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.

The Glycoside Hydrolase Family 65 (GH65) is an enzyme family of inverting α-glucoside phosphorylases and hydrolases that currently contains 10 characterized enzyme specificities. However, its sequence diversity has never been studied in detail. Here, an in-silico analysis of correlated mutations was performed, revealing specificity-determining positions that facilitate annotation of the family’s phylogenetic tree. By searching these positions for amino acid motifs that do not match those found in previously characterized enzymes from GH65, several clades that may harbor new functions could be identified. Three enzymes from across these regions were expressed in E. coli and their substrate profile was mapped. One of those enzymes, originating from the bacterium Mucilaginibacter mallensis, was found to hydrolyze kojibiose and α-1,2-oligoglucans with high specificity. We propose kojibiose glucohydrolase as the systematic name and kojibiose hydrolase or kojibiase as the short name for this new enzyme. This work illustrates a convenient strategy for mapping the natural diversity of enzyme families and smartly mining the ever-growing number of available sequences in the quest for novel specificities.

Background: Gut barrier dysfunction and altered gut microbial metabolism are emerging signatures of chronic gut disorders. Considering growing interest in combining structurally and mechanistically distinct bioactives, we investigated the individual and combined effects of serum-derived bovine immunoglobulin (SBI) and N-acetylglucosamine (NAG) on the gut microbiome and barrier integrity. Methods: The validated ex vivo SIFR^® (Systemic Intestinal Fermentation Research) technology, using microbiota from healthy adults (n = 6), was combined with a co-culture of epithelial/immune (Caco-2/THP-1) cells. Results: While SBI and NAG already significantly improved gut barrier integrity (TEER, transepithelial electrical resistance, +21% and +29%, respectively), the strongest effect was observed for SBI_NAG (+36%). This potent combined effect related to the observation that SBI and NAG each induced distinct, complementary shifts in microbial composition and metabolite output. SBI most selectively increased propionate (~Bacteroidota families) and health-associated indole derivatives (e.g., indole-3-propionic acid), while NAG most specifically boosted acetate and butyrate (~Bifidobacteriaceae, Ruminococcaceae, and Lachnospiraceae). The combination of SBI_NAG displayed effects of the individual ingredients, thus, for instance, enhancing all three short-chain fatty acids (SCFA) and elevating microbial diversity (CMS, community modulation score). Conclusions: Overall, SBI and NAG exert complementary, metabolically balanced effects on the gut microbiota, supporting combined use, particularly in individuals with gut barrier impairment or dysbiosis linked to lifestyle or early-stage gastrointestinal disorders.

Pectin comprises homogalacturonan (HG) and rhamnogalacturonan-I (RG-I). High molecular weight (Mw) pectin can be hydrolyzed into smaller pectic polysaccharides (PPS), offering different solubility and viscosity.We investigated how structural differences influence the bioactivity of potato-derived PPS fractions. Direct immunomodulatory effects were evaluated using a Caco-2/PBMC coculture model, while indirect, microbiome-mediated effects were evaluated via ex vivo colonic fermentation using the ex vivo SIFR® technology, coupled to the same immune model.Both PPS fractions were enriched in RG-I, in contrast to apple pectin, which was HG-rich and characterized by higher galacturonic acid and lower galactose content. High Mw PPS exhibited higher acetylation, whereas low Mw PPS showed more galactan branching. Both PPS fractions induced moderate cytokine responses under basal conditions (healthy state), while attenuating LPS-induced secretion of pro-inflammatory cytokines, effect likely related to their low degree of methyl-esterification. Both PPS fractions were utilized by gut microbes, resulting in increased short-chain fatty acid (SCFA) production and selective enrichment of beneficial taxa. PPS elicited stronger bifidogenic effects than apple pectin. Fermentation-mediated immunomodulatory effects mirrored those of the non-fermented compounds.PPS distinctly shaped the gut microbiome and host immunity depending on their structural features, supporting a structure-guided approach for selecting health targeting ingredients.