Chitin Chains Research Articles

Chitin and Chitosan Fragments Responsible for Plant Elicitor and Growth Stimulator.

Chitin and chitosan are natural polysaccharides with huge application potential in agriculture, such as promoting plant growth, eliciting plant resistance against biotic and abiotic stress, and activating symbiotic signaling between plants and beneficial microorganisms. Chitin and chitosan offer a sustainable alternative for future crop production. The bioactivities of chitin and chitosan closely depend on their structural factors, including molecular size, degree of acetylation, and pattern of acetylation. It is of great significance to identify the key fragments in chitin and chitosan chains that are responsible for these agricultural bioactivities. Herein, we review the recent progress in the structure-function relationship of chitin and chitosan in the field of agriculture application. The preparation of chitin and chitosan fragments and their action mode for plant protection and growth are also discussed.

Gene Cloning, Characterization, and Molecular Simulations of a Novel Recombinant Chitinase from Chitinibacter Tainanensis CT01 Appropriate for Chitin Enzymatic Hydrolysis

Chitin, a polymer of N-acetyl-d-glucosamine (GlcNAc), can be degraded by chitinase, which is produced by higher plants, vertebrates, and bacteria. Chitinases are characterized by the ability to hydrolyze the beta-1,4-linkages in the chitin chain by either an endolytic or an exolytic mechanism. Chitinase 1198 is a novel endochitinase from the genome sequence of Chitinibacter tainanensis CT01. Herein, we report the findings of molecular simulations and bioassays for chitinase 1198. Our experimental results suggest that chitinase 1198 can recognize the nonreducing end of chitin and cleave the second or third glycosidic linkage from the nonreducing end of chitin oligomers. Furthermore, our simulations results revealed that chitinase 1198 is more likely to bind chitin oligomers with the main hydrogen bonds of the Asp440, the second GlcNAc unit of chitin oligomers, and degrade chitin oligomers to (GlcNAc)2 molecules. Moreover, chitinase 1198 is less likely to bind chitin oligomers with the main hydrogen bonds of the Asp440, the third GlcNAc unit of chitin oligomers, and degrade chitin oligomers to (GlcNAc)3 molecules. Lastly, chitinase 1198 can bind (GlcNAc)3 molecules with the main hydrogen bonds of the Asp440, the second GlcNAc of the (GlcNAc)3 molecules, and degrade chitin oligomers to GlcNAc and (GlcNAc)2 molecules.

Chemical-State-Dependent Free Energy Profile from Single-Molecule Trajectories of Biomolecular Motors: Application to Processive Chitinase.

The mechanism of biomolecular motors has been elucidated using single-molecule experiments for visualizing motor motion. However, it remains elusive that how changes in the chemical state during the catalytic cycle of motors lead to unidirectional motions. In this study, we use single-molecule trajectories to estimate an underlying diffusion model with chemical-state-dependent free energy profile. To consider nonequilibrium trajectories driven by the chemical energy consumed by biomolecular motors, we develop a novel framework based on a hidden Markov model, wherein switching among multiple energy profiles occurs reflecting the chemical state changes in motors. The method is tested using simulation trajectories and applied to single-molecule trajectories of processive chitinase, a linear motor that is driven by the hydrolysis energy of a single chitin chain. The chemical-state-dependent free energy profile underlying the burnt-bridge Brownian ratchet mechanism of processive chitinase is determined. The novel framework allows us to connect the chemical state changes to the unidirectional motion of biomolecular motors.

Influence of proteins on mechanical properties of a natural chitin-protein composite

In many biogenic materials, chitin chains are assembled in fibrils that are wrapped by a protein fold. In them, the mechanical properties are supposed to be related to intra- and inter- interactions among chitin and proteins. This hypothesis has been poorly investigated. Here, this research theme is studied using the pen of Loligo vulgaris as a model material of chitin-protein composites. Chemical treatments were used to change the interactions involving only the proteic phase, through unfolding and/or degradation processes. Successively, structural and mechanical parameters were examined using spectroscopy, microscopy, X-ray diffractometry, and tensile tests. The data analysis showed that chemical treatments did not modify the structure of the chitin matrix. This allowed to derive from the mechanical test analysis the following conclusions: (i) the maximum stress (σmax) relies on the presence of the disulfide bonds; (ii) the Young's modulus (E) relies on the overall correct folding of the proteins; (iii) the whole removal of proteins induces a decrease of E (> 90%) and σmax (> 80%), and an increase in the maximum elongation. These observations indicate that in the chitin matrix the proteins act as a strengthener, which efficacy is controlled by the presence of disulfide bridges. This reinforcement links the chitin fibrils avoiding them to slide one on the other and maximizing their resistance and stiffness. In conclusion, this knowledge can explain the physio-chemical properties of other biogenic polymeric composites and inspire the design of new materials. Statement of SignificanceTo date, no study has addressed on how proteins influence chitin-composite material's mechanical properties. Here we show that the Young's modulus and the maximum stress mainly rely on protein disulfide bonds, the inter-proteins ones and those controlling the folding of chitin-binding domains. The removal of protein matrix induce a reduction of Young's modulus and maximum stress, leaving the chitin matrix structurally unaltered. The measure of the maximum elongation shows that the chitin fibrils slide on each other only after removing the protein matrix.In conclusion, this research shows that the proteins act as a stiff matrix reinforced by di-sulfide bridges that link crystalline chitin fibrils avoiding them to slide one on the other.

