Engineering of a chitin deacetylase to generate tailor-made chitosan polymers.

Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions. To understand the molecular basis of these preferences, we analyzed the binding site of a CDA from Pestalotiopsis sp. (PesCDA) using a detailed activity screening of a site-saturation mutagenesis library. In addition, molecular dynamics simulations were conducted to get an in-depth view of crucial interactions along the binding site. Besides elucidating the function of several amino acids, we were able to show that only 3 residues are responsible for the highly specific binding of PesCDA to oligomeric substrates. The preference to bind a GlcNAc unit at subsite -2 and -1 can mainly be attributed to N75 and H199, respectively. Whereas an exchange of N75 at subsite -2 eliminates enzyme activity, H199 can be substituted with tyrosine to increase the GlcN acceptance at subsite -1. This change in substrate preference not only increases enzyme activity on certain substrates and changes composition of oligomeric products but also significantly changes the pattern of acetylation (PA) when N-acetylating polyglucosamine. Consequently, we could clearly show how subsite preferences influence the PA of chitosans produced with CDAs.

Natural photoredox catalysts promote light-driven lytic polysaccharide monooxygenase reactions and enzymatic turnover of biomass.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of crystalline polysaccharides such as cellulose and chitin and are important for biomass conversion in the biosphere as well as in biorefineries. The target polysaccharides of LPMOs naturally occur in copolymeric structures such as plant cell walls and insect cuticles that are rich in phenolic compounds, which contribute rigidity and stiffness to these materials. Since these phenolics may be photoactive and since LPMO action depends on reducing equivalents, we hypothesized that LPMOs may enable light-driven biomass conversion. Here, we show that redox compounds naturally present in shed insect exoskeletons enable harvesting of light energy to drive LPMO reactions and thus biomass conversion. The primary underlying mechanism is that irradiation of exoskeletons with visible light leads to the generation of H2O2, which fuels LPMO peroxygenase reactions. Experiments with a cellulose model substrate show that the impact of light depends on both light and exoskeleton dosage and that light-driven LPMO activity is inhibited by a competing H2O2-consuming enzyme. Degradation experiments with the chitin-rich exoskeletons themselves show that solubilization of chitin by a chitin-active LPMO is promoted by light. The fact that LPMO reactions, and likely reactions catalyzed by other biomass-converting redox enzymes, are fueled by light-driven abiotic reactions in nature provides an enzyme-based explanation for the known impact of visible light on biomass conversion.

Metagenome-derived SusD-homologs affiliated with Bacteroidota bind to synthetic polymers.

Starch utilization system (Sus)D-homologs are well known for their carbohydrate-binding capabilities and are part of the sus operon in microorganisms affiliated with the phylum Bacteroidota. Until now, SusD-like proteins have been characterized regarding their affinity toward natural polymers. In this study, three metagenomic SusD homologs (designated SusD1, SusD38489, and SusD70111) were identified and tested with respect to binding to natural and non-natural polymers. SusD1 and SusD38489 are cellulose-binding modules, while SusD70111 preferentially binds chitin. Employing translational fusion proteins with superfolder GFP (sfGFP), pull-down assays, and surface plasmon resonance (SPR) has provided evidence for binding to polyethylene terephthalate (PET) and other synthetic polymers. Structural analysis suggested that a Trp triad might be involved in protein adsorption. Mutation of these residues to Ala resulted in an impaired adsorption to microcrystalline cellulose (MC), but not so to PET and other synthetic polymers. We believe that the characterized SusDs, alongside the methods and considerations presented in this work, will aid further research regarding bioremediation of plastics. IMPORTANCE: SusD1 and SusD38489 can be considered for further applications regarding their putative adsorption toward fossil-fuel based polymers. This is the first time that SusD homologs from the polysaccharide utilization loci (PUL), largely described for the phylum Bacteroidota, are characterized as synthetic polymer-binding proteins.

Hyaluronic Acid Unveiled: Exploring the Nanomechanics and Water Retention Properties at the Single-Molecule Level.

