Chitinase Activity Research Articles

Antioxidants, Antimicrobial, and Anticancer Activities of Purified Chitinase of Talaromyces funiculosus Strain CBS 129594 Biosynthesized Using Crustacean Bio-Wastes

Talaromyces funiculosus strain CBS 129594 was optimized to promote chitinase activity under solid state fermentation using crustacean bio-wastes. The aim of the study was to use purified chitinase as antioxidant, antimicrobial, and anticancer activities. The results showed that the maximum enzyme yield (2.98 ± 0.2 U/g substrate) was obtained at 1:2 crab shell chitin with the inoculation size (2.5 × 106v/v) after seven days of incubation, pH 6.5, using 0.20% of soybean meal, malt extract, and yeast extract and 100% cane and beet molasses as supplementation. The enzyme was purified with an overall yield of 7.22 purification fold with a specific activity of 9.32 ± 0.3 U/mg protein. The molecular mass of the purified chitinase was 45 kDa. The highest chitinase activity was detected at pH 6.5 and 40 °C. The purified chitinase was activated by Ca2+, Cu2+, Na+, Mn2+, and Mg2+. On the other hand, the enzyme activity was inhibited in the presence of Hg2+, Ag2+, and Li+ at 10 mM, while Zn2+ and Co2+ caused no effect compared to media without any metals. The scavenging of 2.2-diphenyl-1-picrylhydrazyl (DPPH) radicals and 2.2-pheny-l-1-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) increased with increasing the concentrations of the purified chitinase enzyme (100, 200, 300, and 400 µg/mL) which ranged from 48.7% to 57.8% and 8.87% to 63.73%, respectively. The IC50 value of DPPH radicals and ABTS of purified chitinase produced by T. funiculosus strain CBS 129594 was 199 and 306 μg/mL concentration, respectively. The purified chitinase inhibited the growth of Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli), Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus), and fungi (Aspergillus niger, Candida albicans). The highest concentrations of purified chitinase (1000 µg/mL) caused the higher toxicity of cancer cell line MCF7 (97%), HCT116 (88.2%), and HepG2 (97.1%). In conclusion, we can conclude that chitinase can be produced from marine waste and can be used as an antioxidant, antibacterial activity, cancer therapy, and ecofriendly biocontrol agent.

Study on antibacterial mechanism of electron beam radiation on Aspergillus flavus

Contamination with Aspergillus flavus, which produces carcinogenic metabolites, during the post-harvest storage of agricultural products seriously endangers human health and safety. In this study, the efficacy of short-term electron beam irradiation processing with high sterilization efficiency, no contamination and no additives was evaluated for the prevention and control of A. flavus. The effects of irradiation on Aspergillus growth were determined based on analyses of physical and chemical indexes, morphology, mycelial growth, spore germination rates, mycelial dry weights, and toxin-producing ability. The effects of irradiation on the cell membrane and cell wall of A. flavus were investigated based on assays of the chitin content, chitinase activity, protein concentration, and malondialdehyde content. Damage to the antioxidant system was determined by the hydrogen peroxide content, catalase activity, superoxide dismutase activity, superoxide anion radical scavenging activity and rates of 1,1-diphenyl-2-trinitrophenylhydrazine inhibition. The inhibitory effect of irradiation on peanut invasion by A. flavus was verified. After irradiation, the thallus morphology was destroyed, mycelial growth was inhibited, and the spore germination rate, mycelial dry weight, and toxin-producing ability of A. flavus were reduced. Chitinase activity and the malondialdehyde content increased, while the chitin and total protein contents decreased. Catalase activity, superoxide dismutase activity, superoxide anion radical scavenging activity, and the rate of 1,1-diphenyl-2-trinitrophenylhydrazine inhibition decreased, while the hydrogen peroxide content increased after irradiation. These findings suggested that electron beam irradiation can effectively inhibit the normal growth and toxin production of A. flavus. Irradiation increased cell membrane permeability and decreased the integrity of the cell wall. The antioxidant system was damaged to some extent. Compared with that in unirradiated A. flavus, high-dose electron beam irradiation effectively inhibited growth. As a means of postharvest control, electron beam irradiation may have wide applications in the agricultural industry.

