Ligand-Directed Chemistry on Glycoside Hydrolases – A Proof of Concept Study

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.T. M. W. thanks the FWF (Wien, Austria) for financial support (project number P30372-B21). Authors from TU Graz acknowledge support from NAWI Graz.Peer reviewe

Fast Blue RR—Siloxane Derivatized Materials Indicate Wound Infection Due to a Deep Blue Color Development

There is a strong need for simple and fast methods for wound infection determination. Myeloperoxidase, an immune system-derived enzyme was found to be a suitable biomarker for wound infection. Hence, alkoxysilane-derivatized Fast Blue RR was immobilized via simple hydrolytic polymerization. The resulting enzyme-responsive siloxane layers were incubated with myeloperoxidase, wound fluid or hemoglobin. The reaction was monitored via HPLC measurements and the color development quantified spectrophotometrically. Myeloperoxidase was indeed able to oxidize immobilized Fast Blue RR leading to a blue colored product. No conversion was detected in non-infected wound fluids. The visible color changes of these novel materials towards blue enable an easy distinction between infected and non-infected wound fluids

Lysozyme responsive spray-dried chitosan particles for early detection of wound infection

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

Enzymatic systems for cellulose acetate degradation

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