Hydrolysis Of Chitin Research Articles

A novel mechanocatalytic process by vibratory disk mill for efficient hydrolysis of cellulose, chitin and xylan

Polysaccharides are primary sources to manufacture chemical products with low carbon footprints, owing to their carbons from atmospheric carbon dioxide fixed through photosynthesis. Breaking down such polysaccharides into water-soluble saccharides through the hydrolysis of glycosidic bonds is one of key steps to produce valuable chemicals; however, previous chemical and enzymatic processes have been suffered from their fundamental drawbacks (e.g. not environmentally benign, high production cost) and are not regarded as feasible approaches. Recently, mechanocatalytic process, which proceeds hydrolysis of polysaccharides by simply milling with solid acids, attracts attentions owing to its cost-effectiveness and environmental beingness. Herein, we propose the novel mechanocatalytic system to efficiently proceed the hydrolysis of glycosidic bonds in polysaccharides by using “vibratory disk mill”, which can produce strong shear and impact force. By milling polysaccharides (cellulose, chitin and xylan) with solid acid kaolin, the polysaccharides can be efficiently hydrolysed to water-soluble saccharides with a rate twice higher than the previous milling devices, achieving quantitative conversion of polysaccharide to water soluble products (solubility: >99 %). We elucidated the reaction mechanism of polysaccharides with kaolin by X-ray diffractograms: forces produced by the vibratory disk mill can efficiently delaminate layered aluminosilicate nanosheets in kaolin, leading to better accessibility of polysaccharides on the acidic surface of the nanosheets. This novel mechanocatalytic system can also be applicable to produce oligosaccharides directly from crude biomass samples (birchwood chips and shrimp shells) without any chemical purification steps, demonstrating that this system can be used in industrial biomass processing.

Cell wall nanoparticles from hyphae of Alternaria infectoria grown with caspofungin, nikkomycin, or pyroquilon trigger different activation profiles in macrophages.

Alternaria infectoria causes opportunistic human infections and is a source of allergens leading to respiratory allergies. In this work, we prepared cell wall nanoparticles (CWNPs) as a novel approach to study macrophage immunomodulation by fungal hyphal cell walls. A. infectoria was grown in the presence of caspofungin, an inhibitor of β(1,3)-glucan synthesis; nikkomycin Z, an inhibitor of chitin synthases; and pyroquilon, an inhibitor of dihydroxynaphthalene (DHN)-melanin synthesis. Distinct CWNPs were obtained from these cultures, referred to as casCWNPs, nkCWNPs, and pyrCWNPs, respectively. CWNPs are round-shaped particles with a diameter of 70-200 nm diameter particles that when added to macrophages are taken up by membrane ruffling. CWNPs with no DHN-melanin and more glucan (pyrCWNPs) caused early macrophage activation and lowest viability, with the cells exhibiting ultrastructural modifications such as higher vacuolization and formation of autophagy-like structures. CasCWNPs promoted the highest tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) increase, also resulting in the release of partially degraded chitin, an aspect never observed in macrophage-like cells and fungi. After 6 h of interaction with CWNPs, only half were viable, except with control CWNPs. Overall, this work indicates that compounds that modify the fungal cell wall led to CWNPs with new properties that may have implications for the effects of drugs during antifungal therapy. CWNPs provide a new tool to study the interaction of hyphal fungal cell wall components with phagocytic cells and enable to show how the modification of cell wall components in A. infectoria can modulate the response by macrophages.IMPORTANCEAlternaria species are ubiquitous environmental fungi to which the human host can continuously be exposed, through the inhalation of fungal spores but also of fragments of hyphae, from desegregated mycelia. These fungi are involved in hypersensitization and severe respiratory allergies, such as asthma, and can cause opportunistic infections in immunodepressed human host leading to severe disease. The first fungal structures to interact with the host cells are the cell wall components, and their modulation leads to differential immune responses. Here, we show that fungal cells grown with cell wall inhibitors led to cell wall nanoparticles with new properties in their interaction with macrophages. With this strategy, we overcame the limitation of in vitro assays interacting with filamentous fungi and showed that the absence of DNH-melanin leads to higher virulence, while caspofungin leads to cells walls that trigger higher hydrolysis of chitin and higher production of cytokines.

