Plasma-activated water improves the accessibility of chitinase to chitin by decreasing molecular weight and breaking crystal structure.

Chitooligosaccharides, the hydrolysis products of chitin, have superior biological activities and application value to those of chitin itself; however, the ordered and highly crystalline structure of chitin renders its degradation by chitinase difficult. Herein, the effects of plasma-activated water (PAW) pre-treatment on the physicochemical properties, crystal structure, and enzymatic hydrolysis of chitin were investigated. The hydrolysis of PAW-pre-treated chitin (PAW activation time of 5 min) using chitinase from Vibrio harveyi (VhChit2) yielded 71 % more reducing sugar, compared with that from untreated chitin, with the degree of chitin hydrolysis increasing from 13 % without pre-treatment to 23 % post-treatment. Moreover, the amount of VhChit2 adsorbed by chitin increased from 41.7 to 58.2 mg/g. Fourier transform infrared spectrometry revealed that PAW could break the ß-1,4-glycosidic bonds of chitin (but had no effects on the hydrogen and amido bonds), thereby decreasing the molecular weight and crystallinity of the polysaccharide, which caused its structural damage and enhanced its enzymatic hydrolysis by chitinase. Consequently, PAW pre-treatment can be considered a simple, effective, and environmentally-friendly method for the biotransformation of chitin as its easier hydrolysis yields high-value products.

Enzymatic production of diverse N-acetyl chitooligosaccharides employing a novel bifunctional chitinase and its engineered variants.

Bioproduction of diverse N-acetyl chitooligosaccharides from chitin is of great value. In the study, a novel GH family 18 bifunctional chitinase gene (PsChi82) from Paenibacillus shirakamiensis was identified, expressed and biochemically characterized. PsChi82 was most active at pH 5.0, and 55 °C, and displayed remarkable pH stability with the broad pH range of 3.0-12.0. It showed high chitosanase activity of 10.6 U mg-1 and diverse hydrolysis products of GlcNAc, (GlcNAc)2, GlcN-GlcNAc and (GlcN)2-GlcNAc, which may facilitate comprehensively understanding of structure-function relationships of N-acetyl COSs. Three engineered variants were then expressed and characterized. Among them, PsChi82-CBM26 possessed specific activity of 25.1 U mg-1 against colloidal chitin, which was 2.1 folds higher than that of PsChi82. The diverse N-acetyl COSs were subsequently produced by PsChi82-CBM26 with a sugar content of 23.2 g L-1. These excellent properties may make PsChi82-CBM26 potentially useful for N-acetyl COSs production in the food and chemical industries.

Design of Inhibitors Targeting Chitin-Degrading Enzymes by Bioisostere Substitutions and Scaffold Hopping for Selective Control of Ostrinia furnacalis.

Chitin-degrading enzymes are critical components in regulating the molting process of the Asian corn borer and serve as potential targets for controlling this destructive pest of maize. Here, we used a scaffold-hopping strategy to design a series of efficient naphthylimide insecticides. Among them, compound 8c exhibited potent inhibition of chitinase from OfChi-h and OfChtI at low nanomolar concentrations (IC50 = 1.51 and 9.21 nM, respectively). Molecular docking simulations suggested that 8c binds to chitinase by mimicking the interaction of chitin oligosaccharide substrates with chitinase. At low ppm concentrations, compound 8c performed comparably to commercial insecticides in controlling the highly destructive plant pest, the Asian corn borer. Tests on a wide range of nontarget organisms indicate that compound 8c has very low toxicity. In addition, the effect of inhibitor treatment on the expression of genes associated with the Asian corn borer chitin-degrading enzymes was further investigated by quantitative real-time polymerase chain reaction. In conclusion, our study highlights the potential of 8c as a novel chitinase-targeting insecticide for effective control of the Asian corn borer, providing a promising solution in the quest for sustainable pest management.

From Natural Microbe Screening to Sustained Chitinase Activity in Exogenous Hosts.

