Chitin Chains Research Articles

Alkali lignin mediated chitin self-assembly for constructing a fully naturally resourced bioplastic

Exploring green and sustainable alternative materials to address the growing environmental issue caused by petroleum-based plastics has led to increasing attention for the development of degradable bio-based plastics. The synthesis of bioplastics with high strength and toughness still faces a big challenge. Herein, we report a full noncovalent mediated self-assembly strategy for simultaneously improving the strength and toughness of chitin-based bioplastics via plane hot-pressing. The alkali lignin (AL) is introduced to enhance the strength and toughness of the chitinous bioplastics via forming multiple noncovalent interactions with chitin chains. The incorporated dense noncovalent networks and the pressure induced orientated nanofibers significantly enhanced the mechanical properties of the chitinous bioplastic, achieving a tensile strength of 159.0 ± 2.1 MPa and Young’s modulus of 8.4 ± 0.2 GPa at 58 % relative humidity. The alkali lignin improved the moisture stability of the chitinous bioplastic. Moreover, the chitinous bioplastics exhibited superior welding ability, solvent resistance, good UV shileding capability and biodegradability in nature, which demonstrates the developed fabrication design of chitinous bioplastics provides a promising concept for preparing the alternative materials to replace traditional plastics.

Unveiling the anion-specific effect induced structure and behavior variations on a single chitin chain

Ion-specific effects have a profound impact on the macroscopic properties of polysaccharides such as the aggregation and solubility in solutions. However, the underlying molecular mechanisms still require exploration. In this study, we investigated the influence of anions on the structure and behavior of chitin via a combination of single-molecule techniques and macroscopic characterizations. Our findings reveal that the hydration of anions functions as bridging center, leading to the formation of water-ion-water bridges between chitin side groups. Remarkably, this structure exhibits a binding affinity to chitin following the Hofmeister series with the order of F− < Cl− < Br− < I−, resulting in an escalating of left-handed helix order of chitin. Subsequent experiments indicate that the bridging structure affects the size of chitin aggregates in the similar manner, implying the impact of anions on the structure and behavior of chitin across length scales. These results provide valuable insights into the understanding of ion-specific effects on polysaccharides and its applications in food science where high salt environments are involved.

An overview on acylation methods of α-chitin

This article overviews the acylation methods of α-chitin developed over the last four decades. The acylation of polysaccharides has been identified as a useful approach for conferring properties such as thermoplasticity. Owing to the poor solubility of α-chitin, its acylation using acid anhydrides and acyl chlorides has been traditionally investigated under heterogeneous conditions in strong acidic media. Although chitin chains depolymerize under acidic conditions, the resultant derivatives exhibit certain properties and functions. Solvents, such as LiCl/N,N-dimethyladcetamide, ionic liquids, and deep eutectic solvents, are suitable for α-chitin dissolution; therefore, acylation methods for α-chitin under homogeneous conditions have been developed using these solvents as reaction media. The functional materialization of the resultant derivatives was achieved by introducing appropriate substituents and controlling their ratios.

Preparation of Nanochitin Films with Oligochitin Graft Chains

Even nowadays, chitin is mostly unutilized as a biomass resource, although it is abundantly present in nature. To develop an efficient method to use chitin as the component in new functional bio-based materials, in this study, we investigated the preparation of a flexible nanochitin (chitin nanofiber, ChNF) film with oligochitin dihexanoate graft chains. The parent ChNF film was prepared by regeneration of a chitin ion gel with an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), using methanol and subsequent filtration. However, the obtained film showed a quite brittle nature, probably because of the high crystallinity of the chitin chains. To reduce the crystallinity, oligochitin dihexanoate, which was provided by partial depolymerization of the parent chitin dihexanoate under acidic conditions, was modified on the partially deacetylated ChNF film by reductive amination. The introduction of the oligochitin dihexanoate graft chains was supported by 1H NMR and IR measurements. The powder X-ray diffraction (XRD) profile of a film, which was obtained from an aqueous acetic acid suspension of the grafted product, indicated a reduction in chitin crystallinity, which contributes to the disappearance of nanofiber morphology and enhancement of flexibility. The removal of hexanoyl groups from the film was performed by treatment with aqueous NaOH. The IR and XRD measurements of the obtained film suggested the compete dehexanoylation and the reformation of the chitin crystalline structure, respectively. This study provides a method to fabricate new bio-based graft and soft materials entirely comprising chitin moieties.