Hyaluronic acid (HA), a vital glycosaminoglycan in living organisms, possesses remarkable mechanical and viscoelastic properties that have garnered significant attention in therapeutic, biomedical, and cosmetic applications. However, a comprehensive picture of the physicochemical and biocharacterization of HA at the single-molecule level remains elusive. In this work, atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulation were used to investigate the nanomechanics and water retention properties of HA at the single-molecule level. The present study aims to unravel the intricate details of the influence of molecular structure on HA behavior and shed light on its unique attributes. According to the force measurements, the energy used to stretch a HA chain in water is 8.45 kJ/mol, significantly surpassing that of Curdlan (3.45 kJ/mol) and chitin (2.23 kJ/mol), both of which possess molecular structures partially similar to that of HA. Intriguingly, the strength of the intrachain interaction of HA (5.54 kJ/mol) was considerably weaker compared to Curdlan (11.06 kJ/mol) and chitin (or cellulose, 10.76 kJ/mol). This result indicates that HA exhibits a preference for interacting with water rather than with itself, thereby showing enhanced water affinity. Moreover, the force measurements demonstrated that changing the glycosidic bond from ß-(1-3) (Curdlan) or ß-(1-4) (chitin or cellulose) to ß-(1-3) + ß-(1-4) (HA) resulted in polysaccharides displaying improved water affinity and more extended conformation. These conclusions were further verified by molecular dynamics (MD) simulations. Overall, our work sheds new light on the nanomechanics and water retention properties of HA at the single-molecule level, offering valuable insights for future research in this field.

Self-Assembled Hydrogel Membranes with Structurally Tunable Mechanical and Biological Properties.

Using supramolecular self-assembled nanocomposite materials made from protein and polysaccharide components is becoming more popular because of their unique properties, such as biodegradability, hierarchical structures, and tunable multifunctionality. However, the fabrication of these materials in a reproducible way remains a challenge. This study presents a new evaporation-induced self-assembly method producing layered hydrogel membranes (LHMs) using tropocollagen grafted by partially deacetylated chitin nanocrystals (CO-g-ChNCs). ChNCs help stabilize tropocollagen's helical conformation and fibrillar structure by forming a hierarchical microstructure through chemical and physical interactions. The LHMs show improved mechanical properties, cytocompatibility, and the ability to control drug release using octenidine dihydrochloride (OCT) as a drug model. Because of the high synergetic performance between CO and ChNCs, the modulus, strength, and toughness increased significantly compared to native CO. The biocompatibility of LHM was tested using the normal human dermal fibroblast (NHDF) and the human osteosarcoma cell line (Saos-2). Cytocompatibility and cell adhesion improved with the introduction of ChNCs. The extracted ChNCs are used as a reinforcing nanofiller to enhance the performance properties of tropocollagen hydrogel membranes and provide new insights into the design of novel LHMs that could be used for various medical applications, such as control of drug release in the skin and bone tissue regeneration.

Nanochitin: Chemistry, Structure, Assembly, and Applications.

Chitin, a fascinating biopolymer found in living organisms, fulfills current demands of availability, sustainability, biocompatibility, biodegradability, functionality, and renewability. A feature of chitin is its ability to structure into hierarchical assemblies, spanning the nano- and macroscales, imparting toughness and resistance (chemical, biological, among others) to multicomponent materials as well as adding adaptability, tunability, and versatility. Retaining the inherent structural characteristics of chitin and its colloidal features in dispersed media has been central to its use, considering it as a building block for the construction of emerging materials. Top-down chitin designs have been reported and differentiate from the traditional molecular-level, bottom-up synthesis and assembly for material development. Such topics are the focus of this Review, which also covers the origins and biological characteristics of chitin and their influence on the morphological and physical-chemical properties. We discuss recent achievements in the isolation, deconstruction, and fractionation of chitin nanostructures of varying axial aspects (nanofibrils and nanorods) along with methods for their modification and assembly into functional materials. We highlight the role of nanochitin in its native architecture and as a component of materials subjected to multiscale interactions, leading to highly dynamic and functional structures. We introduce the most recent advances in the applications of nanochitin-derived materials and industrialization efforts, following green manufacturing principles. Finally, we offer a critical perspective about the adoption of nanochitin in the context of advanced, sustainable materials.

Wastewater treatment bacteria show differential preference for colonizing natural biopolymers.

Most reduced organic matter entering activated sludge systems is particulate (1-100-µm diameter) or colloidal (0.001-1-µm diameter), yet little is known about colonization of particulate organic matter by activated sludge bacteria. In this study, colonization of biopolymers (chitin, keratin, lignocellulose, lignin, and cellulose) by activated sludge bacteria was compared with colonization of glass beads in the presence and absence of regular nutrient amendment (acetate and ammonia). Scanning electron microscopy and quantitative PCR revealed chitin and cellulose were most readily colonized followed by lignin and lignocellulose, while keratin and glass beads were relatively resistant to colonization. Bacterial community profiles on particles compared to sludge confirmed that specific bacterial phylotypes preferentially colonize different biopolymers. Nitrifying bacteria proved adept at colonizing particles, achieving higher relative abundance on particles compared to bulk sludge. Denitrifying bacteria showed similar or lower relative abundance on particles compared to sludge. KEY POINTS: • Some activated sludge bacteria colonize natural biopolymers more readily than others. • Nitrifying bacteria are overrepresented in natural biopolymer biofilm communities. • Biopolymers in wastewater likely influence activated sludge community composition.

