Characterization, Activity And Purification Of Starch-Debranching Enzymes During Wheat Grain Filling Period

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 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. Under the optimized conditions for the enzyme extracting and activity analysis, we compared the changes of SDBE activity during different stages among different wheat cultivars. Furthermore, SDBE was 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 SDBE activity during different stages among different cultivarsThe changes of DBE activity among six wheat cultivars, named Nuomai 2, Jinan 16, Jining 13 and Lumai 1, Yanyou361 and Lumai 21 were investigated in the paper. As a result, during different stages of filling, the changes of SDBE 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, but the activity of pullulanase appeared as undee-peak curve, and it got another high activity around 25th day after flowering.2 Isolation and purification of SDBEBy the method of ammonium sulfate precipetation and column chromatography on DEAE Cellulose and sephadex G-100 column, SDBE in the endosperm of wheat was purified. Two fractions, named isoamylase and pullulanse, were obtained. Their weight molecular were 83 and 100 kD respectively.Research on the characterization of the purified isoamylase proved that the enzyme showed optimal activity at pH 6.0 and 30℃. It was stable in the pH range from 6.0 to7.0 and temperature range from 28 to 37℃. For purified pullulanase, the optimal activity at pH 5.5 and 50℃. The two types of starch debranching enzymes all be improved by Mn2+, Ca2+ and be iminhibited by Fe2+, Cu2+strongly. The survival rate of enzyme activity remained over 80% when stored at 4℃for a month.3 Digestion research of SDBE on amylopectinUsing amylopectin and pullulan as substrates spectivily, for different types of wheat crude extracts, the chromatograms of HPLC were similar.When amylopecin was hydrolyzed, the retention time of hydrolysation was 4 to 5min and 15 to 16min. While pullulan was hydrolyzed, there was only one peak, its rentention time was 6 to 7min, Glucose and dextrin were identified as the major hydrolysis products of amylopectin by isoamylase and maltotriose was identified as a major hydrolysis product of pullulan by pullulanase.