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Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ⌬malP (encoding maltodextrin phosphorylase), ⌬malQ (encoding amylomaltase), ⌬glgA (encoding glycogen synthase), and ⌬glgA ⌬malP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ⌬malP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ⌬malP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ⌬malQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ⌬glgA and ⌬glgA ⌬malP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.The synthesis of glycogen in bacteria occurs when they are grown with limited nutrients but an abundance of a carbon source (33,34). Escherichia coli accumulates glycogen at levels of more than half of its cell mass under optimal conditions. The glycogen gene cluster in E. coli consists of two operons oriented in tandem, glgBX and glgCAP, encoding enzymes that synthesize and degrade glycogen (12). The encoded enzymes are a branching enzyme (glgB), a debranching enzyme (glgX), an ADP-glucose pyrophosphorylase (glgC), a glycogen synthase (glgA), and a glycogen phosphorylase (glgP). The polymerization of the ␣-1,4-linked glucosyl chain is mediated via the transfer of glucose from ADP-glucose by GlgA, the glycogen synthase, onto the nonreducing ends of linear dextrins that are subsequently branched (formation of ␣-1,6-glycosyl linkage) by GlgB, the branching enzyme. The expression of the glg gene cluster is complicated. It involves the global carbon storage regulator CsrA (2, 53), the cyclic AMP (cAMP)/catabolite gene activator protein (CAP) system (39), and the stringent response (38). In addition, the two-component regulatory system PhoP-PhoQ (29) connects the system to Mg 2ϩ levels, and even the phosphotransferase system appears to affect the glycogen phosphorylase involved in the degradation of glycogen (42,43). glgS, an additional gene involved in glycogen synthesis, is not part of the glg gene cluster. It is not essential for ...

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 alpha-glucanotransferase (TSalphaGT), 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 TSalphaGT-treated waxy rice starch was reduced from 8.9 x 10(8) to 1.2 x 10(5) Da, indicating that the alpha-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 TSalphaGT 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 alpha-amylase greatly decreased. The k cat/ K m 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 k cat/ K m value for HBA was 15.90 s(-1)(mg/mL)(-1), or about three times less than that for branched amylose. The k cat/ K m values of porcine pancreatic alpha-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 alpha-amylase. HBAPC and HBA show potential as novel glucan polymers with low digestibility and high water solubility.