Isoamylase HP (Glycogen 6-glucanohydrolase)

Starch hydrolysis by gut microbiota involves a diverse range of different enzymatic activities. Glucan-branching enzyme GlgB was identified as the most abundant glycosidase in Firmicutes in the swine intestine. GlgB converts α-(1→4)-linked amylose to form α-(1→4,6) branching points. This study aimed to characterize GlgB cloned from a swine intestinal metagenome and to investigate its potential role in formation of α-(1→4,6)-branched α-glucans from starch. The branching activity of purified GlgB was determined with six different starches and pure amylose by quantification of amylose after treatment. GlgB reduced the amylose content of all 6 starches and amylose by more than 85% and displayed a higher preference towards amylose. The observed activity on raw starch indicated a potential role in the primary starch degradation in the large intestine as an enzyme that solubilizes amylose. The oligosaccharide profile showed an increased concentration of oligosaccharide introduced by GlgB that is not hydrolyzed by intestinal enzymes. This corresponded to a reduced in vitro starch digestibility when compared to untreated starch. The study improves our understanding of colonic starch fermentation and may allow starch conversion to produce food products with reduced digestibility and improved quality.

To determine the internal structure of barley starch without amylopectin isolation, whole starch was hydrolyzed using β-amylase to remove the linear amylose and obtain β-limit dextrins (β-LDs). The β-LDs were treated with extensive α-amylase to prepare α-limit dextrins (α-LDs), and the α-LDs were further hydrolyzed with β-amylase into building blocks. The chain-length distribution of β-LD and building block composition were analyzed by size-exclusion chromatography and anion-exchange chromatography. The internal structure of the barley whole starches had similar pattern to barley amylopectins analyzed by conventional methods. The starch of barley amo1-mutated varieties contained more short internal B-chains and less long internal B-chains than that of other varieties. The starch from amo1-mutated varieties had more large building blocks than that from waxy varieties. The simplified method presented in this study can effectively characterize starch internal structure that relates to physicochemical properties of starch, although some details of amylopectin structure are not assessable.

Dextrans and other bacterial α-glucans are versatile and structurally diverse polysaccharides which can be enzymatically synthesized by using glucansucrases. By substituting certain amino acids in the active site of these enzymes, the structure of the synthesized polysaccharides can be modified. In this study, such amino acid substitutions were applied (single and combined) to the dextransucrase from Lactobacillus reuteri TMW 1.106 and the structures of the synthesized polysaccharides were subsequently characterized in detail. Besides methylation analysis, α-glucans were hydrolyzed by several glycoside hydrolases and the liberated oligosaccharides were identified by comparison to standard compounds or by isolation and NMR spectroscopic characterization. Furthermore, two-dimensional NMR spectroscopy was used to analyze the untreated polysaccharides. The results demonstrated that structurally different α-glucans were formed, for example different highly O4-branched dextrans or several reuteran-like polymers with varying fine structures. Consequently, mutant Lactobacillus reuteri TMW 1.106 dextransucrases can be used to form structurally unique polysaccharides.