Pachyman (1,3-β-D-Glucan)

An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked β-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards β-glucan, with a k_m of 7.9 mg/mL and a k_cat of 117.2 s^-1, compared to lichenin which had a k_m of 21.5 mg/mL and a k_cat of 70.0 s^-1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% β-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit.

We previously reported that β-glucosidase BGL1 at low concentration (15 µg mL^-1) from Coprinopsis cinereal exhibited hydrolytic activity only toward laminarioligosaccharides but not toward cellooligosaccharides and gentiobiose. This study shows that BGL1 at high concentration (200 µg mL^-1) also hydrolyzed cellobiose and gentiobiose, which accounted for only 0.83 and 2.05% of its activity toward laminaribiose, respectively. Interestingly, BGL1 at low concentration (1.5 µg mL^-1) showed transglycosylation but BGL1 at high concentration (200 µg mL^-1) did not. BGL1 utilizes only laminarioligosaccharides but not glucose, gentiobiose, and cellobiose to synthesize the higher oligosaccharides. BGL1 transferred one glucosyl residue from substrate laminarioligosaccharide to another laminarioligosaccharide as an acceptor in a β(1 → 3) or β(1 → 6) fashion to produce higher laminarioligosaccharides or 3-O-β-D-gentiobiosyl-D-laminarioligosaccharides. The BGL1-digested laminaritriose exhibited approximately 90% enhancement in the anti-oxidant activity compared to that of untreated laminaritriose, implying a potential application of BGL1-based transglycosylation for the production of high value-added rare oligosaccharides.

(1,3)(1,4)-β-D-Glucan (mixed-linkage glucan or MLG), a characteristic hemicellulose in primary cell walls of grasses, was investigated to determine both its role in cell walls and its interaction with cellulose and other cell wall polysaccharides in vitro. Binding isotherms showed that MLG adsorption onto microcrystalline cellulose is slow, irreversible, and temperature-dependent. Measurements using quartz crystal microbalance with dissipation monitoring showed that MLG adsorbed irreversibly onto amorphous regenerated cellulose, forming a thick hydrogel. Oligosaccharide profiling using endo-(1,3)(1,4)-β-glucanase indicated that there was no difference in the frequency and distribution of (1,3) and (1,4) links in bound and unbound MLG. The binding of MLG to cellulose was reduced if the cellulose samples were first treated with certain cell wall polysaccharides, such as xyloglucan and glucuronoarabinoxylan. The tethering function of MLG in cell walls was tested by applying endo-(1,3)(1,4)-β-glucanase to wall samples in a constant force extensometer. Cell wall extension was not induced, which indicates that enzyme-accessible MLG does not tether cellulose fibrils into a load-bearing network.

A comparative study on preparing dietary fibres (DFs) from three mushroom sclerotia, namely, Pleurotus tuber-regium (PTR), Polyporus rhinocerus (PR) and Wolfiporia cocos (WC), using analytical or industrial enzymes (including α-amylase, protease and amyloglucosidase), was conducted. Apart from enzyme activity and purity, their effects on the yield of sclerotial DF as well as its major components, such as β-glucans, chitin and resistant glycogen (RG), were investigated and compared. The activities of all industrial enzymes were significantly lower than those of their corresponding analytical ones, except for the Fungamyl^® Super MA, which had the highest α-amylase activity (6395 U/g). However, this fungal α-amylase was less able to digest the three sclerotial glycogens when compared with the bacterial alternatives. Amongst all tested enzymes, only analytical and industrial amyloglucosidases were found to have significant amount of contaminating cellulase (7.05–7.07 U/ml) and lichenase (4.62–4.67 U/ml) activities, which would cause endo-depolymerization of the β-glucan-type cell wall components (3.39% reduction in glucose residue after RG correction) of the PTR, leading to a marked α-amylase hydrolysis of its otherwise physically-inaccessible cytoplasmic glycogen (20.3% reduction in RG content). Commercial production of the three novel sclerotial DFs, using the industrial enzymes, would be feasible since, in addition to their economic advantage, both the yield (PTR: 81.2%; PR: 86.5%; WC: 96.2% of sample DM) and total non-starch polysaccharide contents (PTR: 88.0%; PR: 92.5%; WC: 91.1% DF-rich materials of DM) of their resulting sclerotial DFs were comparable to the levels of those prepared using analytical enzymes.