Enhanced degradation of insoluble chitin: Engineering high-efficiency chitinase fusion enzymes for sustainable applications

N-acetyl-D-glucosamine and its dimer are degradation products of chitin waste with great potential in therapeutic and agricultural applications. However, the hydrolysis of insoluble chitin by chitinases remains a major bottleneck. This study investigated the biochemical properties and catalytic mechanisms of PoChi chitinase obtained from Penicillium oxalicum with a focus on enhancing its efficiency during the degradation of insoluble chitin. Recombinant plasmids were engineered to incorporate chitin-binding (ChBD) and/or fibronectin III (FnIII) domains. Notably, PoChi-FnIII-ChBD exhibited the highest substrate affinity (Km = 2.7 mg/mL) and a specific activity of 15.4 U/mg, which surpasses those of previously reported chitinases. These findings highlight the potential of engineered chitinases in advancing industrial biotechnology applications and offer a promising approach to more sustainable chitin waste management.

Hydrolysis of Chitin with Acid and Noble Metal Catalyst

Hydrolysis of Chitin with Acid and Noble Metal Catalyst

Genome-Wide Identification of a Maize Chitinase Gene Family and the Induction of Its Expression by Fusarium verticillioides (Sacc.) Nirenberg (1976) Infection.

Maize chitinases are involved in chitin hydrolysis. Chitinases are distributed across various organisms including animals, plants, and fungi and are grouped into different glycosyl hydrolase families and classes, depending on protein structure. However, many chitinase functions and their interactions with other plant proteins remain unknown. The economic importance of maize (Zea mays L.) makes it relevant for studying the function of plant chitinases and their biological roles. This work aims to identify chitinase genes in the maize genome to study their gene structure, family/class classification, cis-related elements, and gene expression under biotic stress, such as Fusarium verticillioides infection. Thirty-nine chitinase genes were identified and found to be distributed in three glycosyl hydrolase (GH) families (18, 19 and 20). Likewise, the conserved domains and motifs were identified in each GH family member. The identified cis-regulatory elements are involved in plant development, hormone response, defense, and abiotic stress response. Chitinase protein-interaction network analysis predicted that they interact mainly with cell wall proteins. qRT-PCR analysis confirmed in silico data showing that ten different maize chitinase genes are induced in the presence of F. verticillioides, and that they could have several roles in pathogen infection depending on chitinase structure and cell wall localization.

Film-Forming Capability and Antibacterial Activity of Surface-Deacetylated Chitin Nanocrystals: Role of Degree of Deacetylation.

Developing sustainable food-active packaging materials is a major issue in food preservation applications. Chitin nanocrystals (ChNCs) are regarded as unique bioderived nanomaterials due to their inherent nitrogen moiety. By tuning the chemical functionality of this nanomaterial, it is possible to affect its properties, such as film-forming capability and antibacterial activity. In this work, surface-deacetylated chitin nanocrystals (D-ChNCs) with different degrees of deacetylation (DDs) were prepared by partial deacetylation of native chitin and subsequent acid hydrolysis, and their film-forming capability and antibacterial activity were studied systematically. The D-ChNCs showed favorable film-forming ability and antibacterial activity, which are closely related to their DD. With the increase in DD (from 5.7% to 45.4%), the formed transparent films based on ChNCs showed gradually increased elongation at break (from 0.5% to 2.5%) and water contact angle (from 25.5° to 87.0°), but decreased break strength (from 3.13 to 0.89 MPa), Young's modulus (from 0.84 to 0.24 MPa), and water vapor permeability (from 4.7 × 10-10 to 4.1 × 10-10g/m s Pa). Moreover, the antibacterial activity of the D-ChNCs against E. coli and S. aureus also increased with the increase of DD. This study also found that the depolarization and potential dissipation of the bacterial cell membrane induced by the contact between amino-rich D-ChNCs and bacteria through electrostatic attraction are the possible mechanisms causing bacterial cell death. This study provides a basis for understanding the effects of DD on the film-forming capability and antibacterial activity of ChNCs, which is conducive to the design of novel active packaging films based on ChNCs.

Three-Step Enzymatic Remodeling of Chitin into Bioactive Chitooligomers.