Genetic parts and hosts can be sourced from nature to realize new functions for synthetic biology or to improve performance in a particular application environment. Here, we proceed from the discovery and characterization of new parts to stable expression in new hosts with a particular focus on achieving sustained chitinase activity. Chitinase is a key enzyme for various industrial applications that require the breakdown of chitin, the second most abundant biopolymer on the earth. Diverse microbes exhibit chitinase activity, but for applications, the environmental conditions for optimal enzyme activity and microbe fitness must align with the application context. Achieving sustained chitinase activity under broad conditions in heterologous hosts has also proven difficult due to toxic side effects. Toward addressing these challenges, we first screen ocean water samples to identify microbes with chitinase activity. Next, we perform whole genome sequencing and analysis and select a chitinase gene for heterologous expression. Then, we optimize transformation methods for target hosts and introduce chitinase. Finally, to achieve robust function, we optimize ribosome binding sites and discover a beneficial promoter that upregulates chitinase expression in the presence of colloidal chitin in a sense-and-respond fashion. We demonstrate chitinase activity for >21 days in standard (Escherichia coli) and nonstandard (Roseobacter denitrificans) hosts. Besides enhancing chitinase applications, our pipeline is extendable to other functions, identifies natural microbes that can be used directly in non-GMO contexts, generates new parts for synthetic biology, and achieves weeks of stable activity in heterologous hosts.

A Potential Multitarget Insect Growth Regulator Candidate: Design, Synthesis, and Biological Activity of Novel Acetamido Derivatives Containing Hexacyclic Pyrazole Carboxamides.

Insect growth regulators (IGRs) are important green insecticides that disrupt normal growth and development in insects to reduce the harm caused by pests to crops. The ecdysone receptor (EcR) and three chitinases OfChtI, OfChtII, and OfChi-h are closely associated with the molting stage of insects. Thus, they are considered promising targets for the development of novel insecticides such as IGRs. Our previous work identified a dual-target compound 6j, which could act simultaneously on both EcR and OfChtI. In the present study, 6j was first found to have inhibitory activities against OfChtII and OfChi-h, too. Subsequently, taking 6j as a lead compound, 19 novel acetamido derivatives were rationally designed and synthesized by introducing an acetamido moiety into the amide bridge based on the flexibility of the binding cavities of 6j with EcR and three chitinases. Then, their insecticidal activities against Plutella xylostella (P. xylostella), Ostrinia furnacalis (O. furnacalis), and Spodoptera frugiperda (S. frugiperda) were carried out. The bioassay results revealed that most of these acetamido derivatives possessed moderate to good larvicidal activities against three lepidopteran pests. Especially, compound I-17 displayed excellent insecticidal activities against P. xylostella (LC50, 93.32 mg/L), O. furnacalis (LC50, 114.79 mg/L), and S. frugiperda (86.1% mortality at 500 mg/L), significantly better than that of 6j. In addition, further protein validation and molecular docking demonstrated that I-17 could act simultaneously on EcR (17.7% binding activity at 8 mg/L), OfChtI (69.2% inhibitory rate at 50 µM), OfChtII (71.5% inhibitory rate at 50 µM), and OfChi-h (73.9% inhibitory rate at 50 µM), indicating that I-17 is a potential lead candidate for novel multitarget IGRs. This work provides a promising starting point for the development of novel types of IGRs as pest management agents.

Boosting one-step degradation of shrimp shell waste to produce chitin oligosaccharides at smart nanoscale enzyme reactor with liquid-solid system.

