Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity‐based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand‐directed chemistry (LDC) for labelling glycoside hydrolases near – but not in – the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar‐based reversible inhibitors for labelling of two model β‐glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.

The considerable utility of glycoside phosphorylases (GPs) has led to substantial efforts over the past two decades to expand the breadth of known GP activities. Driven largely by the increase of available genomic DNA sequence data, the gap between the number of sequences in the carbohydrate active enzyme database (CAZy DB) and its functionally characterized members continues to grow. This wealth of sequence data presented an exciting opportunity to explore the ever-expanding CAZy DB to discover new GPs with never-before-described functionalities. Utilizing an in silico sequence analysis of CAZy family GH94, we discovered and then functionally and structurally characterized the new GP β-1,3-N-acetylglucosaminide phosphorylase. This new GP was sourced from the genome of the cell-wall-less Mollicute bacterium, Acholeplasma laidlawii and was found to synthesize β-1,3-linked N-acetylglucosaminide linkages. The resulting poly-β-1,3-N-acetylglucosamine represents a new, previously undescribed biopolymer that completes the set of possible β-linked GlcNAc homopolysaccharides together with chitin (β-1,4) and PNAG (poly-β-1,6-N-acetylglucosamine). The new biopolymer was denoted acholetin, a combination of the genus Acholeplasma and the polysaccharide chitin, and the new GP was thus denoted acholetin phosphorylase (AchP). Use of the reverse phosphorolysis action of AchP provides an efficient method to enzymatically synthesize acholetin, which is a new biodegradable polymeric material.

β-N-Acetylhexosaminidases (HexNAcases) are versatile biocatalysts that cleave terminal N-acetylhexosamine units from various glycoconjugates. Established strategies to generate glycoside-forming versions of the wild type enzymes rely on the mutation of their catalytic residues; however, successful examples of synthetically useful HexNAcase mutants are scarce. In order to expand the range of HexNAcases available as targets for enzyme engineering, we functionally screened a metagenomic library derived from a human gut microbiome. From a pool of hits, we characterized four of the more active candidates by sequence analysis and phylogenetic mapping, and found that they all belonged to CAZy family GH20. After detailed kinetic analysis and characterization of their substrate specificities, active site mutants were generated which resulted in the identification of two new thioglycoligases. BvHex E294A and AsHex E301A catalyzed glycosyl transfer to all three of the 3-, 4- and 6-thio-N-acetylglucosaminides (thio-GlcNAcs) that were tested. Both mutant enzymes also catalyzed glycosyl transfer to a cysteine-containing variant of the model peptide Tab1, with AsHex E301A also transferring GlcNAc onto a thiol-containing protein. This work illustrates how large scale functional screening of expressed gene libraries allows the relatively rapid development of useful new glycoside-forming mutants of HexNAcases, expanding the pool of biocatalysts for carbohydrate synthesis.

Sugar nucleotide-dependent (Leloir) glycosyltransferases are synthetically important for oligosac-charides and small molecule glycosides. Their practical use involves one-pot cascade reactions to regenerate the sugar nucleotide substrate. Glycosyltransferase co-immobilization is vital to advance multi-enzyme glycosylation systems on solid support. Here, we show glycosyltransferase chimeras with the cationic binding module Z(basic2) for efficient and well-controllable two-enzyme co-immobilization on anionic (ReliSorb SP400) carrier material. We use the C-glycosyltransferase from rice (Oryza sativa; OsCGT) and the sucrose synthase from soybean (Glycine max; GmSuSy) to synthesize nothofagin, the natural 3'-C-beta-D-glucoside of the dihydrochalcone phloretin, with regeneration of uridine 5'-diphosphate (UDP) glucose from sucrose and UDP. Exploiting enzyme surface tethering via Z(basic2), we achieve programmable loading of the glycosyltransferases (similar to 18 mg/g carrier; 60%-70% yield; similar to 80% effectiveness) in an activity ratio (OsCGT:GmSuSy = similar to 1.2) optimal for the overall reaction rate (similar to 0.2 mmolh(-1) g(-1) catalyst; 30 degrees C, pH 7.5). Using phloretin solubilized at 120 mM as inclusion complex with 2-hydroxypropyl-beta-cyclodextrin, we demonstrate complete substrate conversion into nothofagin (similar to 52 g/L; 21.8 mg product h(-1) g(-1) catalyst) at 4% mass loading of the catalyst. The UDP-glucose was recycled 240 times. The solid catalyst showed excellent reusability, retaining -40% of initial activity after 15 cycles of phloretin conversion (60 mM) with a catalyst turnover number of similar to 273 g nothofaginlg protein used. Our study presents important progress towards applied bio-catalysis with immobilized glycosyltransferase cascades.