Here we describe a complex enzymatic approach to the efficient transformation of abundant waste chitin, a byproduct of the food industry, into valuable chitooligomers with a degree of polymerization (DP) ranging from 6 to 11. This method involves a three-step process: initial hydrolysis of chitin using engineered variants of a novel fungal chitinase from Talaromyces flavus to generate low-DP chitooligomers, followed by an extension to the desired DP using the high-yielding Y445N variant of β-N-acetylhexosaminidase from Aspergillus oryzae, achieving yields of up to 57%. Subsequently, enzymatic deacetylation of chitooligomers with DP 6 and 7 was accomplished using peptidoglycan deacetylase from Bacillus subtilis BsPdaC. The innovative enzymatic procedure demonstrates a sustainable and feasible route for converting waste chitin into unavailable bioactive chitooligomers potentially applicable as natural pesticides in ecological and sustainable agriculture.

Sustainable Production of Chitin Nanowhiskers from Crustacean Biomass Using Cost-Effective Ionic Liquids: Strategies to Avoid Byproduct Formation

Nanochitin, especially in the form of chitin nanowhiskers (ChNWs), represents a significant advance in biopolymer technology due to its high specific surface area, superior tensile strength, and excellent thermal stability. Derived from crustacean waste, which contains 15–40% of chitin, these materials provide a sustainable option that diverts waste from landfills and contributes to environmental conservation. Traditional methods of isolating nanochitin are energy-intensive and generate substantial waste. This study introduces a more sustainable method using inexpensive ionic liquids (ILs) such as [Hmim][HSO4] and [HN222][HSO4], which bypass the costly and destructive steps of traditional procedures. This study also identified the byproduct in IL-mediated chitin hydrolysis reaction as calcium sulfate dihydrate and presented a solution to circumvent the byproduct formation. The effectiveness of the [HN222][HSO4] IL in producing ChNWs from both purified chitin and crustacean biomass was assessed, showing a high yield and maintaining the purity and structural integrity of chitin, thereby demonstrating a significant reduction in the environmental footprint of ChNW production.

Roseateles agri sp. nov., a New Species Isolated from Fresh Soil in Uiwang, South Korea

Two isolates of Roseateles were discovered in soil samples collected from Uiwang-si, Gyeonggi-do, Republic of Korea. These isolates exhibited rod-shaped morphology and were facultatively anaerobic, non-motile, and tested positive for oxidase and catalase. Designated as strains R3-3T and R3-11, their growth was hindered by NaCl concentrations exceeding 0.5%, while their optimal growth conditions were observed at temperatures ranging from 25 °C to 30 °C and pH levels between 7.0 and 9.0. Both strains exhibited positive results for the hydrolysis of Tween 80 and DNA, but tested negative for starch, casein, chitin, and gelatin hydrolysis. Additionally, they assimilated L-Arabinose, D-mannitol, and D-Maltose, while exhibiting negative results for the fermentation of D-glucose, esculin ferric citrate, D-mannose, N-acetyl-glucosamine, potassium gluconate, capric acid, adipic acid, trisodium citrate, and phenylacetic acid. The DNA G+C content of strain R3-3T was measured at 67.5 mol%. Comparative analysis revealed that the average nucleotide identity (ANI) values between R3-3T and the Roseateles type strains ranged from 75.14% to 78.30% while the digital DNA-DNA hybridization (dDDH) values ranged from 20.70% to 22.70%. Consequently, based on comprehensive genomic, chemotaxonomic, phenotypic, and phylogenomic evaluations, the isolated strains have been designated as a new species within the genus Roseateles, named Roseateles agri sp. nov. (with type strain R3-3T = KACC 23678T = NBRC 116681T).

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 β-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.

Biochemical characterization of a novel hyperthermophilic chitinase from a deep-sea Thermotogae bacterium