Chitin oligosaccharides (CTOS) possess potential applications in food, medicine, and agriculture. However, lower mass transfer and catalytic efficiency are the main kinetic limitations for the production of CTOS from shrimp shell waste (SSW) and crystalline chitin. Chemical or physical methods are usually used for pretreatment to improve chitinase hydrolysis efficiency, but this is not eco-friendly and cost-effective. To address this challenge, a chitinase nanoreactor with the liquid-solid system (BcChiA1@ZIF-8) was manufactured to boost the one-step degradation of SSW and crystalline chitin. Compared with free enzyme, the catalytic efficiency of BcChiA1@ZIF-8 on colloidal chitin was significantly improved to 142 %. SSW and crystalline chitin can be directly degraded by BcChiA1@ZIF-8 without any pretreatments. The yield of N, N'-diacetylchitobiose [(GlcNAc)2] from SSW and N-acetyl-D-glucosamine (GlcNAc) from crystalline chitin was 2 times and 3.1 times than that of free enzyme, respectively. The reason was that BcChiA1@ZIF-8 with a liquid-solid system enlarged the interface area, increased the collision frequency between enzyme and substrate, and improved the large-substrates binding activity of chitinase. Moreover, the biphasic system exhibited excellent stability, and the design showed universal applicability. This strategy provided novel guidance for other polysaccharide biosynthesis and the conversion of environmental waste into carbohydrates.

Response surface optimization, purification, characterization and short-chain chitooligosaccharides production from an acidic, thermostable chitinase from Thermomyces dupontii.

Chitin, recovered in huge amounts from coastal waste, may biocatalytically valorized for utilization in food and biotech sectors. Conventional chemical-based conversion makes use of significant volumes of hazardous acid and alkali. Alternatively, enzymes offer better process control and generation of homogeneous products. Process variables were derived to achieve augmented levels of chitinase (3.8809 Ul-1 h-1) productivity from a novel thermophilic fungal strain Thermomyces dupontii, ITCC 9104 following incubation (96 h, 45 °C). An acidic thermostable chitinase TdChiT having molecular mass of 60 kDa has been purified. Optimal TdChiT activity has been demonstrated at 70 °C and pH 5. Notably decreased activity over a broad range of temperature and pH was observed following deglycosylation. Half-life, activation energy, Gibbs free energy, enthalpy and entropy for denaturation of TdChiT at its optimum temperature were 197.40 min, 105.48 kJ mol-1, 100.59 kJ mol-1, 102.64 kJ mol-1 and 5.95 J mol-1 K-1. TdChiT has specificity towards colloidal chitin and (GlcNAc)2-4. Metal ions viz. Mn2+, Ca2+ and Co2+ and nonionic surfactants notably enhanced chitinase activity. Thin layer chromatography analysis has revealed effective hydrolysis of colloidal chitin and (GlcNAc)2-4. TdChiT may potentially be employed for design of better, eco-friendly and less resource-intensive industrial procedures for upcycling of crustacean waste into value-added organonitrogens.

Partitioning recombinant chitinase from Nicotiana benthamiana by an aqueous two-phase system based on polyethylene glycol and phosphate salts.

The objectives of this study were to purify 42 kDa chitinase derived from Trichoderma asperellum SH16 produced in Nicotiana benthamiana by a polyethylene glycol (PEG)/salt aqueous two-phase system (ATPS). The specific activities of the crude chitinase and the partially purified chitinase from N. benthamiana were about 251 unit/mg and 386 unit/mg, respectively. The study found the 300 g/L PEG 6000 + 200 g/L potassium phosphate (PP) and 300 g/L PEG 6000 + 150 g/L sodium phosphate (SP) systems had the highest partitioning efficiency for each salt in primary extraction. However, among the two types of salt, PP displayed higher efficiency than SP, with a partitioning coefficient K of 4.85 vs. 3.89, a volume ratio V of 2.94 vs. 2.68, and a partitioning yield Y of approximately 95 % vs. 83 %. After back extraction, the enzymatic activity of purified chitinase was up to 834 unit/mg (PP) and 492 unit/mg (SP). The purification factors reached 3.32 (PP) and 1.96 (SP), with recovery yields of about 59 % and 61 %, respectively. SDS-PAGE and zymogram analysis showed that the recombinant chitinase was significantly purified by using ATPS. The purified enzyme exhibited high chitinolytic activity, with the hydrolysis zone's diameter being around 2.5 cm-3 cm. It also dramatically reduced the growth of Sclerotium rolfsii; the colony diameter after treatment with 60 unit of enzyme for 104 spores was only about 1 cm, compared to 3.5 cm in the control. The antifungal effect of chitinase suggests that this enzyme has great potential for applications in agricultural production as well as postharvest fruit and vegetable preservation.

