2 CLASSES OF STARCH DEBRANCHING ENZYMES FROM DEVELOPING MAIZE KERNELS

Based on their substrate specificities, two distinct classes of starch debranching enzymes have been isolated from developing maize kernels. One class of enzyme hydrolyzed pullulan most rapidly followed by beta-limit dextrin, amylopectin, and phytoglycogen. This activity was classified as a pullulanase (E.C. # 3.2.1.41). A different class of enzyme debranched amylopectin most rapidly, followed by beta-limit dextrin and phytoglycogen. It, however, would not hydrolyze pullulan and was classified as isoamylase (E.C. # 3.2.1.68). Two forms of isoamylase were isolated which differed in relative molecular mass and eluted separately on anion exchange chromatography, but only one form was characterized. Both classes of enzyme activity caused an increase in starch-iodine staining when incubated in an amylopectin solution. The optimal pH for pullulanase activity was pH 5.5, whereas isoamylase had optimal activity at pH 6.0 to 7.5. Both classes of enzyme required 5 mM dithiothreitol or the equivalent sulfhydryl reducing power for activity. We speculate that pullulanase may be specialized to function in conjunction with beta-amylase in hydrolyzing exposed alpha-1,6 branches, whereas isoamylase appears to be more efficient at hydrolyzing less exposed branches in amylopectin.