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Glycoside Hydrolase family 65 (GH65) is a unique family of carbohydrate-active enzymes. It is the first protein family to bring together glycoside hydrolases, glycoside phosphorylases and glycosyltransferases, thereby spanning a broad range of reaction types. These enzymes catalyze the hydrolysis, reversible phosphorolysis or synthesis of various α-glucosides, typically α-glucobioses or their derivatives. In this review, we present a comprehensive overview of the diverse reaction types and substrate specificities found in family GH65. We describe the determinants that control this remarkable diversity, as well as the applications of GH65 enzymes for carbohydrate synthesis., (© 2024. The Author(s).)

Osmolytes are produced by various microorganisms as a defense mechanism to protect cells and macromolecules from damage caused by external stresses in harsh environments. Due to their useful stabilizing properties, these molecules are applied as active ingredients in a wide range of cosmetics and healthcare products. The metabolic pathways and biocatalytic syntheses of glycosidic osmolytes such as 2-O-α-D-glucosyl-D-glycerate often involve the action of a glycoside phosphorylase. Here, we report the discovery of a glucosylglycerate phosphorylase from carbohydrate-active enzyme family GH13 that is also active on sucrose, which contrasts the strict specificity of known glucosylglycerate phosphorylases that can only use α-D-glucose 1-phosphate as glycosyl donor in transglycosylation reactions. The novel enzyme can be distinguished from other phosphorylases from the same family by the presence of an atypical conserved sequence motif at specificity-determining positions in the active site. The promiscuity of the sucrose-active glucosylglycerate phosphorylase can be exploited for the high-yielding and rapid synthesis of 2-O-α-D-glucosyl-D-glycerate from sucrose and D-glycerate. KEY POINTS: • A Xylanimonas protaetiae glycoside phosphorylase can use both d-glycerate and fructose as glucosyl acceptor with high catalytic efficiency • Biocatalytic synthesis of the osmolyte 2-O-α-d-glucosyl-d-glycerate • Positions in the active site of GH13 phosphorylases act as convenient specificity fingerprints., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Glycosidic osmolytes are widespread natural compounds that protect microorganisms and their macromolecules from the deleterious effects of various environmental stresses. Their protective properties have attracted considerable interest for industrial applications, especially as active ingredients in cosmetics and healthcare products. In that regard, the osmolyte glucosylglycerate is somewhat overlooked. Glucosylglycerate is typically accumulated by certain organisms when they are exposed to high salinity and nitrogen starvation, and its potent stabilizing effects have been demonstrated in vitro. However, the applications of this osmolyte have not been thoroughly explored due to the lack of a cost-efficient production process. Here, we present an overview of the progress that has been made in developing promising strategies for the synthesis of glucosylglycerate and its precursor glycerate, and discuss the remaining challenges. KEY POINTS: • Bacterial milking could be explored for fermentative production of glucosylglycerate • Glycoside phosphorylases of GH13_18 represent attractive alternatives for biocatalytic production • Conversion of glycerol with alditol oxidase is a promising strategy for generating the precursor glycerate., (© 2024. The Author(s).)

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.

Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-D-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.Key points• Cellobiose phosphorylase is engineered for the production of cellotriose.• Cellotriose is synthesised with the highest purity and yield to date.• Both cellobiose and glucose can be used as acceptor for cellotriose production.

Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.

α-Glucan phosphorylases (α-GPs) catalyze the reversible phosphorolysis of α-1,4-linked polysaccharides such as glycogen, starch, and maltodextrins, therefore playing a central role in the usage of storage polysaccharides. The discovery of these enzymes and their role in the course of catalytic conversion of glycogen was rewarded with the Nobel Prize in Physiology or Medicine in 1947. Nowadays, however, thermostable representatives attract special attention due to their vast potential in the enzymatic production of diverse carbohydrates and derivatives such as (functional) oligo- and (non-natural) polysaccharides, artificial starch, glycosides, and nucleotide sugars. One of the most recently explored utilizations of α-GPs is their role in the multi-enzymatic process of energy production stored in carbohydrate biobatteries. Regardless of their use, thermostable α-GPs offer significant advantages and facilitated bioprocess design due to their high operational temperatures. Here, we present an overview and comparison of up-to-date characterized thermostable α-GPs with a special focus on their reported biotechnological applications.