Apple Endophytic fungi and their antagonism against apple scab disease.

Endophytic fungi are microorganisms with the ability to colonize plants for the entire or at least a significant part of their life cycle asymptomatically, establishing a plant-fungus association. They play an important role in balancing ecosystems, as well as benefiting host through increasing plant growth, and protecting the host plants from abiotic and biotic stresses using various strategies. In the present study, endophytic fungi were isolated from wild and endemic apple cultivars, followed by characterizing their antifungal effect against Venturia inaequalis. To characterize the endophytic fungi, 417 fungal strains were separated from 210 healthy fruit, leaf, and branch samples collected from the north of Iran. Among the purified fungal isolates, 33 fungal genera were identified based on the morphological characteristics, of which 38 species were detected according to the morphological features and molecular data of ITS, tef-1α, and gapdh genomic regions (related to the genus). The results represented that most of the endophytic fungi belonged to Ascomycota (67.8%), 31.4% of isolates were mycelia sterilia, while the others were Basidiomycota (0.48%) and Mucoromycota (0.24%). Additionally, Alternaria, Cladosporium, and Nigrospora were determined as the dominant genera. The antifungal properties of the identified isolates were evaluated against V. inaequalis in vitro to determine the release of media-permeable metabolites, Volatile Organic Compounds (VOCs), chitinase, and cellulase as antifungal mechanisms, as well as producing phosphate solubilisation as growth-promoting effect. Based on the results of metabolite and VOC tests, the six isolates of Acremonium sclerotigenum GO13S1, Coniochaeta endophytica 55S2, Fusarium lateritium 61S2, Aureobasidium microstictum 7F2, Chaetomium globosum 2S1 and Ch. globosum 3 L2 were selected for greenhouse tests. Further, Co. endophytica 55S2 and F. lateritium 61S2 could solubilize inorganic phosphate. All isolates except Ch. globosum 3 L2 exhibited cellulase activity, while chitinase activity was observed in Ch. globosum 2S1, Ch. globosum 3 L2, and F. lateritium 61S2. Finally, Co. endophytica 55S2 and Ch. globosum 2S1 completely controlled the disease on the apple seedling leaves under greenhouse conditions.

Transcriptome Profiling of the Resistance Response of Musa acuminata subsp. burmannicoides, var. Calcutta 4 to Pseudocercospora musae.

Banana (Musa spp.), which is one of the world's most popular and most traded fruits, is highly susceptible to pests and diseases. Pseudocercospora musae, responsible for Sigatoka leaf spot disease, is a principal fungal pathogen of Musa spp., resulting in serious economic damage to cultivars in the Cavendish subgroup. The aim of this study was to characterize genetic components of the early immune response to P. musae in Musa acuminata subsp. burmannicoides, var. Calcutta 4, a resistant wild diploid. Leaf RNA samples were extracted from Calcutta 4 three days after inoculation with fungal conidiospores, with paired-end sequencing conducted in inoculated and non-inoculated controls using lllumina HiSeq 4000 technology. Following mapping to the reference M. acuminata ssp. malaccensis var. Pahang genome, differentially expressed genes (DEGs) were identified and expression representation analyzed on the basis of gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes orthology and MapMan pathway analysis. Sequence data mapped to 29,757 gene transcript models in the reference Musa genome. A total of 1073 DEGs were identified in pathogen-inoculated cDNA libraries, in comparison to non-inoculated controls, with 32% overexpressed. GO enrichment analysis revealed common assignment to terms that included chitin binding, chitinase activity, pattern binding, oxidoreductase activity and transcription factor (TF) activity. Allocation to KEGG pathways revealed DEGs associated with environmental information processing, signaling, biosynthesis of secondary metabolites, and metabolism of terpenoids and polyketides. With 144 up-regulated DEGs potentially involved in biotic stress response pathways, including genes involved in cell wall reinforcement, PTI responses, TF regulation, phytohormone signaling and secondary metabolism, data demonstrated diverse early-stage defense responses to P. musae. With increased understanding of the defense responses occurring during the incompatible interaction in resistant Calcutta 4, these data are appropriate for the development of effective disease management approaches based on genetic improvement through introgression of candidate genes in superior cultivars.