Renewable sources: applications in personal care formulations.

A global tendency for products considered environmentally sustainable, and ecologically obtained led the industry related to personal care formulations to fund the research and the development of personal care/cosmetics containing ingredients from natural resources. Furthermore, consumers are aware of environmental and sustainability issueans, thus not harming the environment represents a key consideration when developing a new cosmetic ingredient. In this study we review some examples of active ingredients or raw materials used in cosmetics/personal care/biomedical products that are coming from either through biotechnological systems, or as byproducts of several industries. A skin formulation containing biosynthetic actives, prepared by us and the study regarding its dermocosmetic properties are also described. The need for the standardization processes, the safety assessment tools, the improvement of the exploitation methods of these renewable sources in order the production to be ecologically and economically better are also discussed.

Chitin Organizing and Modifying Enzymes and Proteins Involved InRemodeling of the Insect Cuticle.

Chitin, the extracellular matrix polysaccharide of insects and arthropods is widely distributed in nature in all kingdoms of life and serves a variety of functions. After synthesis by membrane-bound chitin synthases, it is extensively remodeled before incorporation into divergent matrices with wide-ranging physical and biological properties. This chapter discusses the properties of a variety of insect enzymes and proteins involved in this process. Chitin remodeling involves chitin synthases, which make the nascent chitin chains, and chitin deacetylases that partially deacetylate some of the N-acetylglucosamine residues either randomly or sequentially to yield local chitosan-like regions. Other proteins secreted into the procuticle or the midgut help in the assembly of single chitin chains into larger crystalline aggregates that measure in a few 100 nanometers. They are further embedded in a complex matrix of cuticular proteins or become associated with proteins containing chitin-binding domains to constitute the laminar procuticle or the lattice-like peritrophic matrix. During molting, previously formed laminar cuticle or PM are decrystallized/depolymerized to unmask the chitin chains, which then are degraded by a mixture of chitinolytic enzymes consisting of chitinases and N-acetylglucosaminidases present in molting fluid or in gut secretions. Some of the degradation products may be recycled for the synthesis of new matrices. We present a model of chitin synthesis, assembly, and degradation and the roles of these chitin-remodeling enzymes in this overall process.

Chitin in Arthropods: Biosynthesis, Modification, and Metabolism.

Chitin is a structural constituent of extracellular matrices including the cuticle of the exoskeleton and the peritrophic matrix (PM) of the midgut in arthropods. Chitin chains are synthesized through multiple biochemical reactions, organized in several hierarchical levels and associated with various proteins that give their unique physicochemical characteristics of the cuticle and PM. Because, arthropod growth and morphogenesis are dependent on the capability of remodeling chitin-containing structures, chitin biosynthesis and degradation are highly regulated, allowing ecdysis and regeneration of the cuticle and PM. Over the past 20years, much progress has been made in understanding the physiological functions of chitinous matrices. In this chapter, we mainly discussed the biochemical processes of chitin biosynthesis, modification and degradation, and various enzymes involved in these processes. We also discussed cuticular proteins and PM proteins, which largely determine the physicochemical properties of the cuticle and PM. Although rapid advances in genomics, proteomics, RNA interference, and other technologies have considerably facilitated our research in chitin biosynthesis, modification, and metabolism in recent years, many aspects of these processes are still partially understood. Further research is needed in understanding how the structural organization of chitin synthase in plasma membrane accommodate chitin biosynthesis, transport of chitin chain across the plasma membrane, and release of the chitin chain from the enzyme. Other research is also needed in elucidating the roles of chitin deacetylases in chitin organization and the mechanism controlling the formation of different types of chitin in arthropods.

Hierarchical architecture of spider attachment setae reconstructed from scanning nanofocus X-ray diffraction data.