N-Glycosylation is a fundamental protein modification found in both eukaryotes and archaea. Despite lacking N-glycans, many commensal and pathogenic bacteria have developed mechanisms to degrade these isoforms for a variety of functions, including nutrient acquisition and evasion of the immune system. Although much is known about many of the enzymes responsible for N-glycan degradation, the enzymes involved in cleaving the N-glycan core have only recently been discovered. Thus, some of the structural details have yet to be characterized, and little is known about their full distribution among bacterial strains and specifically within potential Gram-positive polysaccharide utilization loci. Here, we report crystal structures for Family 5, Subfamily 18 (GH5_18) glycoside hydrolases from the gut bacterium Bifidobacterium longum (BlGH5_18) and the soil bacterium Streptomyces cattleya (ScGH5_18), which hydrolyze the core Manβ1-4GlcNAc disaccharide. Structures of these enzymes in complex with Manβ1-4GlcNAc reveal a more complete picture of the -1 subsite. They also show that a C-terminal active site cap present in BlGH5_18 is absent in ScGH5_18. Although this C-terminal cap is not widely distributed throughout the GH5_18 family, it is important for full enzyme activity. In addition, we show that GH5_18 enzymes are found in Gram-positive polysaccharide utilization loci that share common genes, likely dedicated to importing and degrading N-glycan core structures.

A prominent attribute of chemical structure in microbial and plant natural products is aromatic C-glycosylation. In plants, various flavonoid natural products have a β-C-d-glucosyl moiety attached to their core structure. Natural product C-glycosides have attracted significant attention for their own unique bioactivity as well as for representing non-hydrolysable analogs of the canonical O-glycosides. The biosynthesis of natural product C-glycosides is accomplished by sugar nucleotide-dependent (Leloir) glycosyltransferases. Here, we provide an overview on the C-glycosyltransferases of microbial, plant and insect origin that have been biochemically characterized. Despite sharing basic evolutionary relationships, as evidenced by their common membership to glycosyltransferase family GT-1 and conserved GT-B structural fold, the known C-glycosyltransferases are diverse in the structural features that govern their reactivity, selectivity and specificity. Bifunctional glycosyltransferases can form C- and O-glycosides dependent on the structure of the aglycon acceptor. Recent crystal structures of plant C-glycosyltransferases and di-C-glycosyltransferases complement earlier structural studies of bacterial enzymes and provide important molecular insight into the enzymatic discrimination between C- and O-glycosylation. Studies of enzyme structure and mechanism converge on the view of a single displacement (SN2)-like mechanism of enzymatic C-glycosyl transfer, largely analogous to O-glycosyl transfer. The distinction between reactions at the O- or C-acceptor atom is achieved through the precise positioning of the acceptor relative to the donor substrate in the binding pocket. Nonetheless, C-glycosyltransferases may differ in the catalytic strategy applied to induce nucleophilic reactivity at the acceptor carbon. Evidence from the mutagenesis of C-glycosyltransferases may become useful in engineering these enzymes for tailored reactivity.