Remote loop evolution reveals a complex biological function for chitinase enzymes beyond the active site.

Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, the evolutionary molecular mechanisms how proteins acquire these loop elements and obtain new function is poorly understood. To address this question, we study glycoside hydrolase family 19 (GH19) chitinase-an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discover that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrate that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that nature applies this strategy at the molecular level to achieve a complex biological function while maintaining the original activity in the catalytic pocket, suggesting an alternative way to design new enzyme function.

Engineering a recombinant chitinase from the marine bacterium Bacillus aryabhattai with targeted activity on insoluble crystalline chitin for chitin oligomer production.

Chitin, an abundant polysaccharide in India, is primary by-product of the seafood industry. Efficiently converting chitin into valuable products is crucial. Chitinase, transforms chitin into chitin oligomers, holds significant industrial potential. However, the crystalline and insoluble nature of chitin makes the conversion process challenging. In this study, a recombinant chitinase from marine bacteria Bacillus aryabhattai was developed. This enzyme exhibits activity against insoluble chitin substrates, chitin powder and flakes. The chitinase gene was cloned into the pET 23a plasmid and transformed into E. coli Rosetta pLysS. IPTG induction was employed to express chitinase, and purification using Ni-NTA affinity chromatography. Optimal chitinase activity against colloidal chitin was observed in Tris buffer at pH 8, temperature 55°C, with the presence of 400 mM sodium chloride. Enzyme kinetics studies revealed a Vmax of 2000 µmole min-1 and a Km of 4.6 mg mL-1. The highest chitinase activity against insoluble chitin powder and flakes reached 875 U mg-1 and 625 U mg-1, respectively. The chitinase demonstrated inhibition of Candida albicans, Fusarium solani, and Penicillium chrysogenum growth. Thin Layer Chromatography (TLC) and LC-MS analysis confirmed the production of chitin oligomers, chitin trimer, tetramer, pentamer, and hexamer, from chitin powder and flakes using recombinant chitinase.

Bioconversion of α-Chitin by a Lytic Polysaccharide Monooxygenase OsLPMO10A Coupled with Chitinases and the Synergistic Mechanism Analysis.

The whole enzymatic conversion of chitin is a green and promising alternative to current strategies, which are based on lytic polysaccharide monooxygenases (LPMOs) and chitinases. However, the lack of LPMOs with high activity toward α-chitin limits the efficient bioconversion of α-chitin. Herein, we characterized a high chitin-active LPMO from Oceanobacillus sp. J11TS1 (OsLPMO10A), which could promote the decrystallization of the α-chitin surface. Furthermore, when coupled with OsLPMO10A, the conversion rate of α-chitin to N-acetyl chitobiose [(GlcNAc)2] by three chitinases (Serratia marcescens, ChiA, -B, and -C) reached 30.86%, which was 2.03-folds that without the addition of OsLPMO10A. Moreover, the results of synergistic reactions indicated that OsLPMO10A and chitinases promoted the degradation of α-chitin each other mainly on the surface. To the best of our knowledge, this study achieved the highest yield of N-acetyl chitooligosaccharides (N-acetyl COSs) among reported LPMOs-driven bioconversion systems, which could be regarded as a promising candidate for α-chitin bioconversion.

Identification and characterization of chitinase producing marine microorganism: Unleashing the potential of chitooligosaccharides for bioethanol synthesis.