Structure and expression of Rhodnius prolixus GH18 chitinases and chitinase-like proteins: Characterization of the physiological role of RpCht7, a gene from subgroup VIII, in vector fitness and reproduction.

Chitinases are enzymes responsible for the hydrolysis of glycosidic linkages within chitin chains. In insects, chitinases are typically members of the multigenic glycoside hydrolase family 18 (GH18). They participate in the relocation of chitin during development and molt, and in digestion in detritivores and predatory insects, and they control the peritrophic membrane thickness. Chitin metabolism is a promising target for developing vector control strategies, and knowledge of the roles of chitinases may reveal new targets and illuminate unique aspects of their physiology and interaction with microorganisms. Rhodnius prolixus is an important vector of Chagas disease, which is caused by the parasite Trypanosoma cruzi. In this study, we performed annotation and structural characterization of nine chitinase and chitinase-like protein genes in the R. prolixus genome. The roles of their corresponding transcripts were studied in more depth; their physiological roles were studied through RNAi silencing. Phylogenetic analysis of coding sequences showed that these genes belong to different subfamilies of GH18 chitinases already described in other insects. The expression patterns of these genes in different tissues and developmental stages were initially characterized using RT-PCR. RNAi screening showed silencing of the gene family members with very different efficiencies. Based on the knockdown results and the general lack of information about subgroup VIII of GH18, the RpCht7 gene was chosen for phenotype analysis. RpCht7 knockdown doubled the mortality in starving fifth-instar nymphs compared to dsGFP-injected controls. However, it did not alter blood intake, diuresis, digestion, molting rate, molting defects, sexual ratio, percentage of hatching, or average hatching time. Nevertheless, female oviposition was reduced by 53% in RpCht7-silenced insects, and differences in oviposition occurred within 14–20 days after a saturating blood meal. These results suggest that RpCht7 may be involved in the reproductive physiology and vector fitness of R. prolixus.

Structural basis for directional chitin biosynthesis

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2–4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a ‘gate lock’ that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.

Probing the Structural Details of Chitin Nanocrystal–Water Interfaces by Three‐Dimensional Atomic Force Microscopy

Chitin is one of the most abundant and renewable natural biopolymers. It exists in the form of crystalline microfibrils and is the basic structural building block of many biological materials. Its surface crystalline structure is yet to be reported at the molecular level. Herein, atomic force microscopy (AFM) in combination with molecular dynamics simulations reveals the molecular-scale structural details of the chitin nanocrystal (chitin NC)-water interface. High-resolution AFM images reveal the molecular details of chitin chain arrangements at the surfaces of individual chitin NCs, showing highly ordered, stable crystalline structures almost free of structural defects or disorder. 3D-AFM measurements with submolecular spatial resolution demonstrate that chitin NC surfaces interact strongly with interfacial water molecules creating stable, well-ordered hydration layers. Inhomogeneous encapsulation of the underlying chitin substrate by these hydration layers reflects the chitin NCs' multifaceted surface character with different chain arrangements and molecular packing. These findings provide important insights into chitin NC structures at the molecular level, which is critical for developing the properties of chitin-based nanomaterials. Furthermore, these results will contribute to a better understanding of the chemical and enzymatic hydrolysis of chitin and other native polysaccharides, which is also essential for the enzymatic conversion of biomass.

Facile route to tri-carboxyl chitin nanocrystals from di-aldehyde chitin modified by selective periodate oxidation

Chitin, a kind of polysaccharide mainly obtained from food waste, has emerged as an important biodegradable biopolymer in composite materials. The difficulty of aldehyde modification, which greatly limited the application of chitin nanocrystals, was addressed by applying a facile route of partial deacetylation followed by periodate oxidation in this study. Deacetylation occurred on the surface of both crystalline and amorphous regions, which were significantly degraded in the following periodate oxidation due to the inevitable cleavage of chitin chains, leading to an increase in the crystallinity index of obtained di-aldehyde chitin. The degree of deacetylation and periodate addition had limited improvement in the aldehyde content of di-aldehyde chitin with a maximum value of around 0.42 mmol/g. With further 2,2,6,6-tetramethylpiperidine-1-oxyl-mediated oxidation, the carboxyl content of tri-carboxyl chitin was improved to 1.58 mmol/g, which played a critical role in the dispersion efficiency and morphology of chitin nanocrystals. The obtained rod-like chitin nanocrystals with a ζ-potential value of −42 mV and an average size of 97 nm have potential application in dye-adsorption and emulsion stabilizers.