Infections are a severe health issue, and the need for an early point-of-care diagnostic approach for wound infections is continuously growing. Lysozyme has shown a great potential as a biomarker for rapid detection of wound infection. In this study, spray-drying of labeled and derivatized chitosans was investigated for the production of small particles responsive to lysozyme. Therefore, various chitosans, differing in their origin (snow crab, Chionoecetes sp., with medium and low molecular weight or shrimp) were N-acetylated, labeled with reactive black 5, and tested for solubility and spray-drying suitability. Reactive black-5-stained N-acetylated chitosan (low molecular weight, origin crab) was successfully spray-dried, and the obtained particles were characterized regarding size, ζ potential, and morphology. The particles showed an average hydrodynamic radius of 612.5 ± 132.8 nm. ζ potential was measured in the context of a later application as an infection detection system for wound infections in artificial wound fluid (−6.14 ± 0.16 mV) and infected wound fluid (−7.93 ± 1.35 mV). Furthermore, the aggregation behavior and surface structure were analyzed by using scanning electron microscopy and confocal laser scanning microscopy revealing spherical-shaped particles with explicit surface topologies. Spray-dried N-acetylated chitosan particles showed a 5-fold increase in lysozyme-responsive release of dyed chitosan fragments due to the enhanced surface area to volume ratio when compared to non-spray-dried N-acetylated chitosan flakes. On the basis of these results, the study showed the improved properties of N-acetylated spray-dried chitosan particles for future applications for early and rapid infection detection.

By reviving an old idea, we demonstrate that alkoxycarbonyl groups can be used in glycosylation reactions to achieve full stereocontrol through participation of a carbonate moiety at O-2. Various benzyloxycarbonyl-protected glycosyl donors were prepared and used for efficient 1,2-trans glycosylation of base-labile compounds and the synthesis of glycosyl esters.

Silicate‐based microporous materials like zeolites are nano enabled particles and used for various applications including pharmaceutical formulations. This study reports on the chemo‐enzymatic functionalization of chitosan‐zeolite particles (CTS‐zeolites) with caffeic acid (CA) and glucose oxidase (GOX) to impart combined antioxidant and antimicrobial properties. CA was grafted on the chitosan moieties by using laccase generating stable particles (zeta potential –36.7 mV) of high antioxidant activity (44% DPPH inhibition). GOX was immobilized both on CTS‐zeolites and on CA modified CTS‐zeolites and creating a hydrogen peroxide generation system continuously and in‐situ producing this oxidative and antimicrobial agent. The system prevented bacterial growth of E. coli and S. aureus over 24 h whereby a steady‐state concentration of around 60 μM hydrogen peroxide in the culture medium was observed. CA and GOX functionalized CTS‐zeolite particles additionally showed combinatorial antioxidant and antimicrobial properties providing a powerful bioactive system for medical applications. These particles proved their suitability for incorporation in bioactive formulations which could be used, inter alia, for topical wound treatments.

Thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can serve as valuable probes for studying the role of glycosides in biological processes. The development of an efficient, enzyme-mediated synthesis of thioglycosides, including S-GlcNAcylated proteins, is reported, using a thioglycoligase derived from a GH20 hexosaminidase from Streptomyces plicatus in which the catalytic acid/base glutamate has been mutated to an alanine (SpHex E314A). This robust, easily-prepared, engineered enzyme uses GlcNAc and GalNAc donors and couples them to a remarkably diverse set of thiol acceptors. Thioglycoligation using 3-, 4-, and 6-thiosugar acceptors from a variety of sugar families produces S-linked disaccharides in nearly quantitative yields. The set of possible thiol acceptors also includes cysteine-containing peptides and proteins, rendering this mutant enzyme a promising catalyst for the production of thio analogues of biologically important GlcNAcylated peptides and proteins.

This work presents electrospun chitosan mats, functionalized with glucose oxidase (GOX) to implement an in-situ hydrogen peroxide (H2O2) generation system. The as spun CTS-PEO mats exhibited a smooth and homogenous morphology in combination with a high specific surface area (5.4 m2/g) providing an excellent basis for further functionalization and subsequent glutaraldehyde crosslinking provided them with superior mechanical stability in aqueous environments. GOX was covalently immobilized, as proven by XPS, and resulted in activity recoveries between 20 and 40%. The functional mats generated a steady state concentration of ∼60 μM H2O2 per cm2 which resulted in growth inhibition of E. coli and of S. aureus already after two hours of incubation. Additional cytotoxicity tests of the modified mats against mouse fibroblasts did not show an influence on the viability of the cells which proved it a functional biomaterial of great potential for biomedical applications.