The dwindling supply of the petroleum product and its carbon footprint has initiated search for a sustainable fuel and alternate feed-stocks. One such underexplored feedstock is chitin, a waste derived from sea food processing. The limitation of insolubility and crystallinity inherent in chitin is addressed with the chitin hydrolysates. In the present study, a chitinases producing marine isolate was isolated from the sediments of Arabian Sea from a depth of 20 m. In order to increase the expression of the chitinases, sequential optimisation using one factor at a time and Taguchi experimental designs were employed which resulted in a yield of 13.46 U/mL which was 2.62 fold higher than the initial bioprocess condition values. In a two-step refinery protocol, Candida albicans was evolved towards chitooligosaccharides using chemically synthesized hydrolysates. In a fed -batch fermentation design the Candida yielded a 12.8 % conversion of these commercial chitin oligosaccharides into bioethanol in a run time of 48 h. This is the first report demonstrating the potential of Candida to utilise chitin oligosaccharides for the production of bioethanol.

pH/chitinase dual stimuli-responsive essential oil-delivery system based on mesoporous silica nanoparticles for control of rice blast.

BACKGROUND: Owing to their surface modifiability, smart mesoporous silica nanoparticles (MSNs) can be designed to respond to plant disease-microenvironmental stimuli, thereby achieving on-demand release of active ingredients to control disease by effectively improving citral (CT) stability. RESULTS: A pH/chitinase dual stimuli-responsive essential oil-delivery system (CT@HMS@CH/TA) was successfully fabricated by encapsulating CT in hollow mesoporous silica (HMS), and coating with tannic acid (TA) and chitosan (CH) within HMS by using the layer-by-layer assembly technique (LbL). CT@HMS@CH/TA with an average particle size of 125.12 ± 0.12 nm and a hollow mesoporous nanostructure showed high CT-loading efficiency (16.58% ± 0.17%). The photodegradation rate of CT@HMS@CH/TA under UV irradiation (48 h) was only 15.31%, indicating a 3.34-fold UV stability improvement. CT@HMS@CH/TA exhibited a higher CT release rate in response to acidic pH and the presence of chitinase, simulating the prevailing conditions as Magnaporthe oryzae infection. Furthermore, CT@HMS@CH/TA exhibited better adhesion without affecting normal rice growth, significantly upregulating chitinase gene expression and enhancing chitinase activity on M. oryzae, thus enhancing CT antifungal activity. CONCLUSION: CT@HMS@CH/TA improved CT stability and showed intelligent, controlled release-performance and higher antifungal efficacy, thus providing a new strategy for efficient application of essential oils for green control of rice blast disease. © 2024 Society of Chemical Industry.

Novel Butenolide Derivatives as Dual-Chitinase Inhibitors to Arrest the Growth and Development of the Asian Corn Borer.

OfChtI and OfChi-h are considered potential targets for the control of Asian corn borer (Ostrinia furnacalis). In this work, the previously reported OfChtI inhibitor 5f was found to show certain inhibitory activity against OfChi-h (Ki = 5.81 µM). Two series of novel butenolide derivatives based on lead compound 5f were designed with the conjugate skeleton, contributing to the π-binding interaction to chitinase, and then synthesized. Compounds 4a-l and 7a-p displayed excellent inhibitory activities against OfChtI and OfChi-h, respectively, at a concentration of 10 µM. Compound 4h was found to be a good dual-Chitinase inhibitor, with Ki values of 1.82 and 2.00 µM against OfChtI and OfChi-h, respectively. The inhibitory mechanism studies by molecular docking suggested that π-π stacking interactions were crucial to the inhibitory activity of novel butenolide derivatives against two different chitinases. A preliminary bioassay indicated that 4h exhibited certain growth inhibition effects against O. furnacalis. Butenolide-like analogues should be further studied as promising novel dual-chitinase inhibitor candidates for the control of O. furnacalis.

Chitinous material bioconversion by three new chitinases from the yeast Mestchnikowia pulcherrima.