In the carbohydrate-active enzyme database, GH13_18 is a family of retaining glycoside phosphorylases that act on α-glucosides. In this work, we explored the functional diversity of this family by comparing distinctive sequence motifs in different branches of its phylogenetic tree. A glycoside phosphorylase from Marinobacter adhaerens HP15 that was predicted to have a novel function was expressed and characterised. The enzyme was found to catalyse the reversible phosphorolysis of 2-O-α-D-glucosylglycerol with retention of the anomeric configuration, a specificity that has never been described before. Homology modelling, docking and mutagenesis were performed to pinpoint particular acceptor site residues (Tyr194, Ala333, Gln336) involved in the binding of glycerol. The new enzyme specificity provides additional insights into bacterial metabolic routes, being the first report of a phosphorolytic route for glucosylglycerol in a glucosylglycerol-producing organism. Furthermore, glucosylglycerol phosphorylase might be an attractive biocatalyst for the production of the osmolyte glucosylglycerol, which is currently produced on industrial scale by exploiting a side activity of the closely related sucrose phosphorylase. Family GH13_18 has clearly proven to be more diverse than was initially assumed, and the analysis of specificity-determining sequence motifs has shown to be a straightforward and fruitful tool for enzyme discovery.

Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into fructose and nucleotide (NDP)-glucose. To date, only SuSy's from plants and cyanobacteria, both photosynthetic organisms, have been characterized. Here, four prokaryotic SuSy enzymes from the nonphotosynthetic organisms Nitrosomonas Europaea (SuSyNe), Acidithiobacillus caldus (SuSyAc), Denitrovibrio acetiphilus (SusyDa), and Melioribacter roseus (SuSyMr) were recombinantly expressed in Escherichia coli and thoroughly characterized. The purified enzymes were found to display high-temperature optima (up to 80 °C), high activities (up to 125 U/mg), and high thermostability (up to 15 min at 60 °C). Furthermore, SuSyAc, SuSyNe, and SuSyDa showed a clear preference for ADP as nucleotide, as opposed to plant SuSy's which prefer UDP. A structural and mutational analysis was performed to elucidate the difference in NDP preference between eukaryotic and prokaryotic SuSy's. Finally, the physiological relevance of this enzyme specificity is discussed in the context of metabolic pathways and genomic organization.

Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55 °C), and with a melting temperature of 79 °C and a half-life of 60 h at the industrially relevant temperature of 60 °C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6'-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6'-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6'-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis.

UDP-hexose 4-epimerases are important enzymes that play key roles in various biological pathways, including lipopolysaccharide biosynthesis, galactose metabolism through the Leloir pathway, and biofilm formation. Unfortunately, the determinants of their substrate specificity are not yet fully understood. They can be classified into three groups, with groups 1 and 3 preferring non-acetylated and acetylated UDP-hexoses, respectively, whereas members of group 2 are equally active on both types of substrates. In this study, the UDP-Glc(NAc) 4-epimerase from Marinithermus hydrothermalis (mGalE) was functionally expressed in Escherichia coli and thoroughly characterized. The enzyme was found to be thermostable, displaying its highest activity at 70 °C and having a half-life of 23 min at 60 °C. Activity could be detected on both acetylated and non-acetylated UDP-hexoses, meaning that this epimerase belongs to group 2. This observation correlates well with the identity of the so-called "gatekeeper" residue (Ser279), which has previously been suggested to influence substrate specificity (Schulz et al., J Biol Chem 279:32796-32803, 2004). Furthermore, substituting this serine to a tyrosine brings about a significant preference for non-acetylated sugars, thereby demonstrating that a single residue can determine substrate specificity among type 1 and type 2 epimerases. In addition, two consecutive glycine residues (Gly118 and Gly119) were identified as a unique feature of GalE enzymes from Thermus species, and their importance for activity as well as affinity was confirmed by mutagenesis. Finally, homology modeling and mutational analysis has revealed that the enzyme's catalytic triad contains a threonine residue (Thr117) instead of the usual serine.