The aging population and accompanying diseases like diabetes resulted in an increased occurrence of chronic wounds. Topical wound treatment with antimicrobial agents to inhibit bacterial invasion and promote wound healing is often associated with difficulties. Here, we investigated the potential of succinyl chitosan (SC)-carboxymethyl cellulose (CMC) hydrogels which constantly release clinically relevant levels of hydrogen peroxide (H2O2). CMC hydrogel matrix was in situ converted by limited hydrolysis by a cellulase into substrates accepted by cellobiose dehydrogenase (CDH) for continuous production of H2O2 (30 μM over 24 h). This dual-enzyme catalyzed in situ H2O2 generation system proved its antimicrobial activity in a zone of inhibition (ZOI) assay best simulating the application as wound dressing and was found to be biocompatible toward mouse fibroblasts (95% viability). The hydrogels were thoroughly characterized regarding their rheological properties indicating fast gel formation (<3 min) and moderate cross-linking (1.5% strain, G' = 10 Pa). Cooling (fridge conditions) was found to be the simple on/off switch of the enzymatic machinery which is of great importance regarding storage and applicability of the bioactive hydrogel. This robust and bioactive antimicrobial hydrogel system overcomes dosing issues of common topical wound treatments and constitutes a promising wound healing approach for the future.

Chitosan hydrogels are gaining increasing interest for biomedical applications due to attractive properties such as biocompatibility. In order to replace toxic chemical cross-linkers for hydrogel formation, we investigated the cross-linking potential of laccase oxidized phenolics. HPLC-TOF-MS and ATR-FTIR demonstrated that phenolics were bond to glucosamine as chitosan model substrate. Phenolics concentrations required for hydrogel formation varied from 500 μM for catechol to 5000 μM for sinapic acid. The hydrogels showed different swelling and release properties assessed using methylene blue release as a model. Laccase oxidized caffeic acid and pyrogallol-chitosan hydrogels showed excellent behavior in up-taking water with a swelling of 208.7% for caffeic acid. Biocompatibility results did not show any significant inhibition of growth of HEK293 cell line when phenolics like catechol or eugenol were used. Therefore, this study demonstrates that laccase oxidized phenolics are potential cross-linking agents of chitosan as a novel green approach to synthesizing chitosan hydrogels.

Cellulose acetate (CA)-based materials, like cigarette filters, contribute to landscape pollution challenging municipal authorities and manufacturers. This study investigates the potential of enzymes to degrade CA and to be potentially incorporated into the respective materials, enhancing biodegradation. Deacetylation studies based on Liquid Chromatography-Mass Spectrometry-Time of Flight (LC-MS-TOF), High Performance Liquid Chromatography (HPLC), and spectrophotometric analysis showed that the tested esterases were able to deacetylate the plasticizer triacetin (glycerol triacetate) and glucose pentaacetate (cellulose acetate model compound). The most effective esterases for deacetylation belong to the enzyme family 2 (AXE55, AXE 53, GAE), they deacetylated CA with a degree of acetylation of up to 1.8. A combination of esterases and cellulases showed synergistic effects, the absolute glucose recovery for CA 1.8 was increased from 15% to 28% when an enzymatic deacetylation was performed. Lytic polysaccharide monooxygenase (LPMO), and cellobiohydrolase were able to cleave cellulose acetates with a degree of acetylation of up to 1.4, whereas chitinase showed no activity. In general, the degree of substitution, chain length, and acetyl group distribution were found to affect CA degradation. This study shows that, for a successful enzyme-based deacetylation system, a cocktail of enzymes, which will randomly cleave and generate shorter CA fragments, is the most suitable.