When sitting and walking, the feet of wandering spiders reversibly attach to many surfaces without the use of gluey secretions. Responsible for the spiders' dry adhesion are the hairy attachment pads that are built of specially shaped cuticular hairs (setae) equipped with approximately 1 µm wide and 20 nm thick plate-like contact elements (spatulae) facing the substrate. Using synchrotron-based scanning nanofocus X-ray diffraction methods, combining wide-angle X-ray diffraction/scattering and small-angle X-ray scattering, allowed substantial quantitative information to be gained about the structure and materials of these fibrous adhesive structures with 200 nm resolution. The fibre diffraction patterns showed the crystalline chitin chains oriented along the long axis of the attachment setae and increased intensity of the chitin signal dorsally within the seta shaft. The small-angle scattering signals clearly indicated an angular shift by approximately 80° of the microtrich structures that branch off the bulk hair shaft and end as the adhesive contact elements in the tip region of the seta. The results reveal the specific structural arrangement and distribution of the chitin fibres within the attachment hair's cuticle preventing material failure by tensile reinforcement and proper distribution of stresses that arise upon attachment and detachment.

Priming and elongation of chitin chains: Implications for chitin synthase mechanism

Most fungi have multiple chitin synthases (CSs) that may make chitin at different sites on the cell surface, at different times during growth, and in response to cell wall stress. The structure-based model for CS function is for transfer of GlcNAc from UDP-GlcNAc at the cytoplasmic face of the plasma membrane to the non-reducing end of a growing chitin chain, which is concomitantly translocated through a transmembrane channel formed by the synthase. Two aspects of CS mechanism are investigated: how chains might be initiated, and what governs how long they can get. First, it is shown that CSs incorporate free GlcNAc into di-N-acetylchitobiose and into insoluble chitin in a UDP-GlcNAc-dependent manner, and therefore that GlcNAc primes chitin synthesis. Second, average lengths of insoluble chitin chains were measured by determining the molar ratio of priming GlcNAc to chain-extending, UDP-GlcNAc-derived GlcNAc, and showed dependence on UDP-GlcNAc concentration, approaching a maximum at higher concentrations of substrate. These results, together with previous findings that 2-acylamido GlcN analogues prime formation of chitin oligosaccharides and stimulate chitin synthesis are discussed in the context of the structure-based model, and lead to the following proposals. 1) CSs may “self-prime” by hydrolyzing UDP-GlcNAc to yield GlcNAc. 2) A CS’s active site is not continuously occupied by a nascent chitin chain, rather, CSs can release chitin chains, then re-initiate, and therefore synthesize chitin chains in bursts. 3) The length of chitin chains made by a given CS will impact that CS’s contribution to construction of the fungal cell wall.

The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size.

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N-acetyl-glucosamine) oligomers, we here identify six-subunit-long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size-dependent system of immuno-modulation that appears conserved in plants and humans. Since blocking of the chitin-TLR2 interaction effectively prevents chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin-TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.

Processive chitinase is Brownian monorail operated by fast catalysis after peeling rail from crystalline chitin

Processive chitinase is a linear molecular motor which moves on the surface of crystalline chitin driven by processive hydrolysis of single chitin chain. Here, we analyse the mechanism underlying unidirectional movement of Serratia marcescens chitinase A (SmChiA) using high-precision single-molecule imaging, X-ray crystallography, and all-atom molecular dynamics simulation. SmChiA shows fast unidirectional movement of ~50 nm s−1 with 1 nm forward and backward steps, consistent with the length of reaction product chitobiose. Analysis of the kinetic isotope effect reveals fast substrate-assisted catalysis with time constant of ~3 ms. Decrystallization of the single chitin chain from crystal surface is the rate-limiting step of movement with time constant of ~17 ms, achieved by binding free energy at the product-binding site of SmChiA. Our results demonstrate that SmChiA operates as a burnt-bridge Brownian ratchet wherein the Brownian motion along the single chitin chain is rectified forward by substrate-assisted catalysis.

Effect of sex on the nutritional value of house cricket, Acheta domestica L.

Since January 2018, insects have been recognised as novel foods in the EU, but their nutritional value varies, and factors affecting their nutritional composition have been debated. We investigated the effect of sex on the nutritional value and chemical composition of the house cricket (Acheta domestica L.). Both sexes were rich in protein and lipids. The proximate composition was partly influenced by sex; females contained a significantly higher amount of lipids (18.3–21.7 vs 12.9–16.1 g/100 g dry matter, p = 0.0001) and fewer proteins than males (61.2–64.9 vs 66.3–69.6 g/100 g dry matter, p = 0.0001). Males contained more chitin (p = 0.0015) and nitrogen chains (p = 0.0003) than females. Only the ash (p = 0.4314) and nitrogen-free extract (p = 0.4871) were uninfluenced by sex. Furthermore, nutrient quality expressed as essential amino acid (72.3–77.1), thrombogenicity (1.22–1.45), and atherogenicity indices (0.53–0.58) did not differ between sexes (p > 0.05).

