Cyclic β-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic β-1,2-glucans (CβGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that β-glucosidase-resistant compounds are produced by TiCGSCy with linear β-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CβGs. Next, action pattern analyses using β-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate β-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CβGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of β-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. KEY POINTS: • It was clearly evidenced that cyclization domain alone produces cyclic β-1,2-glucans. • The domain exclusively catalyzes transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189.

β-Glucan phosphorylases are carbohydrate-active enzymes that catalyze the reversible degradation of β-linked glucose polymers, with outstanding potential for the biocatalytic bottom-up synthesis of β-glucans as major bioactive compounds. Their preference for sugar phosphates (rather than nucleotide sugars) as donor substrates further underlines their significance for the carbohydrate industry. Presently, they are classified in the glycoside hydrolase families 94, 149, and 161 ( www.cazy.org ). Since the discovery of β-1,3-oligoglucan phosphorylase in 1963, several other specificities have been reported that differ in linkage type and/or degree of polymerization. Here, we present an overview of the progress that has been made in our understanding of β-glucan and associated β-glucobiose phosphorylases, with a special focus on their application in the synthesis of carbohydrates and related molecules. KEY POINTS: • Discovery, characteristics, and applications of β-glucan phosphorylases. • β-Glucan phosphorylases in the production of functional carbohydrates.

Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-D-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-D-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.

Biological (or reductive) soil disinfestation (BSD or RSD) is a bioremediation process to control soil-borne plant pathogens using activities of indigenous bacteria in the soil. Three obligate anaerobic bacterial strains (TW1, TW10, and TB10), which were isolated from anoxic soil subjected to BSD treatments, were examined for their abilities to produce anti-fungal enzymes. All strains were affiliated with the different lineages of the genus Clostridium. The three strains decomposed β-1,3-glucans (curdlan and laminarin), and β-1,3-glucanase activities were detected from their culture supernatants with these glucans. The three strains also produced the enzyme with wheat bran as a growth substrate and killed the Fusarium pathogen (Fusarium oxysporum f. sp. spinaciae) in the anaerobic co-incubation conditions. Observation by fluorescence microscopy of the pathogen cells showed that the three strains had degraded the fungal cells in different manners upon co-incubation with wheat bran. When the three strains were cultivated with the dead cells or the cell wall samples prepared from the Fusarium pathogen, strain TW1 utilized these materials as easily decomposable substrates by releasing β-1,3-glucanase. When observed by fluorescence microscopy, it appeared that strain TW1 degraded the mycelial cell wall nearly thoroughly, with the septa remaining as undecomposed luminous rings. In contrast, the other two strains decomposed neither the dead cells nor the cell wall samples directly. The results indicate that the various anaerobic bacteria proliferated in the soil under the BSD treatments should play key roles as an organized bacterial community to eliminate fungal pathogens, namely by release of anti-fungal enzymes with different properties.Key points•Three clostridial strains isolated from BSD-treated soils produced β-1,3-glucanase.•All strains killed the Fusarium pathogen in the anaerobic co-incubation conditions.•One of the strains produced β-1,3-glucanase with the fungal cell wall as a substrate.•The strain degraded the cell wall almost completely, except for the mycelial septa.

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic alpha-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering.

Grifola frodosa polysaccharides, especially β-D-glucans, possess significant anti-tumor, antioxidant and immunostimulatory activities. However, the synthesis mechanism remains to be elucidated. A newly discovered glycosyltransferase UGT88A1 was found to extend glucan chains in vitro. However, the role of UGT88A1 in the growth and polysaccharide synthesis of G. frondosa in vivo remains unclear. In this study, the overexpression of UGT88A1 improved mycelial growth, increased polysaccharide production, and decreased cell wall pressure sensitivity. Biomass and polysaccharide production decreased in the silenced strain, and the pressure sensitivity of the cell wall increased. Overexpression and silencing of UGT88A1 both affected the monosaccharide composition and surface morphology of G. frondosa polysaccharides and influenced the antioxidant activity of polysaccharides from different strains. The messenger RNA expression of glucan synthase (GLS), UTP-glucose-1-phosphate uridylyltransferase (UGP), and UDP-xylose-4-epimerase (UXE) related to polysaccharide synthesis, and genes related to cell wall integrity increased in the overexpression strain. Overall, our study indicates that UGT88A1 plays an important role in the growth, stress, and polysaccharide synthesis of G. frondosa, providing a reference for exploring the pathway of polysaccharide synthesis and metabolic regulation. KEY POINTS: •UGT88A1 plays an important role in the growth, stress response, and polysaccharide synthesis in G. frondosa. •UGT88A1 affected the monosaccharide composition, surface morphology and antioxidant activity of G. frondosa polysaccharides. •UGT88A1 regulated the mRNA expression of genes related to polysaccharide synthesis and cell wall integrity.