Design of Protease-Responsive Antifungal Liposomal Formulation Decorated with a Lipid-Modified Chitin-Binding Domain.

Candida albicans is a prevalent fungal pathogen that displays antibiotic resistance. The polyene antifungal amphotericin B (AmB) has been the gold standard because of its broad antifungal spectra, and its liposomal formulation, AmBisome, has been used widely and clinically in treating fungal infections. Herein, we explored enhancing the antifungal activity of AmBisome by integrating a small chitin-binding domain (LysM) of chitinase A derived from Pteris ryukyuensis. LysM conjugated with a lipid (LysM-lipid) was initially prepared through microbial transglutaminase (MTG)-mediated peptide tag-specific conjugation of LysM with a lipid-peptide substrate. The AmBisome formulation modified with LysM-lipid conjugates had a size distribution that was comparable to the native liposomes but an increased zeta potential, indicating that LysM-lipid conjugates were anchored to AmBisome. LysM-lipid-modified AmBisome exhibited long-term stability at 4 °C while retaining the capacity to bind chitin. Nevertheless, the antifungal efficacy of LysM-lipid-modified AmBisome against C. albicans was modest. We then redesigned a new LysM-lipid conjugate by introducing a peptide linker containing a thrombin digestion (TD) site at the C-terminus of LysM (LysM-TD linker-lipid), thereby facilitating the liberation of the LysM domain from AmBisome upon the addition of thrombin. This new AmBisome formulation anchored with LysM-TD linker-lipid exhibited superior performance in suppressing C. albicans growth in the presence of thrombin compared with the LysM-lipid formulation. These results provide a platform to design stimuli-responsive AmBisome formulations that respond to external environments and thus advance the treatment of pathogenic fungi infections.

Single-channel characterization of the chitooligosaccharide transporter chitoporin (SmChiP) from the opportunistic pathogen Serratia marcescens.

Serratia marcescens is an opportunistic pathogen that can utilize chitin as a carbon source, through its ability to produce chitin-degrading enzymes to digest chitin and membrane transporters to transport the degradation products (chitooligosaccharides) into the cells. Further characterization of these proteins is important to understand details of chitin metabolism. Here, we investigate the properties and function of the S. marcescens chitoporin, namely SmChiP, a chitooligosaccharide transporter. We show that SmChiP is a monomeric porin that forms a stable channel in artificial phospholipid membranes, with an average single-channel conductance of 0.5 ± 0.02 nS in 1 M KCl electrolyte. Additionally, we demonstrated that SmChiP allowed the passage of small molecules with a size exclusion limit of <300 Da and exhibited substrate specificity toward chitooligosaccharides, both in membrane and detergent-solubilized forms. We found that SmChiP interacted strongly with chitopentaose (Kd = 23 ± 2.0 µM) and chitohexaose (Kd = 17 ± 0.6 µM) but did not recognize nonchitose oligosaccharides (maltohexaose and cellohexaose). Given that S. marcescens can use chitin as a primary energy source, SmChiP may serve as a target for further development of nutrient-based antimicrobial therapies directed against multidrug antibiotic-resistant S. marcescens infections.

Estimate of the degradation potentials of cellulose, xylan, and chitin across global prokaryotic communities.

Complex polysaccharides (e.g. cellulose, xylan, and chitin), the most abundant renewable biomass resources available on Earth, are mainly degraded by microorganisms in nature. However, little is known about the global distribution of the enzymes and microorganisms responsible for the degradation of cellulose, xylan, and chitin in natural environments. Through large-scale alignments between the sequences released by the Earth Microbiome Project and sequenced prokaryotic genomes, we determined that almost all prokaryotic communities have the functional potentials to degrade cellulose, xylan, and chitin. The median abundances of genes encoding putative cellulases, xylanases, and chitinases in global prokaryotic communities are 0.51 (0.17-1.01), 0.24 (0.05-0.57), and 0.33 (0.11-0.71) genes/cell, respectively, and the composition and abundance of these enzyme systems are environmentally varied. The taxonomic sources of the three enzymes are highly diverse within prokaryotic communities, and the main factor influencing the diversity is the community's alpha diversity index rather than gene abundance. Moreover, there are obvious differences in taxonomic sources among different communities, and most genera with degradation potentials are narrowly distributed. In conclusion, our analysis preliminarily depicts a panorama of cellulose-, xylan-, and chitin-degrading enzymatic systems across global prokaryotic communities.