Polyphenol-driving assembly for constructing chitin-polyphenol-metal hydrogel as wound dressing

The metal-polyphenol networks have attracted appealing attention for diverse biomedical applications due to their remarkable characteristics. Though various of metal-polyphenolic materials have been prepared, the homogeneous metal-polyphenol based hydrogel fabrication remains a challenge (e.g., quick aggregation). Herein, a facile and low-cost polyphenol-mediating non-covalent driven assembly strategy was developed for fabricating homogeneous chitin-polyphenol-metal hydrogels. Polyphenols not only noncovalently crosslinked chitin chains, but simultaneously captured metal nanomaterials from metal substrates and immobilized in chitin-polyphenol networks. A range of metal (Fe, Cu, Ti, Zn) and polyphenol (tannic acid, gallic acid, quercetin, pyrogallic acid) could be incorporated into this hydrogel framework. As a demonstration, the chitin-tannic acid-Cu hydrogel showed excellent antibacterial properties and significantly enhanced infected wound repair via promoting the cell proliferation and angiogenesis, showing the potential in wound dressing. The low cost, versatility and flexibility assembly process can be used to fabricate diverse polymer-polyphenol-metal hydrogel, thereby enabling their use in various applications.

Anisotropic and robust hydrogels combined osteogenic and angiogenic activity as artificial periosteum

Periosteum possesses unique characteristics such as anisotropic structure and superior osteogenic and angiogenic ability, which plays vital roles in the formation, remodeling and fracture repair of bone. However, the development of artificial periosteum with such highly desired characteristics is still a challenge. Here, we exhibit the strong and anisotropic chitin composite hydrogels that are composed of chitin chains and maleation chitin whiskers (mCHWs) via chemical and physical dual cross-linking strategy and the stretching-drying process. Moreover, bioactive deferoxamine (DFO) was chosen to further functionalize this anisotropic composite hydrogel to strengthen its ability of osteogenesis and vascularization. This design endows the as-prepared composite hydrogels with remarkable tensile strength (17.6 ± 1.4 MPa) and Young's modulus (28.1 ± 2.2 MPa), which are obviously superior to those of polysaccharide hydrogels according to the most references. More importantly, such composite hydrogels not only can effectively enhance cells adhesion and proliferation, but also have the outstanding capability to facilitate the osteogenesis and angiogenesis of cells. Thus, these anisotropic and robust chitin composite hydrogels show promising application for the artificial periosteum.

Anisotropic Hybrid Hydrogels Constructed via the Noncovalent Assembly for Biomimetic Tissue Scaffold

AbstractAnisotropic structure is key for exploring the biomimetic functions of anisotropic hydrogels. However, the anisotropic hydrogel study should not be limited to its architecture design but must include the understanding and improvement of the internal interaction among their components. Herein, a noncovalent mediated assembly strategy is proposed to simultaneously improve the chitin chain mobility and enhance the interfacial interaction, for achieving anisotropic chitin/2D material (molybdenum disulfide and brushite as example) hydrogels via mechanical deformation. Tannic acid (TA) is used to i) introduce the dynamic noncovalent crosslinking structure among the chitin chains for affording considerable molecular mobility to allow chitin chains alignment under mechanical deformation; ii) enhance chitin–2D interfacial interaction for benefiting 2D materials orientation under the chitin chains driving. The design concept achieves multiple noncovalent assembly crosslinks (chitin–chitin, chitin–TA, and chitin–TA–2D) and biomimetic anisotropic nanofibrous morphology, leading to the superior mechanical performance. The anisotropic chitin–TA/brushite hydrogel effectively accelerates bone regeneration by promoting cell osteogenic differentiation and directional migration, showing potential in tissue engineering. It is anticipated that the noncovalent mediated assembly concept could be used to fabricate other polymer based composite anisotropic hydrogels for diverse applications.

Non-conventional expression of recombinant chitinase A originated from Bacillus licheniformis DSM8785, in Saccharomyces cerevisiae INVSc1