Crystal structures of an archaeal chitinase ChiD and its ligand complexes.

Chitinase D (designated as Pc-ChiD) was found in a hyperthermophilic archaeon, Pyrococcus chitonophagus (previously described as Thermococcus chitonophagus), that was isolated from media containing only chitin as carbon source. Pc-ChiD displays chitinase activity and is thermostable at temperatures up to 95°C, suggesting its potential for industrial use. Pc-ChiD has a secretion signal peptide and two chitin-binding domains (ChBDs) in the N-terminal domain. However, the C-terminal domain shares no sequence similarity with previously identified saccharide-degrading enzymes and does not contain the DXDXE motif conserved in the glycoside hydrolase (GH) 18 family chitinases. To elucidate its overall structure and reaction mechanism, we determined the first crystal structures of Pc-ChiD, both in the ligand-free form and in complexes with substrates. Structure analyses revealed that the C-terminal domain of Pc-ChiD, Pc-ChiD(ΔBD), consists of a third putative substrate-binding domain, which cannot be predicted from the amino acid sequence, and a catalytic domain structurally similar to that found in not the GH18 family but the GH23 family. Based on the similarity with GH23 family chitinase, the catalytic residues of Pc-ChiD were predicted and confirmed by mutagenesis analyses. Moreover, the specific C-terminal 100 residues of Pc-ChiD are important to fix the putative substrate-binding domain next to the catalytic domain, contributing to the structure stability as well as the long chitin chain binding. Our findings reveal the structure of a unique archaeal chitinase that is distinct from previously known members of the GH23 family.

Understanding dissolution process of chitin crystal in ionic liquids: theoretical study.

Chitin is a promising biomass resource and has high potential for industrial applications owing to its huge annual production in nature. However, it exhibits poor processability and solubility due to its very stable and crystalline character. Recently, ionic liquids (ILs) have attracted attention as solvents for structural polysaccharides - for example, 1-allyl-3-methylimidazolium bromide (AMIMBr) has been found to dissolve chitin. As few ILs are known to dissolve chitin, little research has been conducted on the dissolution mechanism involved. In this study, we have adopted a molecular dynamics (MD) approach to study the dissolution of chitin crystals in imidazolium-based ILs. The MD simulation in AMIMBr has demonstrated that the dissolution process involved peeling of chitin chains from the crystal surface, with Br- cleaving the chitin hydrogen bonds, and AMIM+ preventing a return to the crystalline phase after the peeling. By contrast, in imidazolium acetates, which has also been reported to dissolve chitin, although the molecular chains are peeled off, the peeled chains occasionally return to the crystalline phase. Furthermore, the MD trajectory analysis has revealed that the solubility of chitin is well correlated with the number of intermolecular hydrogen bonds by acetamido groups in the chitin crystal. It has been experimentally proven that mixing a small amount of 2-bromoethyl acetate, as a bromide generator, with 1-allyl-3-methylimidazolium chloride can enhance chitin solubility, which supports the dissolution mechanism indicated by the above theoretical results.

Structure and function of a broad-specificity chitin deacetylase from Aspergillus nidulans FGSC A4

Enzymatic conversion of chitin, a β-1,4 linked polymer of N-acetylglucosamine, is of major interest in areas varying from the biorefining of chitin-rich waste streams to understanding how medically relevant fungi remodel their chitin-containing cell walls. Although numerous chitinolytic enzymes have been studied in detail, relatively little is known about enzymes capable of deacetylating chitin. We describe the structural and functional characterization of a 237 residue deacetylase (AnCDA) from Aspergillus nidulans FGSC A4. AnCDA acts on chito-oligomers, crystalline chitin, chitosan, and acetylxylan, but not on peptidoglycan. The Km and kcat of AnCDA for the first deacetylation of penta-N-acetyl-chitopentaose are 72 µM and 1.4 s−1, respectively. Combining mass spectrometry and analyses of acetate release, it was shown that AnCDA catalyses mono-deacetylation of (GlcNAc)2 and full deacetylation of (GlcNAc)3–6 in a non-processive manner. Deacetylation of the reducing end sugar was much slower than deacetylation of the other sugars in chito-oligomers. These enzymatic characteristics are discussed in the light of the crystal structure of AnCDA, providing insight into how the chitin deacetylase may interact with its substrates. Interestingly, AnCDA activity on crystalline chitin was enhanced by a lytic polysaccharide monooxygenase that increases substrate accessibility by oxidative cleavage of the chitin chains.