Chitin represents the most abundant source of renewable biomass in marine ecosystems, and its degradation by chitinase holds potential for numerous industrial applications. In this study, a novel hyperthermophilic chitinase, TbChi52, derived from a deep-sea bacterium of the Thermotogae phylum was introduced. TbChi52 exhibits optimal enzymatic activity at 90 ℃ and pH 5.5. Furthermore, it shows exceptional thermal stability characterized by a prolonged thermal denaturation half-life and a significant melting temperature. Functionally, TbChi52 displays dual activities, acting as both an exo-chitinase and a β-N-acetylaminoglucosidase (NAGase). It yields N-acetyl-D-glucosamine (GlcNAc) as the primary product during the hydrolysis of colloidal chitin. Leveraging the robust thermo-stability of TbChi52, a streamlined, one-step purification protocol was developed. A mere 10-minute thermal treatment at 75 ℃ suffices to yield a highly pure TbChi52 preparation with minimal enzyme activity loss in chitin hydrolysis, achieving an 87.4 % hydrolysis rate of colloidal chitin and a 92.7 % purity of GlcNAc. Collectively, the outstanding thermal properties and the simple preparation of TbChi52 highlight its potential for industrial applications. It provides a sustainable and eco-friendly approach to harnessing the world's most abundant marine biomass, along with serving as a potential enzyme-engineered template tailored for specific applications.

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.

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.

Mechanochemical Hydrolysis of Polysaccharide Biomass: Scope and Mechanistic Insights.

Mechanical forces can affect chemical reactions in a way that thermal reactions cannot do, which may have a variety of applications. In biomass conversion, the selective conversion of cellulose and chitin is a grand challenge because they are the top two most abundant resources and recalcitrant materials that are insoluble in common solvents. However, recent works have clarified that mechanical forces enable the depolymerization of these polysaccharides, leading to the selective production of corresponding monomers and oligomers. This article reviews the mechanochemical hydrolysis of cellulose and chitin, particularly focusing on the scope and mechanisms to show a landscape of this research field and future subjects. We introduce the background of mechanochemistry and biomass conversion, followed by recent progress on the mechanochemical hydrolysis of the polysaccharides. Afterwards, a considerable space is devoted to the mechanistic consideration on the mechanochemical reactions.

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

BackgroundChitinases 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.ResultsThree 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.ConclusionsThree 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.

Genome-Wide Identification and Expression Analysis of Chitinase Genes in Watermelon under Abiotic Stimuli and Fusarium oxysporum Infection.

Chitinases, which catalyze the hydrolysis of chitin, the primary components of fungal cell walls, play key roles in defense responses, symbiotic associations, plant growth, and stress tolerance. In this study, 23 chitinase genes were identified in watermelon (Citrullus lanatus [Thunb.]) and classified into five classes through homology search and phylogenetic analysis. The genes with similar exon-intron structures and conserved domains were clustered into the same class. The putative cis-elements involved in the responses to phytohormone, stress, and plant development were identified in their promoter regions. A tissue-specific expression analysis showed that the ClChi genes were primarily expressed in the roots (52.17%), leaves (26.09%), and flowers (34.78%). Moreover, qRT-PCR results indicate that ClChis play multifaceted roles in the interaction between plant/environment. More ClChi members were induced by Race 2 of Fusarium oxysporum f. sp. niveum, and eight genes were expressed at higher levels on the seventh day after inoculation with Races 1 and 2, suggesting that these genes play a key role in the resistance of watermelon to Fusarium wilt. Collectively, these results improve knowledge of the chitinase gene family in watermelon species and help to elucidate the roles played by chitinases in the responses of watermelon to various stresses.

Soil enzyme activities in heavily manured and waterlogged soil cultivated with ryegrass (Lolium multiflorum)

Extended waterlogging (WL) conditions can alter soil enzyme activities and their role in maintaining healthy soils. We assessed the effects of soil moisture regimes (field capacity [FC] and WL) and phosphorus (P) rates (0, 15, 30, 45 kg available P ha–1) on ( i) soil enzymes and microbial biomass carbon (MBC), microbial biomass nitrogen (MBN), and microbial biomass P (MBP); and ( ii) dissolved organic carbon (DOC) and total dissolved nitrogen (TDN). The treatments were tested in a 4-month greenhouse experiment using intact soil columns under annual ryegrass ( Lolium multiflorum). WL decreased the activity of β-glucosidase and acid phosphomonoesterase but increased N-acetyl-β-glucosaminidase in soils. These changes were associated with changes in MBC, DOC, MBN, and TDN, but not MBP. Anoxic conditions in WL soil promote the activity of anaerobes and contribute to the reduction of Fe oxyhydroxides and the release of DOC, TDN, and P in the soil solution. The activity of the extracellular enzymes decreased in WL with additions of slurry indicating adequate supply of C, N, and P. Our results also showed that both enzyme activities and microbial biomass were restricted in the upper soil layer with limited downward movement along the soil profile. We can conclude that since these enzymes control the hydrolysis of cellulose, phosphomonoester, and chitin, soil moisture influences the direction and magnitude of C, N, and P in manured and waterlogged soil cultivated with ryegrass.