Over-expression of PR proteins with chitinase activity in transgenic plants for alleviation of fungal pathogenesis

Over-expression of PR proteins with chitinase activity in transgenic plants for alleviation of fungal pathogenesis

Biological control of blue mold rot in apple by Kluyveromyces marxianus XZ1 and the possible mechanisms of action

The yeast Kluyveromyces marxianus was permitted for direct addition to food and shown antagonistic ability against some pathogens. However, the effect and detailed mechanism of K. marxianus on control blue mold disease and patulin accumulation in apple remain unknown. In our study, K. marxianus XZ1 reduced the disease incidence and patulin content in apple caused by Penicillium expansum at 3 d after incubation compared with the control, which is decreased by 51% and 52%, respectively. Studies on the potential mechanisms of action revealed that (1) K. marxianus XZ1 adhered well to the apple wound to gain a competitive advantage; (2) antibacterial substances produced by K. marxianus XZ1 under the stimulation of P. expansum to inhibit the growth of this fungus; (3) activity of catalase, peroxidase, polyphenoloxidase, chitinase, and phenylalanine ammonialyase was activated to participate in the defense of fruit against P. expansum; (4) the expression levels of some genes involved in plant hormone signal transduction, including salicylic acid, jasmonate acid and ethylene signaling pathway, were upregulated to strengthen the fruit's defense against mold. Taken together, our results indicated that K. marxianus XZ1 may serve as a potential biological agent against postharvest decay and patulin contamination caused by P. expansum in apple fruit, mainly attributed to its ability of competition for space and nutrients, secretion of antifungal components, parasitism and activation of plant hormone signal transduction.

Functional analysis of the short splicing variant encoded by CHI3L1/YKL-40 in glioblastoma.

The glycoprotein YKL-40 has been well studied as a serum biomarker of prognosis and disease status in glioblastoma. YKL-40 is a chitinase-like protein with defective chitinase activity that plays an important role in promoting cell proliferation, migration, and metastasis in glioblastoma multiforme (GBM). The short variant (SV) of YKL-40, generated by an alternative splicing event that splices out exon 8, was reported in the early developing human musculoskeletal system, although its role in GBM is still unknown. Our results showed that individual glioblastoma cell lines displayed increased expression of the short variant of YKL-40 after low serum treatment. In addition, unlike the full-length (FL) version, which was localized to all cell compartments, the short isoform could not be secreted and was localized only to the cytoplasm. Functionally, FL YKL-40 promoted cell proliferation and migration, whereas SV YKL-40 suppressed them. Transcriptome analysis revealed that these opposing roles of the two isoforms may be modulated by differentially regulating several oncogenic-related pathways, including p53, the G2/M checkpoint, and MYC-related signaling. This study may provide new ideas for the development of targeted anti-YKL-40 therapy in GBM treatment.

Chitinase inhibits growth of human breast and colorectal cancer cells in vitro and Ehrlich ascites carcinoma cells in vivo

Trichosanthes dioica seed extract was loaded on a QA-cellulose column and the unbound fraction with the chitinase activity was run on SDS-PAGE. Multiple bands were observed and were separated by a Sephadex G-50 column. The combination of the 6 and 33 kDa masses supported the degradation of chitinase as purified earlier. Only the 33 kDa fraction contained sugar and showed chitinase activity. The chitinase was also isolated by using a chitin column. At 200 µg/ml protein concentration, the chitinase inhibited 49.1 %, 48.8 % and 38.12 % of Ehrlich ascites carcinoma, HCT-116 and MCF-7 cells growth, respectively, in a dose-dependent manner. Exactly, 46 % and 82 % EAC cell growth inhibition were observed after treating the EAC cells bearing Swiss albino mice with the chitinase at the doses of 1.0 and 2.0 mg/Kg/day respectively. EAC, HCT-116 and MCF-7 cells growth inhibitions were due to the induction of apoptosis. ROS was accumulated in HCT-116 and MCF-7 cells. After treatment of HCT-116 cells, the expression level of p53 and TNFα genes increased and PARP gene decreased. On the other hand, elevated expression was observed for PARP, MAPK, NFκB, FAS, FADD, and Caspase-8 genes in MCF-7 cells. The induction of apoptosis in HCT-116 was further confirmed by caspase protein expression. The chitinase causes ‘S’ cell cycle arrest in MCF-7 and HCT-116 cells. T. dioica seed chitinase inhibited EAC, HCT-116 and MCF-7 cells by inducing apoptosis in vitro and EAC in vivo in mice. These promising results indicated that T. dioica seed chitinase can be an anticancer agent.

Berberine as a promising natural compound to control Penicillium italicum causing blue mold of citrus fruit

The blue mold caused by Penicillium italicum is one of the most common diseases in citrus fruit. Berberine from Chinese herb has predominate bioactive effects. This study investigated the inhibitory effect of berberine on P. italicum and the underlying mechanisms. Results showed that berberine displayed excellent efficacy to inhibit mycelial growth and spore germination of P. italicum in vitro dose-dependently. Moreover, berberine damaged the plasma membrane integrity, indicated by increased relative conductivity but decreased contents of soluble protein and reducing sugar of P. italicum mycelia. Meanwhile, higher H2O2 content was also observed in berberine-treated P. italicum mycelia. Additionally, berberine suppressed the disease development in citrus fruit through increasing the enzymatic activities of polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and chitinase (CHI) as well as contents of total phenols and flavonoids in citrus peel. Furthermore, gene expression analysis suggested that genes encoding these defense-related enzymes were up-regulated by berberine. These results suggested that berberine could be expected as a new natural strategy to control postharvest diseases of citrus fruit.

Screening of chitin degrading bacteria from the gut of Asian common toad Duttaphrynus melanostictus: Implication for valorization of chitin rich seafood waste

Chitin is a structural component of many invertebrates and the second most abundant biopolymer after cellulose. Owing to its high insolubility, waste management of chitin rich seafood has severe environmental consequences. Despite the value added bioconversion of waste chitin into commodity products like reducing sugars, its biodegradation by bacteria is still lacking. To this end, we have isolated and characterized chitin degrading bacteria from the gut of common Asian toad, Duttaphrynus melanostictus . The isolates, FG4 and ND3 demonstrated highest chitin degradation activities of 1.44 ± 0.129 U/mL and 1.269 ± 0.138 U/mL respectively. Molecular phylogeny using 16S rRNA gene sequence affiliated FG4 to Stenotrophomonas maltophila and ND3 to Bacillus paramycoides , respectively. Among the tested substrates, highest chitinase activities of 4.210 ± 0.070 U/mL and 1.724 ± 0.494 U/mL were exhibited by S. maltophila FG4 on prawn shells (PS) and chitin flakes (CF), respectively. Similarly, the highest substrate degradation ratio of prawn shells and chitin flakes were 49.52% and 38.15% by S. maltophila FG4. Furthermore, scanning electron microscopy revealed adhesion of bacteria and creation of pores on the substrates. The Fourier transform infrared spectroscopy (FTIR) and X - ray diffraction analysis revealed hydrolysis of prawn shells and chitin flakes by bacteria. Further, the high-resolution mass spectrometric (HRMS) analysis confirmed production of N-acetyl D glucosamine by S. maltophila FG4 on prawn shells. Thus, the present study identifies D. melanostictus gut as a unique reservoir to isolate chitinolytic bacteria that may have potential industrial and therapeutic applications. • First report of bioprospection of amphibian guts for bacteria capable of chitin hydrolysis. • Gut chitinolytic isolates could utilize sea food wastes as sole carbon source. • Applications in production of valuable byproducts such as N-acetyl-D-glucosamine.

Effects of triazole plant growth regulators on molting mechanism in Chinese mitten crab (Eriocheir sinensis)

Rice crab co-culture is a new integrated farming model in China. The application of triazole plant growth regulators (PRGs) is often used as an advantageous option to combat rice lodging. However, there is still a gap regarding the toxicity of these PRGs on the growth and development of the Chinese mitten crab (Eriocheir sinensis, E. sinensis). Here the effect of triazoles (paclobutrazol and uniconazole) on the molting mechanism of E. sinensis was investigated. Monitoring of regulatory genes associated with molting showed that the two PRGs were found to inhibit the expression of ecdysteroid hormone (EH), ecdysteroid receptors gene (EcR), and retinoid X receptors gene (RXR) and induce secretion of molt-inhibiting hormone (MIH) gene. In addition, the activities of chitinase (CHIA) and N-acetyl-β-d-aminoglucosidase (β-NAGase) were also inhibited by exposure to PRGs. Exposure to PRGs also elevated the mRNA expression of the growth-related myostatin gene (MSTN). These results revealed that there is a long-term risk of exposure to triazoles PRGs that may inhibit molting and affect normal development and immune system of E. sinensis.

Bioefficacy of fungal chitin oligomers in the control of postharvest decay in tomato fruit

Tomato is one of the most commercialised and consumed fruits worldwide. However, tomatoes are highly susceptible to Alternaria rot. Among the safe strategies proposed to control Alternaria rot is the induction of defence mechanisms through biological elicitors, such as chitin. Chitin and its oligosaccharides are an activate plant defence mechanisms, but studies of fruits exposed to fungal chitin fragments are scarce. Therefore, the present work aimed to obtain and partially characterise chitin oligomers of Alternaria alternata, and evaluate their effect on the defence mechanism of tomato fruits and their tolerance to Alternaria rot. The chitin oligomers obtained had a molecular weight of ≤ 1 kDa, 12% N-acetyl-glucosamine, 0.2% residual protein, and were 94% acetylated. These oligomers markedly increased the enzymatic activity of chitinase and β-1,3-glucanase in tomato fruits, and the development of Alternaria rot was inhibited by 78%. Chitin oligomers of A. alternata represent a promising alternative to attenuate Alternaria rot in tomato fruits through an enzymatic defence mechanism.

Transcriptome analysis reveals mechanisms involved in the enhanced antagonistic efficacy of Sporidiobolus pararoseus Y16 treated by γ-aminobutyric acid

Our previous study showed that γ-aminobutyric acid (GABA) can significantly enhance the biological control efficacy of Sporidiobolus pararoseus Y16 on postharvest diseases of grapes. The possible physiological biocontrol mechanisms of the yeast have been reported previously, however, the associated molecular mechanisms were unclear. This study aimed to investigate the possible molecular mechanisms behind the enhanced biological control efficacy of S. pararoseus Y16 cultured with GABA based on transcriptome analysis. In addition, the activities of catalase (CAT) and chitinase (CHI) and the content of malondialdehyde (MDA) in S. pararoseus Y16 were investigated. The results showed that after the addition of 4 mg/mL GABA, the activities of CAT and CHI in S. pararoseus Y16 were significantly increased, and the content of MDA accumulation was decreased. Transcriptome analysis found that GABA-treated S. pararoseus Y16 can improve the ability of yeast ion transport, cell wall remodeling, antioxidants, growth and reproduction, resistance to stress, amino acid synthesis, etc., resulting in increased antagonistic efficacy of S. pararoseus Y16. These results provide a reference for future research on the use of antagonistic yeasts to prevent and control postharvest diseases of fruits and vegetables, and provide a theoretical basis for the practical application of antagonistic yeasts.

Pathological examination of Ym1, a chitinase family protein, in Mesocestoides corti-infected mice.

Mammals express a set of chitinase family proteins, comprising chitinases, which can hydrolyze chitin, and chitinase-like proteins without the chitinase activity but possessing chitin-binding properties. They act as endogenous lectins, regulating various physiological/pathological events. Ym1, originally identified as an eosinophil chemotactic factor or a macrophage-derived protein in parasite-infected mice, is a rodent-specific chitinase-like protein. Ym1 is also purified from eosinophilic crystals formed in the lung and urinary system in various disease models. We previously reported that major cellular sources of murine Ym1 are alveolar macrophages in the lung and neutrophils/monocytes lineage cells of the spleen and bone marrow under normal conditions. We here analyzed the detailed cellular expression of Ym1 in Mesocestoides corti (M. corti)-infected mice. Ym1 was significantly increased in the liver containing the larvae, lung, and peritoneal exudate cells in M. corti-infected mice, where activated macrophages expressed Ym1. Characteristic needle-shaped eosinophilic crystals appeared in the larvae-free lung, and Ym1 was localized to endoplasmic reticulum of activated alveolar macrophages. Moreover, swollen mesothelial cells covering the liver, spleen, and heart expressed Ym1 abundantly. Although the role of Ym1 in parasitic infection remains unclear, our findings focusing on an endogenous lectin may help in better understanding defense mechanism against parasites.

Histological and molecular responses of Vigna angularis to Uromyces vignae infection

BackgroundTo advance the understanding of adzuki bean (Vigna angularis) resistance to infection with the rust-causing fungus Uromyces vignae (Uv), we comprehensively analyzed histological events and the transcriptome of Uv-infected adzuki bean.ResultsCompared with the susceptible cv. Baoqinghong (BQH), the resistant cv. QH1 showed inhibition of uredospore germination and substomatal vesicle development, intense autofluorescence of cells around the infection site, and cell wall deposit formation in response to Uv infection. In cv. QH1, gene set enrichment analysis (GSEA) showed enrichment of chitin catabolic processes and responses to biotic stimuli at 24 h post-inoculation (hpi) and cell wall modification and structural constituent of cytoskeleton at 48 hpi. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated enrichment of WRKY transcription factors (TFs), the calcium binding protein cml, and hydroquinone glucosyltransferase at both 24 and 48 hpi. In total, 1992 and 557 differentially expressed genes (DEGs) were identified at 24 and 48 hpi, respectively. Cell surface pattern-recognition receptors (PRRs), WRKY TFs, defense-associated pathogenesis-related (PR) proteins, and lignin and antimicrobial phenolic compound biosynthesis were significantly induced. Finally, we detected the chitinase (CHI) and phenylalanine ammonia-lyase (PAL) activity were higher in QH1 and increased much earlier than in BQH.ConclusionIn cv. QH1, cell-surface PRRs rapidly recognize Uv invasion and activate the corresponding TFs to increase the transcription of defense-related genes and corresponding enzymatic activities to prevent fungal development and spread in host tissues.

Combination of ethrel and antiseptic medium flow of micro‐circulating improves shelf‐life quality of kiwifruit after long‐term controlled atmosphere storage

The uneven maturity and severe internal rot of kiwifruit were rapidly accelerated when transferred to room temperature after long-term controlled atmosphere storage. In this study, the effects of ethrel combined with different flow microcirculation of antiseptic mediums on the shelf-life quality of “Qinmei” kiwifruit stored at 20°C were evaluated. Results indicated that the combined aerosolization application of 500 mg/L natamycin, 7.5% w/w H2O2 and 0.5 mg/L atomized ozone could control the decay incidence and respiratory rate of ethrel-treated kiwifruit. Further studies indicated that ethrel and O3 treatment greatly increased the activities of chitinase, β-1,3-glucanase, catalase, and superoxide dismutase and produced higher ascorbic acid levels (151.16 mg·100 g−1/FW) to enhance disease-resistant of kiwifruit. This treatment could inhibit the activity of polyphenol oxidase and delay the process of membrane lipid peroxidation, contributing to preserving the integrity of cell structures well. Moreover, ethrel + O3 treatment could effectively reduce ethylene production (3.12 μl/kg/h) to delay the ripening and senescence of fruit stored at 20°C. More importantly, this treatment exhibited the lowest contents of decay incidence (9.75%), total bacterial count (4.07 log cfu/g), and yeasts and molds (4.18 log cfu/g), and thus extended the shelf life for up to 16 days. Therefore, a combination of ethrel and ozone is a potentially effective method for the maintenance of the shelf-life properties of kiwifruit after long-term cold storage. Novelty impact statement In this study, the treatment of ethrel-treated with aerosolized ozone of flow microcirculation of 0.5 mg/L O3 could effectively suppress ripening and senescence, enhance antioxidant capacity and disease resistance, reduce the degree of membrane lipid peroxidation, and considerably preserve the integrity of cell structures, which contributed to preventing the penetration of pathogens, retarding the decay incidence, and delaying the shelf-life quality deterioration of kiwifruit transferred to room temperature after long-term controlled atmosphere storage.

Antifungal Chitinase Production by Bacillus paramycoides B26 using Squid Pen Powder as a Carbon Source

This study aimed to optimize the medium compositions and cultural conditions for improved chitinase production by a potential strain of Bacillus isolated from the marine environment and determine the antifungal activity of its chitinase against plant pathogenic fungi. Five potential isolates were cultured for chitinase production by submerged fermentation using colloidal chitin in a liquid medium. In this study, chitinase activity was determined by measuring reducing sugars, which were determined by the 3,5-dinitrosalicylic acid (DNS) assay. The most potential isolate, B26, showed similarity to Bacillus paramycoides based on the 16S rRNA gene sequence. The maximum chitinase production was achieved at 6.52±0.02 U/mL after 72 h of incubation in a medium containing 2% squid pen powder, supplemented with 0.5% sodium nitrate and 2% NaCl, with an initial pH of 7. It was observed that the optimization of cultural conditions resulted in 2.83 times higher chitinase production than an unoptimized medium. The antifungal activity of crude chitinase against phytopathogenic fungi was evaluated by a well-diffusion method. The chitinase of B. paramycoides B26 effectively inhibited the growth of Fusarium solani TISTR 3436 (83.4%) and Penicillium chrysogenum TISTR 3554 (80.12%).

A Rhizobacterium, Streptomyces albulus Z1-04-02, Displays Antifungal Activity against Sclerotium Rot in Mungbean.

Sclerotium rot causes damping-off and stem rot in seedlings and mature mungbeans, which negatively impacts cultivation. The use of a rhizobacterium to control soil-borne diseases is an alternative method to the excess use of synthetic fungicides; therefore, this study aims to screen rhizosphere actinobacteria with fungicidal activities against Sclerotium rolfsii, the pathogen that causes sclerotium rot in mungbeans. Primary screening showed that the Streptomyces sp. isolate Z1-04-02 displayed the highest effectiveness against S. rolfsii in dual culture plates, with a percentage inhibition of 74.28%. An assay containing enzymes that degrade cell walls, of the cell-free culture filtrate (CF) of Z1-04-02, showed that the activities of chitinase and β-1,3-glucanase were 0.0209 and 1.0210 U/mL, respectively, which was significantly higher than that of the control (media alone). The cell-free CF of Z1-04-02, incubated at 37 °C and 100 °C, using agar well diffusion, effectively inhibited the growth of S. rolfsii with inhibition percentages of 37.78% and 27.78%, respectively. Solid-phase microextraction (SPME) was applied to trap volatiles released from Z1-04-02 and gas chromatography–mass spectrometry (GC/MS); volatile antifungal compounds were tentatively identified as bicyclic monoterpene (1R)-(-)-myrtenal. The application of the cell-free CF, and the spore suspension of Z1-04-02, showed disease severity indexes (DSIs) of 12.5% and 8.25%, respectively, which were significantly lower than those showing inoculation by S. rolfsii alone. The identification of this strain by morphology, biochemistry tests, and 16s rDNA sequences revealed that Z1-04-02 was Streptomyces albulus. This finding revealed that S. albulus Z1-04-02 displayed diverse fungicidal activities against S. rolfsii, and it has the potential to act as a biological control agent in terms of inhibiting sclerotium rot in mungbeans.