Extremely Strong and Transparent Chitin Films: A High‐Efficiency, Energy‐Saving, and “Green” Route Using an Aqueous KOH/Urea Solution

Crystalline polysaccharides are useful for important and rapidly growing applications ranging from advanced energy storage, green electronics, and catalyst or enzyme supports to tissue engineering and biological devices. However, the potential value of chitin in such applications is currently neglected because of its poor swellability, reactivity, and solubility in most commonly used solvents. Here, a high‐efficiency, energy‐saving, and “green” route for the fabrication of extremely strong and transparent chitin films is described in which chitin is dissolved in an aqueous KOH/urea solution and neutralized in aqueous ethanol solution. The neutralization temperature, ethanol concentration, and chitin solution deacetylation time are critical parameters for the self‐assembly of chitin chains and for tuning the morphology and aggregate structures of the resulting chitin hydrogels and films. Moreover, the drawing orientation can produce extremely strong and tough chitin films with a tensile strength, Young's modulus, and work of fracture of 226 MPa, 7.2 GPa, and 20.3 MJ m−3, respectively. The method developed here should contribute to the utilization of seafood waste and, thereby, to the sustainable use of marine resources.

Crab and prawn shell utilization as a source of bio-based thermoplastics through graft polymerization with acrylate monomers

To recycle crab and prawn shell wastes and prepare environment friendly thermoplastics, various acrylate monomers—methyl (meth)acrylates, ethyl (meth)acrylates, and butyl (meth)acrylates were grafted onto molecular chains of chitin and crude protein in shells. The acrylates were proved to be successfully grafted onto the demineralized shells through FTIR characterization and grafting parameters of the shell-g-polyacrylates were obtained through titration and Soxhlet extraction. Then, effects of grafting ratio and molecular structure of acrylate monomers on tensile properties and water resistance of the grafted shell thermoplastic films were surveyed. With the increase in grafting ratio of the demineralized shell-g-PMMA, tensile strength of the thermoplastic films initially increased, reached the maximum when the ratio was 31.86%, and then leveled off while both the elongation and water resistance of the film showed an increasing trend. In addition, with the increase in the alkyl chain length of ester group in acrylate unit, tensile strength and water resistance of the grafted shell film decreased continuously while tensile elongation kept increasing. As for α-methyl in methacrylate unit, the film prepared from the shells grafted with methacrylates had higher tensile strength and water resistance but lower elongation than the ones grafted with corresponding acrylates.

Knocking down chitin synthase 2 by RNAi is lethal to the cotton boll weevil

The cotton boll weevil (Anthonomus grandis) is the most destructive cotton insect pest in Brazil. The endophytic habit of this insect makes difficult its chemical control. Chitin synthase (CHS) is an integral membrane glycosyltransferase that is essential for chitin chain polymerization and deposition in insect chitinous structures, such as the peritrophic membrane (PM). Because it is not present in plants or vertebrates, CHS can be considered a promising target for eco-friendly biotechnological approaches, such as RNA interference (RNAi)-mediated gene silencing. Considering the relevance of CHS genes in the chitin biosynthetic pathway in insects, we report here the molecular cloning of the full-length CHS2 cDNA from the cotton boll weevil, and its functional validation via RNAi. The AgraCHS2 cDNA sequence is 4,869bp, with a 4,446bp open reading frame that encodes a predicted protein with 1,482 amino acid residues. Predicted protein has high similarity (53 to 78%) with other insects CHS. Moreover, only one copy is present in A. grandis genome. Transcriptional analysis showed that AgraCHS2 transcripts are restricted to the insect midgut at the third-larval instar and adult stages, which are considered the main feeding stages. RNAi-mediated knockdown of the AgraCHS2 affected A. grandis development, resulting in oviposition reduction of 93% and leading to 100% adult mortality. These data, in addition to the observation of PM severe disorganization in the midgut after AgraCHS2 knock-down, suggest AgraCHS2 as a promising target for developing RNAi-based biotechnological alternatives to specifically control the cotton boll weevil.

A broad pH range and processive chitinase from a metagenome library.

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions

Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (α/β)8 TIM barrel catalytic domain; and iii) the α+β insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBDVhChiA. The recombinant ChBDVhChiA was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBDVhChiA contained β-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBDVhChiA bound to both α- and β-chitins, with the greatest affinity for β-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions.