Gene Cloning, Heterologous Expression, and In Silico Analysis of Chitinase B from Serratia marcescens for Biocontrol of Spodoptera frugiperda Larvae Infesting Maize Crops.

Spodoptera frugiperda, the fall armyworm (FAW), is a highly invasive polyphagous insect pest that is considered a source of severe economic losses to agricultural production. Currently, the majority of chemical insecticides pose tremendous threats to humans and animals besides insect resistance. Thus, there is an urgent need to develop new pest management strategies with more specificity, efficiency, and sustainability. Chitin-degrading enzymes, including chitinases, are promising agents which may contribute to FAW control. Chitinase-producing microorganisms are reported normally in bacteria and fungi. In the present study, Serratia marcescens was successfully isolated and identified from the larvae of Spodoptera frugiperda. The bacterial strain NRC408 displayed the highest chitinase enzyme activity of 250 units per milligram of protein. Subsequently, the chitinase gene was cloned and heterologously expressed in E. coli BL21 (DE3). Recombinant chitinase B was overproduced to 2.5-fold, driven by the T7 expression system. Recombinant chitinase B was evaluated for its efficacy as an insecticidal bioagent against S. frugiperda larvae, which induced significant alteration in subsequent developmental stages and conspicuous malformations. Additionally, our study highlights that in silico analyses of the anticipated protein encoded by the chitinase gene (ChiB) offered improved predictions for enzyme binding and catalytic activity. The effectiveness of (ChiB) against S. frugiperda was evaluated in laboratory and controlled field conditions. The results indicated significant mortality, disturbed development, different induced malformations, and a reduction in larval populations. Thus, the current study consequently recommends chitinase B for the first time to control FAW.

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.

Effect of four chitinase genes on the female fecundity in Sogatella furcifera (Horváth).

BACKGROUND: The white-backed planthopper (WPH), Sogatella furcifera (Horváth), is a destructive rice pest with strong reproductive capacity. To gain insights into the roles of chitinases in the reproductive process of this insect species, this study represents the first-ever endeavor to conduct an in-depth exploration into the reproductive functions of four chitinase genes. RESULTS: In this study, it was observed that four chitinase genes were expressed in female adults, with a relatively high expression level in the ovaries. SfCht2 and SfIDGF1 were highly expressed during later ovarian development. while SfENGase increased and then decreased with ovarian development. SfCht2, SfCht6-2 and SfENGase were highly expressed in fat body on the first and second days after eclosion, whereas SfIDGF1 highest on day 7. Compared with control group, Silencing four chitinase genes inhibited ovarian development and significantly shortened the oviposition period of S. furcifera, reducing egg-laying capacity but not affecting egg hatching. The detection demonstrated that the expression levels of SfVg, SfVgR and 70-90% juvenile hormone (JH) signaling pathway-related reproductive genes was significantly down-regulated. Moreover, SfCht6-2 and SfENGase significantly affected the expression levels of Target of Rapamycin (TOR) signaling pathway genes. SfENGase had the ability to impact nutrient signaling pathways and fatty acid metabolism, repressing vitellogenin synthesis and ultimately influencing ovarian development of S. furcifera. CONCLUSIONS: Overall, this study provides insight into the function of chitinases in insect fecundity and is of great significance for enriching the cognition of insect chitinase function. They will become the suitable target genes for controlling the most destructive rice planthoppers. © 2023 Society of Chemical Industry.

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.

Isolation and selection of autochthonous strains of Trichoderma spp. with inhibitory activity against Sporisorium reilianum.

Head smut is a worldwide disease caused by the fungus Sporisorium reilianum. In Mexico, this phytosanitary problem has been described in the central part of the country, specifically in the Mezquital Valley in the state of Hidalgo, where this basidiomycete causes significant economic losses. In this work, seven strains of Trichoderma spp. were isolated from corn rhizospheres collected from crops in the affected zone. The isolates were identified as Trichoderma asperellum MH1, T. asperellum T4H1, T. harzianum T1H1, T. harzianum T1H3, T. atrobrunneum T1H2, T. tomentosum T2H4, and T. brevicompactum T3H1. All strains showed the ability to grow on the phytopathogen but with distinct degrees of mycoparasitism. SEM observations demonstrated the ability of T. asperellum T4H1 to invade the S. reilianum yeast growth. All the strains produced volatile compounds with antifungal activity. With the exception of T. asperellum MH1, all strains inhibited the development of the pathogen by means of non-volatile compounds. Production of the extracellular enzymes (lipase, cellulase, chitinase, protease, and laccase) was evaluated, with most strains presenting high lipolytic activity and low proteolytic activity. The production of cellulase and chitinase was observed only in five strains. Laccase production was found in three isolates. Evaluations at the greenhouse of the sequential application of three mixtures of the isolates were conducted in a greenhouse; findings showed that the phytopathogen was not detected by specific PCR in the plants that received the treatment.

Serendipita indica chitinase protects rice from the blast and bakanae diseases.

Serendipita indica, a multifunctional and useful endophyte fungus, has been intensively investigated in promoting plant growth and resistance towards biotic and abiotic stress. Multiple chitinases from microorganisms or plants have been identified to have a high antifungal activity as a biological control. However, chitinase of S. indica still needs to be characterized. We functionally characterized a chitinase (SiChi) in S. indica. The result showed that the purified SiChi protein confers high chitinase activity; importantly, SiChi inhibits the conidial germination of Magnaporthe oryzae and Fusarium moniliforme. After the successful colonization of rice roots by S. indica, both the rice blast disease and bakanae disease were significantly reduced. Interestingly, the purified SiChi could promptly induce rice disease resistance towards M. oryzae and F. moniliforme pathogens when sprayed on rice leaves. Like S. indica, SiChi could upregulate rice pathogen-resistant proteins and defense enzymes. In conclusion, chitinase of S. indica has direct antifungal activity and indirect induced resistance activity, implying an efficient and economic strategy for rice disease control by applying S. indica and SiChi.

Comparison of in vitro Antifungal Activity Methods Using Extract of Chitinase-producing Aeromonas sp. BHC02.

Biological control to prevent fungal plant diseases offers an alternative approach to facilitate sustainable agriculture. Since the chitin in fungal cell walls is a target for biocontrol agents, chitinases are one of the important antifungal molecules. In this study, the aim was to investigate a new chitinase isolated from a fluvial soil bacterium and to show the antifungal activity of the characterized chitinase by comparing the three common methods. The bacterium with the highest chitinase activity was identified as Aeromonas sp. by 16 S rRNA sequence analysis. Following the determination of the optimum enzyme production time, the enzyme was partially purified, and the physicochemical parameters of the enzyme were investigated. In the antifungal studies, direct Aeromonas sp. BHC02 cells or partially purified chitinase were used. As a result, in the first method in which the Aeromonas sp. BHC02 cells were spread on the surface of petri dishes, no zone formation was observed around the test fungi spotted on the surface. However, zone formation was observed in the methods in which the antifungal activity was investigated using the partially purified chitinase enzyme. For example, in the second method, the enzyme was spread on the surface of PDA, and zone formation was observed only around Penicillum species among the test fungi spotted on the surface. In the third method, in which the necessary time was given for the formation of mycelium of the test fungi, it was observed that the growth of Fusarium solani, Alternaria alternata and Botrytis cinerea was inhibited by the partially purified chitinase. This study concludes that the results of the antifungal activities depend on the method used and all fungal chitins cannot be degraded with one strain's chitinase. Depending on the variety of chitin, some fungi can be more resistant.

Chitinase is a Potent Insecticidal Molecular Target of Camptothecin and Its Derivatives.

Camptothecin (CPT) is a prominent molecule in natural product research because of its application prospects in medicine and agriculture. In this study, CPT and its derivatives were discovered to be competitive inhibitors of group II and group h insect chitinases, both of which are key components of insect chitinolytic systems. CPT and 7-ethyl-10-hydroxycamptothecin (SN-38) inhibited group II chitinase from Ostrinia furnacalis (OfChtII) with Ki values of 5.1 and 2.0 µM, respectively. Results from tryptophan fluorescence spectroscopy, molecular docking analysis, and molecular dynamics simulations revealed that both CPT and SN-38 inhibit OfChtII-C1 by interacting with solvent-exposed tryptophan residues in a substrate-binding cleft. CPT exhibited high insecticidal activity toward the orthopteran pest Locusta migratoria, possibly because of the midgut metabolism of CPT, with only moderate activities toward lepidopteran pests. Even though SN-38 exhibited much lower insecticidal activities than CPT, it still showed higher inhibitory activity toward chitinase. This study reports a new molecular target of CPT and provides insights into molecular design of CPT-based insecticides against different kinds of pests.

Thermo-neutrophilic cellulases and chitinases characterized from a novel putative antifungal biocontrol agent: Bacillus subtilis TD11.

Cellulose and chitin are the most abundant naturally occurring biopolymers synthesized in plants and animals and are used for synthesis of different organic compounds and acids in the industry. Therefore, cellulases and chitinases are important for their multiple uses in industry and biotechnology. Moreover, chitinases have a role in the biological control of phytopathogens. A bacterial strain Bacillus subtilis TD11 was previously isolated and characterized as a putative biocontrol agent owing to its significant antifungal potential. In this study, cellulase and chitinase produced by the strain B. subtilis TD11 were purified and characterized. The activity of the cellulases and chitinases were optimized at different pH (2 to 10) and temperatures (20 to 90°C). The substrate specificity of cellulases was evaluated using different substances including carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), and crystalline substrates. The cellulase produced by B. subtilis TD11 had a molecular mass of 45 kDa while that of chitinase was 55 kDa. The optimal activities of the enzymes were found at neutral pH (6.0 to 7.0). The optimum temperature for the purified cellulases was in the range of 50 to 70°C while, purified chitinases were optimally active at 50°C. The highest substrate specificity of the purified cellulase was found for CMC (100%) followed by HEC (>50% activity) while no hydrolysis was observed against the crystalline substrates. Moreover, it was observed that the purified chitinase was inhibitory against the fungi containing chitin in their hyphal walls i.e., Rhizoctonia, Colletotrichum, Aspergillus and Fusarium having a dose-effect relationship.

Purification and characterization of Stenotrophomonas maltophilia chitinase with antifungal and insecticidal properties.

This study aimed to determine the ability of bacteria to produce the chitinase enzyme, purify, and characterize the enzyme from the isolate with the best activity, and determine the use of this purified enzyme as a biocontrol agent. The chitinolytic bacterium was identified as Stenotrophomonas maltophilia. The chitinase enzyme was purified 1.4 times at a 30% ammonium sulfate concentration with a yield of 40.7%. Following partial purification, the enzyme was purified by ion-exchange chromatography using HiPrep Q XL 16/10 column and HiPrep™ 26/10 desalting column with 25.34% and 18.12% yields, respectively. It was calculated that the purified enzyme had a molecular weight of 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme was determined at 50 °C and pH 7.0. Enzyme activity was most induced by Fe2+, while it was most inhibited by Zn2+ at 5 mM concentration. Km and Vmax values of the enzyme for colloidal chitin were calculated as 1.6419 mg/mL and 16.129 U/mg, respectively. The purified chitinase was used as a biocontrol agent against the fungus Fusarium oxysporum and potato beetle Leptinotarsa decemlineata. The enzyme was shown to be effective in reducing the growth of fungus and causing disruption of the chitin structure of potato beetle.

Chronic interleukin-13 expression in mouse olfactory mucosa results in regional aneuronal epithelium.

BACKGROUND: Olfactory dysfunction is highly associated with chronic rhinosinusitis with nasal polyps (CRSwNP), and the severity of loss has been linked with biomarkers of type 2 inflammation. The ability of dupilumab to rapidly improve the sense of smell prior to improvement in polyp size suggests a direct role of IL-4/IL-13 receptor signaling in the olfactory epithelium (OE). METHODS: We created a transgenic mouse model in which IL-13 is inducibly expressed specifically within the OE. Gene expression analysis and immunohistology were utilized to characterize the effect of IL-13 on the structure of the OE. RESULTS: After induction of olfactory IL-13 expression, there is a time-dependent loss of neurons from OE regions, accompanied by a modest inflammatory infiltrate. Horizontal basal cells undergo morphologic changes consistent with activation and demonstrate proliferation. Mucus production and increased expression of eotaxins is observed, with marked expression of Ym2 by sustentacular cells. DISCUSSION: Chronic IL-13 exposure has several effects on the OE that are likely to affect function. The neuronal loss is in keeping with other models of allergic type 2 nasal inflammation. Future studies are needed to correlate cellular and molecular alterations in olfactory cell populations with findings in human CRSwNP, as well as to assess olfactory function in behavioral model systems.

Identification, cloning, and characterization of a novel chitinase from leaf-cutting ant Atta sexdens: An enzyme with antifungal and insecticidal activity.

Chitinases are enzymes that degrade chitin, a polysaccharide found in the exoskeleton of insects, fungi, yeast, and internal structures of other vertebrates. Although chitinases isolated from bacteria, fungi and plants have been reported to have antifungal or insecticide activities, chitinases from insects with these activities have been seldomly reported. In this study, a leaf-cutting ant Atta sexdens DNA fragment containing 1623 base pairs was amplified and cloned into a vector to express the protein (AsChtII-C4B1) in Pichia pastoris. AsChtII-C4B1, which contains one catalytic domain and one carbohydrate-binding module (CBM), was secreted to the extracellular medium and purified by ammonium sulfate precipitation followed by nickel column chromatography. AsChtII-C4B1 showed maximum activity at pH 5.0 and 55 °C when tested against colloidal chitin substrate and maintained >60% of its maximal activity in different temperatures during 48 h. AsChtII-C4B1 decreased the survival of Spodoptera frugiperda larvae fed with an artificial diet that contained AsChtII-C4B1. Our results have indicated that AsChtII-C4B1 has a higher effect on larva-pupa than larva-larva molts. AsChtII-C4B1 activity targets more specifically the growth of filamentous fungus than yeast. This work describes, for the first time, the obtaining a recombinant chitinase from ants and the characterization of its insecticidal and antifungal activities.

Biological Characteristics of Recombinant Arthrobotrys oligospora Chitinase AO-801.

Chitinase AO-801 is a hydrolase secreted by Arthrobotrys oligospora during nematode feeding, while its role remained elusive. This study analyzed the molecular characteristics of recombinant chitinase of Arthrobotrys oligospora (reAO-801). AO-801 belongs to the typical glycoside hydrolase 18 family with conserved chitinase sequence and tertiary structure of (α/ß)8 triose-phosphate isomerase (TIM) barrel. The molecular weight of reAO-801 was 42 kDa. reAO-801 effectively degraded colloidal and powdered chitin, egg lysate, and stage I larval lysate of Caenorhabditis elegans. The activity of reAO-801 reached its peak at 40˚C and pH values between 4-7. Enzyme activity was inhibited by Zn2+, Ca2+, and Fe3+, whereas Mg2+ and K+ potentiated its activity. In addition, urea, sodium dodecyl sulfate, and 2-mercaptoethanol significantly inhibited enzyme activity. reAO-801 showed complete nematicidal activity against C. elegans stage I larvae. reAO-801 broke down the C. elegans egg shells, causing them to die or die prematurely by hatching the eggs. It also invoked degradation of Haemonchus contortus eggs, resulting in apparent changes in the morphological structure. This study demonstrated the cytotoxic effect of reAO-801, which laid the foundation for further dissecting the mechanism of nematode infestation by A. oligospora.

An eco-friendly approach of biocontrol of aphid (Aphis gossypii Glover) by Trichoderma harzianum.

Aphid (Aphis gossypii) is one of the important pests of papaya crop. In this work, applications of Trichoderma harzianum and Beauveria bassiana (biocontrol agents) and malathion (insecticide) were conducted in vitro and in agrifields for testing their anti-aphid efficacy and compared their efficacy. Furthermore, the enzymatic mechanism of T. harzianum with respect to biocontrolling the pest was unearthed. The LD50 dose of T. harzianum and B. bassiana was 1.2 × 105 spores mL-1 and 1.0 × 106 spores mL-1 respectively after 48 h of administration. The LT50 of T. harzianum also exhibited a lower effective time (47.70 h) than B. bassiana (57.53 h) for the same concentration of spores applied (1 × 105 spores mL-1). The pooled data analysis of two years (2019-2020) showed that the application of T. harzianum spores in agrifields exhibited 31.75 ± 13.00a percentage of reduction of aphid population whereas malathion exhibited 23.93 ± 1.30a%, in comparison to control. The statistical analysis indicated that the application of malathion exhibited the same efficacy as T. harzianum isolate and placed in the same category. In plate detection assay, T. harzianum produced a higher hydrolytic zone for chitinase (8.0 ± 0.4 cm diameter) and protease (7.0 ± 0.4 cm diameter) enzymes, than B. bassiana (1.3 ± 0.2 cm and 1.1 ± 0.2 cm respectively). Quantitative estimation of enzymes exhibited that T. harzianum produced 299 ± 11a µg mL-1 of chitinase, 519 ± 19a µg mL-1 of protease, and 65 ± 12a µg mL-1 of PR1, and on the other hand, B. bassiana yielded 124 ± 12b, 361 ± 23b, and 29 ± 18b µg mL-1 of chitinase, protease, and PR1 respectively. It indicated that T. harzianum was superior over the B. bassiana in terms of production capacity of all three enzymes. In conclusion, all the above experimental results suggested that T. harzianum showed better aphid-killing efficacy than B. bassiana. It also suggested that T. harzianum should replace hazardous chemical pesticide (malathion) for eco-friendly biocontrol of aphids.