BACKGROUND: Chitinases are widely distributed enzymes that perform the biotransformation of chitin, one of the most abundant polysaccharides on the biosphere, into useful value-added chitooligosaccharides (COS) with a wide variety of biotechnological applications in food, health, and agricultural fields. One of the most important group of enzymes involved in the degradation of chitin comprises the glycoside hydrolase family 18 (GH18), which harbours endo- and exo-enzymes that act synergistically to depolymerize chitin. The secretion of a chitinase activity from the ubiquitous yeast Mestchnikowia pulcherrima and their involvement in the post-harvest biological control of fungal pathogens was previously reported. RESULTS: Three new chitinases from M. pulcherrima, MpChit35, MpChit38 and MpChit41, were molecularly characterized and extracellularly expressed in Pichia pastoris to about 91, 90 and 71 mU ml- 1, respectively. The three enzymes hydrolysed colloidal chitin with optimal activity at 45 ºC and pH 4.0-4.5, increased 2-times their activities using 1 mM of Mn2+ and hydrolysed different types of commercial chitosan. The partial separation and characterization of the complex COS mixtures produced from the hydrolysis of chitin and chitosan were achieved by a new anionic chromatography HPAEC-PAD method and mass spectrometry assays. An overview of the predicted structures of these proteins and their catalytic modes of action were also presented. Depicted their high sequence and structural homology, MpChit35 acted as an exo-chitinase producing di-acetyl-chitobiose from chitin while MpChit38 and MpChit41 both acted as endo-chitinases producing tri-acetyl-chitotriose as main final product. CONCLUSIONS: Three new chitinases from the yeast M. pulcherrima were molecularly characterized and their enzymatic and structural characteristics analysed. These enzymes transformed chitinous materials to fully and partially acetylated COS through different modes of splitting, which make them interesting biocatalysts for deeper structural-function studies on the challenging enzymatic conversion of chitin.

The gastric mucosa of Atlantic salmon (Salmo salar) is abundant in highly active chitinases.

Atlantic salmon (Salmo salar) possesses a genome containing 10 genes encoding chitinases, yet their functional roles remain poorly understood. In other fish species, chitinases have been primarily linked to digestion, but also to other functions, as chitinase-encoding genes are transcribed in a variety of non-digestive organs. In this study, we investigated the properties of two chitinases belonging to the family 18 glycoside hydrolase group, namely Chia.3 and Chia.4, both isolated from the stomach mucosa. Chia.3 and Chia.4, exhibiting 95% sequence identity, proved inseparable using conventional chromatographic methods, necessitating their purification as a chitinase pair. Biochemical analysis revealed sustained chitinolytic activity against ß-chitin for up to 24 h, spanning a pH range of 2 to 6. Moreover, subsequent in vitro investigations established that this chitinase pair efficiently degrades diverse chitin-containing substrates into chitobiose, highlighting the potential of Atlantic salmon to utilize novel chitin-containing feed sources. Analysis of the gastric matrix proteome demonstrates that the chitinases are secreted and rank among the most abundant proteins in the gastric matrix. This finding correlates well with the previously observed high transcription of the corresponding chitinase genes in Atlantic salmon stomach tissue. By shedding light on the secreted chitinases in the Atlantic salmon's stomach mucosa and elucidating their functional characteristics, this study enhances our understanding of chitinase biology in this species. Moreover, the observed capacity to effectively degrade chitin-containing materials implies the potential utilization of alternative feed sources rich in chitin, offering promising prospects for sustainable aquaculture practices.

Product inhibition slow down the moving velocity of processive chitinase and sliding-intermediate state blocks re-binding of product.

Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 µM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 µM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 µM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.

Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation.

Chitinases are widely studied enzymes that have already found widespread application. Their continued development and valorisation will be driven by the identification of new and improved variants and/or novel applications bringing benefits to industry and society. We previously identified a novel application for chitinases wherein the Candida albicans cell wall surface chitinase 3 (Cht3) was shown to have potential in vaccine applications as a subunit antigen against fungal infections. In the present study, this enzyme was investigated further, developing production and purification protocols, enriching our understanding of its properties, and advancing its application potential. Cht3 was heterologously expressed in Pichia pastoris and a 4-step purification protocol developed and optimised: this involves activated carbon treatment, hydrophobic interaction chromatography, ammonium sulphate precipitation, and gel filtration chromatography. The recombinant enzyme was shown to be mainly O-glycosylated and to retain the epitopes of the native protein. Functional studies showed it to be highly specific, displaying activity on chitin, chitosan, and chito-oligosaccharides larger than chitotriose only. Furthermore, it was shown to be a stable enzyme, exhibiting activity, and stability over broad pH and temperature ranges. This study represents an important step forward in our understanding of Cht3 and contributes to its development for application.

