Enzymatic Synthesis and Properties of Highly Branched Rice Starch Amylose and Amylopectin Cluster

We enzymatically modified rice starch to produce highly branched amylopectin and amylose and analyzed the resulting structural changes. To prepare the highly branched amylopectin cluster (HBAPC), we first treated waxy rice starch with Thermus scotoductus α-glucanotransferase (TSαGT), followed by treatment with Bacillus stearothermophilus maltogenic amylase (BSMA). Highly branched amylose (HBA) was prepared by incubating amylose with Bacillus subtilis 168 branching enzyme (BBE) and subsequently treating it with BSMA. The molecular weight of TSαGT-treated waxy rice starch was reduced from 8.9 x 10⁸ to 1.2 x 10⁵ Da, indicating that the α-1,4 glucosidic linkage of the segment between amylopectin clusters was hydrolyzed. Analysis of the amylopectin cluster side chains revealed that a rearrangement in the side-chain length distribution occurred. Furthermore, HBAPC and HBA were found to contain significant numbers of branched maltooligosaccharide side chains. In short, amylopectin molecules of waxy rice starch were hydrolyzed into amylopectin clusters by TSαGT in the enzymatic modification process, and then further branched by transglycosylation using BSMA. HBAPC and HBA showed higher water solubility and stability against retrogradation than amylopectin clusters or branched amylose. The hydrolysis rates of HBAPC and HBA by glucoamylase and α-amylase greatly decreased. The kcat/Km value of glucoamylase acting on the amylopectin cluster was 45.94 s-1(mg/mL)-1 and that for glucoamylase acting on HBAPC was 11.10 s-1(mg/mL)-1, indicating that HBAPC was 4-fold less susceptible to glucoamylase. The kcat/Km value for HBA was 15.90 s-1(mg/mL)-1, or about three times less than that for branched amylose. The kcat/Km values of porcine pancreatic α-amylase for HBAPC and HBA were 496 and 588 s-1(mg/mL)-1, respectively, indicating that HBA and HBAPC are less susceptible to hydrolysis by glucoamylase and α-amylase. HBAPC and HBA show potential as novel glucan polymers with low digestibility and high water solubility.