The Ustilago maydis AA10 LPMO is active on fungal cell wall chitin.

Lytic polysaccharide monooxygenases (LPMOs) can perform oxidative cleavage of glycosidic bonds in carbohydrate polymers (e.g., cellulose, chitin), making them more accessible to hydrolytic enzymes. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. The AA10 LPMOs are active on chitin and/or cellulose and mostly found in bacteria and in some viruses and archaea. Interestingly, AA10-encoding genes are also encountered in some pathogenic fungi of the Ustilaginomycetes class, such as Ustilago maydis, responsible for corn smut disease. Transcriptomic studies have shown the overexpression of the AA10 gene during the infectious cycle of U. maydis. In fact, U. maydis has a unique AA10 gene that codes for a catalytic domain appended with a C-terminal disordered region. To date, there is no public report on fungal AA10 LPMOs. In this study, we successfully produced the catalytic domain of this LPMO (UmAA10_cd) in Pichia pastoris and carried out its biochemical characterization. Our results show that UmAA10_cd oxidatively cleaves α- and β-chitin with C1 regioselectivity and boosts chitin hydrolysis by a GH18 chitinase from U. maydis (UmGH18A). Using a biologically relevant substrate, we show that UmAA10_cd exhibits enzymatic activity on U. maydis fungal cell wall chitin and promotes its hydrolysis by UmGH18A. These results represent an important step toward the understanding of the role of LPMOs in the fungal cell wall remodeling process during the fungal life cycle. IMPORTANCE Lytic polysaccharide monooxygenases (LPMOs) have been mainly studied in a biotechnological context for the efficient degradation of recalcitrant polysaccharides. Only recently, alternative roles and paradigms begin to emerge. In this study, we provide evidence that the AA10 LPMO from the phytopathogen Ustilago maydis is active against fungal cell wall chitin. Given that chitin-active LPMOs are commonly found in microbes, it is important to consider fungal cell wall as a potential target for this enigmatic class of enzymes.

Synthesis and antimicrobial activity of new glucosamine derivatives

AbstractIn this study, a series of reactions were used to protect and deprotect hydroxyl and amine groups of D‐glucosamine which was obtained by the hydrolysis of chitin in concentrated HCl. Amidation of this protected monomer with aromatic and heteroaromatic acids was carried out in dichloromethane in the presence of DCC as a coupling reagent to produce new amides in good yields. Their structures were determined by NMR and MS spectra while their antifungal and antibacterial activities were screened.

Overexpression of chitinase PbChia1 from Plasmodiophora brassicae improves broad-spectrum disease resistance of Arabidopsis

ABSTRACT Chitinase plays an important role in plant resistance against chitin-containing pathogens through hydrolysis of chitin. Clubroot caused by Plasmodiophora brassicae is a major disease for cruciferous crops and vegetables worldwide. The cell wall of P. brassicae resting spores contains chitin. Chitinase is regarded as capable of improving plant resistance to fungal diseases. However, there has been no report about the function of chitinase in P. brassicae. Here, wheat germ agglutinin staining and commercial chitinase treatment demonstrated that chitin is a functional component in P. brassicae. In addition, Chitinase PbChia1 was identified by chitin pull-down assay combined with LC-MS/MS. PbChia1 was found to be a typical secreted chitinase, which could bind chitin with chitinase activity in vitro. PbChia1 could significantly decrease the resting spores of P. brassicae and therefore relieve the severity of clubroot symptom, with a biocontrol effect of 61.29%. Overexpression of PbChia1 in Arabidopsis thaliana improved its resistance to P. brassicae, increased host survival rate and seed yield, enhanced PAMPs-triggered reactive oxygen species burst, MAPK activation and expression of immune-related genes. PbChia1 transgenic plants also showed resistance to other pathogens, such as biotrophic bacterium Pst DC3000, necrotrophic fungi Sclerotinia sclerotiorum and Rhizoctonia solani. These findings indicate that chitinase PbChia1 is a candidate gene that can confer broad-spectrum disease resistance in breeding.