis, the activity of isoamylase appeared as a single-peak curve.2 Type and expression of the isoamylase isozymesNative-PAGE showed that wheat endosperm has a number of different bands with isoamylase activity during grain filling period. This result prove that isoamylase isozymes exist in the wheat grain throughout the grouting process to ensure production transportation and accumulation.3 Purification and enzymatic characterization of isoamylase-oligomerBy the method of ammonium sulfate precipetation and column chromatography on DEAE Cellulose and sephadex G-200 column, isoamylase-oligomer in the endosperm of wheat was purified. Two fractions were obtained: presumably a homo-oligomer of ISA1 and a hetero-oligomer composed of four SA1 and one ISA2. The molecular sizes of the homo- and hetero-oligomers, were approximately 320 and 380 kDa, respectively. The fractions exhibiting the first ISA peak contained two protein band as visualized by Coomassie brilliant blue staining of the SDS-PAGE gel with sizes of approximately 83 and 90 kDa, while those exhibiting the second ISA peak contained only a single 83 kDa protein bands.The ISA1 homo-oligomer optimum temperature for the 30℃, after 10min 40℃treatment completely lost activity, 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 exist separate activity, but When the ISA1 homo-oligomer was incubated with the ISA2, it had the same bands with ISA1-ISA2 hetro-oligomer, indicating that ISA1 and ISA2 combine to form a hetero-oligomer.