Chitinases are glycosyl hydrolases, that cleave the ?-1,4 linkage between N-acetyl glucosamines present in chitin chains. Chitin is the second most abundant polysaccharide on Earth after cellulose, and it is produced in the exoskeleton of crustaceans and insects, and in some parts of the cell walls of fungi. Enzymatic development and the extraction of superior derivatives from chitin wastes ? such as chitooligosaccharides with vast importance in the medical and biofuels industry ? lead to the necessity of creating chitinases using different strains of organisms. In this paper, the chiA gene from the Bacillus licheniformis DSM8785 encoding chitinase A (ChiA) with C-terminal hexahistidine tag was cloned and expressed in the extracellular expression system pYES2 from Saccharomyces cerevisiae INVSc1 as a hyperglycosylated enzyme. The production of recombinant ChiA was successfully confirmed by dot blotting, using anti-His antibodies. The optimal time of expression was identified to be 24 h when galactose was added only at the beginning of fermentation, the chitinase activity starting to decrease after this threshold. Nevertheless, in another experiment, when galactose was added every 24 h for 72 h, the expression continued for the entire period. The purified enzyme was detected, using sodium dodecyl sulphate?polyacrylamide gel electrophoresis (SDS-PAGE), as a heterogeneous diffuse band between 80 and 180 kDa. The molecular mass of the same ChiA enzyme expressed in Pichia pastoris KM71H and Escherichia coli BL21 (DE3) was compared using SDS-PAGE with ChiA expressed in S. cerevisiae INVSc1. The activity of ChiA was determined using the fluorogenic substrate, 4-methylumbelliferyl ?-D-N,N,N-triacetylchitotrioside (4MUTC). Using a bioinformatics simulation, the number of the glycolsylation sites of the ChiA gene sequence and the proximity of these sites to the alpha factor sequence were hypothesized to be a possible reason for which ChiA enzyme was internally expressed.

Synthesis of thermoplastic chitin hexanoate-graft-poly(ε-caprolactone)

Herein, we report that chitin hexanoate-graft-poly(ε-caprolactone) (ChHex-g-PCL) is thermoplastic, as confirmed by the formation of a melt-pressed film. Chitin hexanoates with degrees of substitution (DSs) of 1.4–1.8 and bearing free hydroxy groups were first prepared by the hexanoylation of chitin using adjusted feed equivalents of hexanoyl chloride in the presence of pyridine and N,N-dimethyl-4-aminopyridine in 1-allyl-3-methylimidazolium bromide, an ionic liquid. Surface-initiated ring-opening graft polymerization of ε-caprolactone from the hydroxy groups of the chitin hexanoates was conducted in the presence of tin(II) 2-ethylhexanoate as the catalyst at 100 °C to produce (ChHex-g-PCL)s. The feed equivalent of the catalyst, reaction time, and DS value were found to affect the molar substitution and degree of polymerization of the PCL graft chains. Longer PCL graft chains formed their crystalline structures and the (ChHex-g-PCL)s largely contained uncrystallized chitin chains. Accordingly, these (ChHex-g-PCL)s exhibited melting points associated with the PCL graft chains, leading to thermoplasticity.

Heterologous expression and characterization of thermostable chitinase and β-N-acetylhexosaminidase from Caldicellulosiruptor acetigenus and their synergistic action on the bioconversion of chitin into N-acetyl-d-glucosamine

The bioconversion of chitin into N-acetyl-d-glucosamine (GlcNAc) using chitinolytic enzymes is one of the important avenues for chitin valorization. However, industrial applications of chitinolytic enzymes have been limited by their poor thermostability. Therefore, it is necessary to discover thermostable chitinolytic enzymes for GlcNAc production from chitin. In this study, two chitinolytic enzyme-encoding genes CaChiT and CaHex from Caldicellulosiruptor acetigenus were identified and heterologously expressed in Escherichia coli. The purified recombinant CaChiT and CaHex showed optimal activities at 70 °C and 90 °C respectively, and exhibited good thermostability over a range of temperature below 70 °C and broad pH stability at pH range of 3.0-8.0. CaChiT and CaHex were active on colloidal chitin, pNP-(GlcNAc)2, pNP-(GlcNAc)3, and pNP-GlcNAc, pNP-(GlcNAc)2, pNP-(GlcNAc)3, pNP-Glc respectively. Besides, the chitin oligosaccharides and colloidal chitin hydrolysis profiles revealed that CaChiT degraded chitin chains through exo-mode of action. Furthermore, CaChiT and CaHex exhibited a synergistic effect in the degradation of colloidal chitin, reaching 0.60 mg/mL of GlcNAc production after 1 h incubation. These results suggested that a combination of CaChiT and CaHex have great potential for industrial applications in the enzymatic production of GlcNAc from chitin-containing biowastes.

