β-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 β-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66^T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied β-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc β-1,4 and β-1,3 linkages in chitobiose (GlcNAc)₂ and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: k_cat/K_M being 341 and 344 mM⁻¹·s⁻¹, respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide β-Gal-1,4-β-Glc-1,1-β-GlcNAc, and in low amounts the β-1,2- or β-1,3-linked trisaccharide β-Gal-1,4(β-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 β-N-acetylhexosaminidases.

A new Ba SP variant Q345F/P134D controls (+)-catechin glucosylation regioselectivity by modifying stabilisation interactions in the catalytic pocket of the enzyme.

Polydatin (3,4',5-trihydroxystilbene-3-β-D-glucoside, piceid), a natural stilbenoid found in different plant sources, has gained increasing attention for its potential health benefits. However, prior to its widespread adoption in human therapeutics and consumer products, a comprehensive investigation of its toxicological effects is crucial. In this study, the toxicity of polydatin was investigated in a developmental toxicity test using zebrafish (Danio rerio) as a valuable model for preclinical assessments. We employed the Fish Embryo Test (FET test - OECD n°236) to investigate the effects of polydatin on survival, hatchability, development, and behavior of zebrafish embryo-larval stage. Remarkably, the results demonstrated that polydatin up to 435 μM showed no toxicity. Throughout the exposure period, zebrafish embryos exposed to polydatin exhibited normal development, with no significant mortality observed. Furthermore, hatching success and heartbeat rate were unaffected, and no morphological abnormalities were identified, signifying a lack of teratogenic effects and cardiotoxicity. Locomotion activity assessment revealed normal swimming patterns and response to stimuli, indicating no neurotoxic effects. Our study provides valuable insights into the toxicological profile of polydatin, suggesting that it may offer potential therapeutic benefits under a considerable concentration range. In addition, zebrafish model proves to be an efficient system for early-stage toxicological screening, guiding further investigations into the secure utilization of polydatin for human health and wellness.

Sucrose phosphorylases, through transglycosylation reactions, are interesting enzymes that can transfer regioselectively glucose from sucrose, the donor substrate, onto acceptors like flavonoids to form glycoconjugates and hence modulate their solubility and bioactivity. Here, we report for the first time the structure of sucrose phosphorylase from the marine bacteria Alteromonas mediterranea (AmSP) and its enzymatic properties. Kinetics of sucrose hydrolysis and transglucosylation capacities on (+)-catechin were investigated. Wild-type enzyme (AmSP-WT) displayed high hydrolytic activity on sucrose and was devoid of transglucosylation activity on (+)-catechin. Two variants, AmSP-Q353F and AmSP-P140D catalysed the regiospecific transglucosylation of (+)-catechin: 89 % of a novel compound (+)-catechin-4'-O-α-d-glucopyranoside (CAT-4') for AmSP-P140D and 92 % of (+)-catechin-3'-O-α-d-glucopyranoside (CAT-3') for AmSP-Q353F. The compound CAT-4' was fully characterized by NMR and mass spectrometry. An explanation for this difference in regiospecificity was provided at atomic level by molecular docking simulations: AmSP-P140D was found to preferentially bind (+)-catechin in a mode that favours glucosylation on its hydroxyl group in position 4' while the binding mode in AmSP-Q353F favoured glucosylation on its hydroxyl group in position 3'.

Sucrose phosphorylases, through transglycosylation reactions, are interesting enzymes that can transfer regioselectively glucose from sucrose, the donor substrate, onto acceptors like flavonoids to form glycoconjugates and hence modulate their solubility and bioactivity. Here, we report for the first time the structure of sucrose phosphorylase from the marine bacteria Alteromonas mediterranea ( Am SP) and its enzymatic properties. Kinetics of sucrose hydrolysis and transglucosylation capacities on (+)-catechin were investigated. Wild-type enzyme (AmSP-WT) displayed high hydrolytic activity on sucrose and was devoid of transglucosylation activity on (+)-catechin. Two variants, Am SP-Q353F and Am SP-P140D catalysed the regiospecific transglucosylation of (+)-catechin: 89% of a novel compound (+)-catechin-4′-O-α-D-glucopyranoside (CAT-4’) for AmSP-P140D and 92% of (+)-catechin-3′-O-α-D-glucopyranoside (CAT-3’) for AmSP-Q353F. The compound CAT-4’ was fully characterized by NMR and mass spectrometry. An explanation for this difference in regiospecificity was provided at atomic level by molecular docking simulations: Am SP-P140D was found to preferentially bind (+)-catechin in a mode that favours glucosylation on its hydroxyl group in position 4’ while the binding mode in AmSP-Q353F favoured glucosylation on its hydroxyl group in position 3’.