Converting Bulk Sugars into Functional Fibers: Discovery and Application of a Thermostable ß-1,3-Oligoglucan Phosphorylase.

Despite their broad application potential, the widespread use of ß-1,3-glucans has been hampered by the high cost and heterogeneity associated with current production methods. To address this challenge, scalable and economically viable processes are needed for the production of ß-1,3-glucans with tailorable molecular mass distributions. Glycoside phosphorylases have shown to be promising catalysts for the bottom-up synthesis of ß-1,3-(oligo)glucans since they combine strict regioselectivity with a cheap donor substrate (i.e., α-glucose 1-phosphate). However, the need for an expensive priming substrate (e.g., laminaribiose) and the tendency to produce shorter oligosaccharides still form major bottlenecks. Here, we report the discovery and application of a thermostable ß-1,3-oligoglucan phosphorylase originating from Anaerolinea thermophila (AtßOGP). This enzyme combines a superior catalytic efficiency toward glucose as a priming substrate, high thermostability, and the ability to synthesize high molecular mass ß-1,3-glucans up to DP 75. Coupling of AtßOGP with a thermostable variant of Bifidobacterium adolescentis sucrose phosphorylase enabled the efficient production of tailorable ß-1,3-(oligo)glucans from sucrose, with a near-complete conversion of >99 mol %. This cost-efficient process for the conversion of renewable bulk sugar into ß-1,3-(oligo)glucans should facilitate the widespread application of these versatile functional fibers across various industries.

Sweet Biotechnology: Enzymatic Production and Digestibility Screening of Novel Kojibiose and Nigerose Analogues.

In view of the global pandemic of obesity and related metabolic diseases, there is an increased interest in alternative carbohydrates with promising physiochemical and health-related properties as a potential replacement for traditional sugars. However, our current knowledge is limited to only a small selection of carbohydrates, whereas the majority of alternative rare carbohydrates and especially their properties remain to be investigated. Unraveling their potential properties, like digestibility and glycemic content, could unlock their use in industrial applications. Here, we describe the enzymatic production and in vitro digestibility of three novel glycosides, namely, two kojibiose analogues (i.e., d-Glcp-α-1,2-d-Gal and d-Glcp-α-1,2-d-Rib) and one nigerose analogue (i.e., d-Glcp-α-1,3-l-Ara). These novel sugars were discovered after an intensive acceptor screening with a sucrose phosphorylase originating from Bifidobacterium adolescentis (BaSP). Optimization and upscaling of this process led to roughly 100 g of these disaccharides. Digestibility, absorption, and caloric potential were assessed using brush border enzymes of rat origin and human intestinal Caco-2 cells. The rare disaccharides showed a reduced digestibility and a limited impact on energy metabolism, which was structure-dependent and even more pronounced for the three novel disaccharides in comparison to their respective glucobioses, translating to a low-caloric potential for these novel rare disaccharides.

Exploration of GH94 Sequence Space for Enzyme Discovery Reveals a Novel Glucosylgalactose Phosphorylase Specificity.

The substantial increase in DNA sequencing efforts has led to a rapid expansion of available sequences in glycoside hydrolase families. The ever-increasing sequence space presents considerable opportunities for the search for enzymes with novel functionalities. In this work, the sequence-function space of glycoside hydrolase family 94 (GH94) was explored in detail, using a combined approach of phylogenetic analysis and sequence similarity networks. The identification and experimental screening of unknown clusters led to the discovery of an enzyme from the soil bacterium Paenibacillus polymyxa that acts as a 4-O-ß-d-glucosyl-d-galactose phosphorylase (GGalP), a specificity that has not been reported to date. Detailed characterization of GGalP revealed that its kinetic parameters were consistent with those of other known phosphorylases. Furthermore, the enzyme could be used for production of the rare disaccharides 4-O-ß-d-glucosyl-d-galactose and 4-O-ß-d-glucosyl-l-arabinose. Our current work highlights the power of rational sequence space exploration in the search for novel enzyme specificities, as well as the potential of phosphorylases for rare disaccharide synthesis.

ß-Glucan phosphorylases in carbohydrate synthesis.

ß-Glucan phosphorylases are carbohydrate-active enzymes that catalyze the reversible degradation of ß-linked glucose polymers, with outstanding potential for the biocatalytic bottom-up synthesis of ß-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 ß-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 ß-glucan and associated ß-glucobiose phosphorylases, with a special focus on their application in the synthesis of carbohydrates and related molecules. KEY POINTS: • Discovery, characteristics, and applications of ß-glucan phosphorylases. • ß-Glucan phosphorylases in the production of functional carbohydrates.