Structural and functional variation of chitin-binding domains of a lytic polysaccharide monooxygenase from Cellvibrio japonicus

Among the extensive repertoire of carbohydrate-active enzymes, lytic polysaccharide monooxygenases (LPMOs) have a key role in recalcitrant biomass degradation. LPMOs are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides such as cellulose and chitin. Several LPMOs contain carbohydrate-binding modules (CBMs) that are known to promote LPMO efficiency. However, structural and functional properties of some CBMs remain unknown, and it is not clear why some LPMOs, like CjLPMO10A from the soil bacterium Cellvibrio japonicus, have multiple CBMs (CjCBM5 and CjCBM73). Here, we studied substrate binding by these two CBMs to shine light on their functional variation and determined the solution structures of both by NMR, which constitutes the first structure of a member of the CBM73 family. Chitin-binding experiments and molecular dynamics simulations showed that, while both CBMs bind crystalline chitin with Kd values in the micromolar range, CjCBM73 has higher affinity for chitin than CjCBM5. Furthermore, NMR titration experiments showed that CjCBM5 binds soluble chitohexaose, whereas no binding of CjCBM73 to this chitooligosaccharide was detected. These functional differences correlate with distinctly different arrangements of three conserved aromatic amino acids involved in substrate binding. In CjCBM5, these residues show a linear arrangement that seems compatible with the experimentally observed affinity for single chitin chains. On the other hand, the arrangement of these residues in CjCBM73 suggests a wider binding surface that may interact with several chitin chains. Taken together, these results provide insight into natural variation among related chitin-binding CBMs and the possible functional implications of such variation.

Full-Length Transcriptome of Thalassiosiraweissflogii as a Reference Resource and Mining of Chitin-Related Genes.

β-Chitin produced by diatoms is expected to have significant economic and ecological value due to its structure, which consists of parallel chains of chitin, its properties and the high abundance of diatoms. Nevertheless, few studies have functionally characterised chitin-related genes in diatoms owing to the lack of omics-based information. In this study, we first compared the chitin content of three representative Thalassiosira species. Cell wall glycosidic linkage analysis and chitin/chitosan staining assays showed that Thalassiosira weissflogii was an appropriate candidate chitin producer. A full-length (FL) transcriptome of T. weissflogii was obtained via PacBio sequencing. In total, the FL transcriptome comprised 23,362 annotated unigenes, 710 long non-coding RNAs (lncRNAs), 363 transcription factors (TFs), 3113 alternative splicing (AS) events and 3295 simple sequence repeats (SSRs). More specifically, 234 genes related to chitin metabolism were identified and the complete biosynthetic pathways of chitin and chitosan were explored. The information presented here will facilitate T. weissflogii molecular research and the exploitation of β-chitin-derived high-value enzymes and products.

Chaperone-assisted soluble expression and characterization of chitinase chiZJ408 in Escherichia coli BL21 and the chitin degradation by recombinant enzyme

The chaperone-assisted soluble expression and characterization of high molecular weight chitinase chiZJ408 in Escherichia coli BL21 were investigated. Chitin degradation products by chitinase chiZJ408 were analyzed. The results indicated that the introduction of the chaperone GroELS promoted the correct folding of chitinase chiZJ408 and increased its soluble expression by 14.9% (p < 0.05) in E. coli BL21. The optimal pH and temperature of chitinase chiZJ408 were respectively 6.0 and 50 °C. Chitinase chiZJ408 was stable at pHs of 4.0 ∼ 7.0 and at below 40 °C. Mg2+and Ca2+ had a significant impact on improving the activity of chitinase chiZJ408. Chitinase chiZJ408 was demonstrated to be exochitinase that cleaved the β-1,4-glycosidic bond of the chitin chain to generate only N,N’-diacetylchitobiose. This study broadens our understanding of chitinase and provides a basis for solving the problem of inclusion body formed by long fragment gene expression in E. coli.

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 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.

Rapid dissolution of β-chitin and hierarchical self-assembly of chitin chains in aqueous KOH/urea solution

β-chitin allows for rapid penetration of solvent molecules, followed by swelling and dissolution. During neutralization, the chitin chains self-assembled into nanofibrils, and even the formation of hydrogels of crystalline α-chitin.

Chromatographic Assays for the Enzymatic Degradation of Chitin.

Chitin is an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product types that require different analytical methods for detection and quantitation. While soluble chromogenic substrates are readily available for chitinases, proper insight into the activity of these enzymes can only be obtained by measuring activity toward their polymeric, insoluble substrate, chitin. For LPMOs, only assays using insoluble chitin are possible and relevant. Working with insoluble substrates complicates enzyme assays from substrate preparation to product analysis. Here, we describe typical set-ups for chitin degradation reactions and the chromatographic methods used for product analysis. Graphical abstract: Overview of chromatographic methods for assessing the enzymatic degradation of chitin.