Biochemical purification and characterization of a truncated acidic, thermostable chitinase from marine fungus for N-acetylglucosamine production.

N-acetylglucosamine (GlcNAc) is widely used in nutritional supplement and is generally produced from chitin using chitinases. While most GlcNAc is produced from colloidal chitin, it is essential that chitinases be acidic enzymes. Herein, we characterized an acidic, highly salinity tolerance and thermostable chitinase AfChiJ, identified from the marine fungus Aspergillus fumigatus df673. Using AlphaFold2 structural prediction, a truncated Δ30AfChiJ was heterologously expressed in E. coli and successfully purified. It was also found that it is active in colloidal chitin, with an optimal temperature of 45°C, an optimal pH of 4.0, and an optimal salt concentration of 3% NaCl. Below 45°C, it was sound over a wide pH range of 2.0–6.0 and maintained high activity (≥97.96%) in 1–7% NaCl. A notable increase in chitinase activity was observed of Δ30AfChiJ by the addition of Mg2+, Ba2+, urea, and chloroform. AfChiJ first decomposed colloidal chitin to generate mainly N-acetyl chitobioase, which was successively converted to its monomer GlcNAc. This indicated that AfChiJ is a bifunctional enzyme, composed of chitobiosidase and β-N-acetylglucosaminidase. Our result suggested that AfChiJ likely has the potential to convert chitin-containing biomass into high-value added GlcNAc.

Chitinase (CHI) of Spodoptera frugiperda affects molting development by regulating the metabolism of chitin and trehalose

Chitin is the main component of insect exoskeleton and midgut peritrophic membrane. Insect molting is the result of the balance and coordination of chitin synthesis and degradation in chitin metabolism under the action of hormones. In this study, a 678 bp dsRNA fragment was designed and synthesized according to the known CHI (Chitinase) sequence of Spodoptera frugiperda. It was injected into the larvae to observe the molting and development of S. frugiperda. At the same time, the activities of trehalase and chitinase, the contents of trehalose, chitin and other substances were detected, and the expression of related genes in the chitin synthesis pathway was determined. The results showed that CHI gene was highly expressed at the end of each instar, prepupa and pupal stage before molting; At 12 and 24 h after dsRNA injection of CHI gene of S. frugiperda, the expression of CHI gene decreased significantly, and the chitinase activity decreased significantly from 12 to 48 h. The expression of chitin synthase (CHSB) gene decreased significantly, and the chitin content increased significantly. Some larvae could not molt normally and complete development, leading to certain mortality. Secondly, after RNAi of CHI gene, the content of glucose and glycogen increased first and then decreased, while the content of trehalose decreased significantly or showed a downward trend. The activities of the two types of trehalase and the expression levels of trehalase genes decreased first and then increased, especially the trehalase activities increased significantly at 48 h after dsCHI injection. And trehalose-6-phosphate synthase (TPS), glutamine: fructose-6-phosphate amidotransferase (GFAT), UDP-N-acetylglucosamine pyrophosphorylases (UAP), hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and phosphoacetylglucosamine mutase (PAGM) all decreased significantly at 24 h, and then increased or significantly increased at 48 h. These results indicated that when the expression of chitinase gene of S. frugiperda was inhibited, it affected the degradation of chitin in the old epidermis and the formation of new epidermis, and the content of chitin increased, which led to the failure of larvae to molt normally. Moreover, the chitin synthesis pathway and trehalose metabolism were also regulated. The relevant results provide a theoretical basis for screening target genes and developing green insecticides to control pests by using the chitin metabolism pathway.