Band 5, Heft 1

Pozol is a beverage made with maize dough that is prepared after boiling the kernels in limewater, causing a decrease in soluble sugars, with starch being the main fermentable carbohydrate in the dough. Previously, Streptococcus infantarius ssp. infantarius 25124 ( Sii -25124) was identified as the most amylolytic bacteria isolated in this product. Analysis of Sii -25124 amylolytic enzymes revealed two amylases, a cytoplasmic α-amylase of 55.7 kDa and an extracellular amylopullulanase of 246.3 kDa, with two catalytic domains, one typical of an α-amylase and another typical of a pullulanase/glycogen debranching enzyme. Characterization of the joint activity of both enzymes using Sii -25124 cell lysate supernatant demonstrated stability between 30 °C and 45°C, and pH stability in a range between 6.8 and 8.0. The joint activity of Sii -25124 amylases showed a fast production of reducing sugars when starch was used as the substrate. In contrast, reducing sugar production from amylopectin was lower, but it steadily increased throughout the reaction time. The amylopullulanase produced by Sii -25124 hydrolyzes the starch in the dough to produce low molecular weight oligosaccharides, which may be transported into Sii -25124 cells, so that intracellular α-amylase hydrolyzes them to mono- and disaccharides. Amylopullulanase production by Sii -25124 could be an example of a specialized enzyme that successfully dominates starchy food fermentation.

The 4,6-α-glucanotransferases of the glycoside hydrolase family 70 can convert starch into isomaltooligosaccharides (IMOs). However, no thermostable 4,6-α-glucanotransferases have been reported to date, limiting their applicability in the starch conversion industry. Here we report the identification and characterization of a thermostable 4,6-α-glucanotransferase from Bacillus coagulans DSM 1. The gene was cloned and the recombinant protein, called BcGtfC, was produced in Escherichia coli . BcGtfC is stable up to 66 °C in the presence of substrate. It converts debranched starch into an IMO product with a high percentage of α-1,6-glycosidic linkages and a relatively high molecular weight compared to commercially available IMOs. Importantly, the product is only partly and very slowly digested by rat intestine powder, suggesting that the IMO will provide a low glycaemic response in vivo when applied as food ingredient. Thus, BcGtfC is a thermostable 4,6-α-glucanotransferase suitable for the industrial production of slowly digestible IMOs from starch.

Cyclodextrin-hydrolyzing enzymes are widespread in bacteria and archaea where they play their roles in carbohydrates metabolism. They were previously characterized as cyclodextrinases, neopullulanases and maltogenic amylases. In the Carbohydrate-Active enZyme (CAZy) database, most of these enzymes are grouped into the GH13_20 subfamily of the α-amylase family GH13. Here, we have summarized the information available on the substrate specificity, structural features, physiological roles and applications of cyclodextrin-preferring glycoside hydrolases. These enzymes form a distinct group in the α-amylase family. Members of this distinct group possess an extra extension at the N-terminus, which causes a modification of the active site geometry thus making these enzymes more specific for smaller molecules like cyclodextrins than for macromolecules such as starches or pullulan. Multi-substrate specificity, hydrolytic as well as transglycosylation activities make these enzymes attractive for applications in the food and pharmaceutical industries. We have tried here to collect information available on their biochemical properties, three-dimensional structures, physiological roles and potential applications.

The industrial thermostable Bacillus licheniformis α-amylase (BLA) has wide applications, including in household detergents, and efforts to improve its performance are continuously ongoing. BLA during the industrial production is deamidated and glycated resulting in multiple forms with different isoelectric points. Forty modified positions were identified by tandem mass spectrometric peptide mapping of BLA forms separated by isoelectric focusing. These modified 12 asparagine, 9 glutamine, 8 arginine and 11 lysine residues are mostly situated on the enzyme surface and several belong to regions involved in stability, activity and carbohydrate binding. Eight residues presumed to interact with starch at the active site and surface binding sites (SBSs) were subjected to mutational analysis. Five mutants mimicking deamidation (N→D, Q→E) at the substrate binding cleft showed moderate to no effect on thermostability and k cat and K M for maltoheptaose and amylose. Notably, the mutations improved laundry wash efficiency in detergents at pH 8.5 and 10.0. Replacing three reducing sugar reactive side chains (K→M, R→L) at a distant substrate binding region and two SBSs enhanced wash performance especially in liquid detergent at pH 8.5, slightly improved enzymatic activity and maintained thermostability. Wash performance was most improved (5-fold) for the N265D mutant near substrate binding subsite +3.

Glycogen is a natural polysaccharide with a dendrimer structure, in which glucose is frequently branched and polymerized. Functionalizing the numerous non-reducing ends on the molecular surface of glycogen could be expected to enable its use in various fields. We developed a method for enzymatically synthesizing a suitable form of glycogen from sucrose by using sucrose phosphorylase and branching enzyme, both of which belong to the α-amylase family, as well as glucan phosphorylase. We refer to this enzymatically synthesized glycogen as the glucan dendrimer (GD). We then selectively modified the non-reducing ends on the surface of GD particles by using the reaction of glucan phosphorylase with various hexose 1-phosphates. Modifying the non-reducing ends of GD with glucuronic acid or glucosamine added negative and positive charges to the GD particles. In addition, we found that glucuronic acid and/or glucosamine residues at the non-reducing ends can be used to covalently conjugate functional substances, such as sugar chains, proteins, and peptides to the surface of GD particles. GD and modification of its non-reducing ends represent versatile platforms for pharmaceutical applications of polysaccharides.