Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168

Abstract

The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and ␤-cyclodextrin (␤-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and ␤-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched ␤-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the ␣-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.Bacillus subtilis can utilize polysaccharides such as starch, glycogen, and amylose as carbon sources by hydrolyzing them into smaller maltodextrins via the action of extracellular ␣-amylase (AmyE) (14). In B. subtilis, ␣-glucosidase encoded by malL has been known to contribute to maltodextrin metabolism in the cell (40, 41). Schönert et al. (42) reported that maltose is transported by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in B. subtilis. They also reported that maltodextrins with degrees of polymerization (DP) of 3 to 7 (G3 to G7) are taken up via a maltodextrin-specific (Mdx) ATP-binding cassette (ABC) transport system (42).This system is made up of a maltodextrin-binding protein (MdxE) and two membrane proteins (MdxF and MdxG), as well as an ATPase (MsmX). The basic model proposed for the transport and metabolism of maltooligosaccharides includes a series of carbohydrate-hydrolyzing and -transferring enzymes. However, the enzymatic hydrolysis of maltodextrins and glycogen, providing a major energy reservoir ...

Size, structure and scaling relationships in glycogen from various sources investigated with asymmetrical flow field-flow fractionation and 1H NMR

“…The lowest scattering angle, 14.4 • , was not included, as the data obtained were imprecise. A dn/dc value of 0.146 ml g −1 was used [23] and the second virial coefficient was assumed to be negligible.…”

Role of Maltose Enzymes in Glycogen Synthesis by Escherichia coli

“…The weight-average molecular weight (M w ) of the sample was calculated using ASTRA V4.90.07 software (Wyatt Technology), using the Berry extrapolation method for curve fitting and a dn/dc value of 0.146 ml/g. The details of the method were described by Shim et al (45).…”

“…The dried glycogen was resuspended in 1 ml of sterile distilled water and stored at Ϫ18°C until further analysis. The quantification of the glycogen was carried out as described previously by Shim et al (45).…”

Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

“…Moreover, a limited dextrin containing a branch chain of DP4 is produced via the action of glycogen phosphorylase and is also conveniently studied in relation to mammalian debranching enzyme. The average branch chain lengths of glycogen in E. coli (28), S. solfataricus (35), and B. subtilis (36) are DP7 to -11, and the structures are expected to be similar to that of mammalian glycogen (DP13). Meléndez-Hevia et al (34) and Meléndez et al (37) developed a mathematical model of the mammalian glycogen molecule.…”