The chitin-binding domain of Bacillus thuringiensis ChiA74 inhibits gram-negative bacterial and fungal pathogens of humans and plants.

The chitinase ChiA74 is synthesized by Bacillus thuringiensis and possesses a modular organization composed of four domains. In the C-terminal of the enzyme is located the chitin-binding domain (CBD), which has not been isolated as a single unit or characterized. Here, we aimed to isolate the ChiA74's CBD as a single unit, determine the binding properties, and evaluate its antimicrobial and hemolytic activities. We cloned the ChiA74's CBD and expressed it in Escherichia coli BL21. The single domain was purified, analyzed by SDS-PAGE, and characterized. The recombinant CBD (rCBD) showed a molecular mass of ∼14 kDa and binds strongly to α-chitin, with Kd and Bmax of ∼4.7 ± 0.9 µM and 1.5 ± 0.1 µmoles/g chitin, respectively. Besides, the binding potential (Bmax/Kd) was stronger for α-chitin (∼0.31) than microcrystalline cellulose (∼0.19). It was also shown that the purified rCBD inhibited the growth of the clinically relevant Gram-negative bacteria (GNB) Vibrio cholerae, and V. parahemolyticus CVP2 with minimum inhibitory concentrations (MICs) of 121 ± 9.9 and 138 ± 3.2 µg/mL, respectively, and of one of the most common GNB plant pathogens, Pseudomonas syringae with a MIC of 230 ± 13.8 µg/mL. In addition, the rCBD possessed antifungal activity inhibiting the conidia germination of Fusarium oxysporum (MIC = 192 ± 37.5 µg/mL) and lacked hemolytic and agglutination activities against human erythrocytes. The significance of this work lies in the fact that data provided here show for the first time that ChiA74's CBD from B. thuringiensis has antimicrobial activity, suggesting its potential use against significant pathogenic microorganisms. Future works will be focused on testing the inhibitory effect against other pathogenic microorganisms and elucidating the mechanism of action.

A novel bi-functional cold-adaptive chitinase from Chitinilyticum aquatile CSC-1 for efficient synthesis of N-acetyl-D-glucosaminidase.

In order to better utilize chitinolytic enzymes to produce high-value N-acetyl-D-glucosamine (GlcNAc) from chitinous waste, there is an urgent need to explore bi-functional chitinases with exceptional properties of temperature, pH and metal tolerance. In this study, we cloned and characterized a novel bi-functional cold-adaptive chitinase called CaChi18A from a newly isolated strain, Chitinilyticum aquatile CSC-1, in Bama longevity village of Guangxi Province, China. The activity of CaChi18A at 50 °C was 4.07 U/mg. However, it exhibited significant catalytic activity even at 5 °C. Its truncated variant CaChi18A_ΔChBDs, containing only catalytic domain, demonstrated significant activity levels, exceeding 40 %, over a temperature range of 5-60 °C and a pH range of 3 to 10. It was noteworthy that it displayed tolerance towards most metal ions at a final concentration of 0.1 mM, including Fe3+ and Cu2+ ions, retaining 122.52 ± 0.17 % and 116.42 ± 1.52 % activity, respectively. Additionally, it exhibited favorable tolerance towards organic solvents with the exception of formic acid. Interestedly, CaChi18A and CaChi18A_ΔChBDs had a low Km value towards colloidal chitin (CC), 0.94 mg mL-1 and 2.13 mg mL-1, respectively. Both enzymes exhibited chitobiosidase and N-acetyl-D-glucosaminidase activities, producing GlcNAc as the primary product when hydrolyzing CC. The high activities across a broader temperature and pH range, strong environmental adaptability, and hydrolytic properties of CaChi18A_ΔChBDs suggested that it could be a promising candidate for GlcNAc production.