Chitinase from the Latex of Ficus benjamina L. Displays Antifungal Activity by Inducing ROS Generation and Structural Damage to the Fungal Cell Wall and Plasma Membrane.

BACKGROUND: Chitinases are plant defense-related proteins with a high biotechnological potential to be applied in agriculture. OBJECTIVES: This study aimed to purify a chitinase from the latex of Ficus benjamina. METHODS: An antifungal class I chitinase, named FbLx-Chi-1, was purified from the latex of Ficus benjamina after precipitation with 30-60% ammonium sulfate and affinity chromatography on a chitin column and antifungal potential assay against phytopathogenic fungi important to agriculture. RESULTS: FbLx-Chi-1 has 30 kDa molecular mass, as estimated by SDS-PAGE and the optimal pH and temperature for full chitinolytic activity were 5.5 and 60ºC, respectively. FbLx-Chi-1 is a high pH-, ion-tolerant and thermostable protein. Importantly, FbLx-Chi-1 hindered the growth of the phytopathogenic fungi Colletotrichum gloeosporioides, Fusarium pallidoroseum, and Fusarium oxysporum. The action mode of FbLx-Chi-1 to hamper F. pallidoroseum growth seems to be correlated with alterations in the morphology of the hyphal cell wall, increased plasma membrane permeability, and overproduction of reactive oxygen species. CONCLUSION: These findings highlight the biotechnological potential of FbLx-Chi-1 to control important phytopathogenic fungi in agriculture. In addition, FbLx-Chi-1 could be further explored to be used in industrial processes such as the large-scale environmentally friendly enzymatic hydrolysis of chitin to produce its monomer N-acetyl-ß-D-glucosamine, which is employed for bioethanol production, in cosmetics, in medicine, and for other multiple applications.

Efficacy of Entomopathogenic Fungal Formulations against Elasmolomus pallens (Dallas) (Hemiptera: Rhyparochromidae) and Their Extracellular Enzymatic Activities.

Elasmolomus pallens are post-harvest insect pests of peanuts that are becoming resistant to chemical insecticides. In this, we study evaluated the effect of conidial formulations on entomopathogenic fungi against E. pallens to reduce the adverse effects. Fungal conidia were formulated and applied on sterile filter papers at varying concentrations (1 × 104-1 × 108 conidia mL-1) inside plastic containers. The test insects were exposed and maintained in a relative humidity of 80 ± 10% for 10 d at room temperature (25 ± 2 °C). Mortality was recorded every 24 h. Dose-response bioassay (LC50 and LC90) values for Aspergillus flavus formulated in oil were 1.95 × 106 and 3.66 × 109 conidia/mL, whereas formulations in Tween 80 had 9.36 × 107 and 6.50 × 109 conidia/mL. However, oil-formulated Metarhizium anisopliae had 3.92 × 106 and 2.57 × 108 conidia/mL, with 6.85 × 106 and 5.37 × 108, for formulations in Tween 80. A. flavus had LT50 values of 3.3 and 6.6 days, whereas M. anisopliae had LT50 values of 3.6 and 5.7 d. Maximum protease, chitinase, and lipase activities of 2.51, 0.98, and 3.22 U/mL, respectively, were recorded for A. flavus, whereas values of 2.43, 0.93, and 3.46 were recorded for M. anisopliae. The investigated pathogens demonstrate potential against E. pallens; therefore, their applicability under field conditions requires further investigation.

A marine chitinase from Bacillus aryabhattai with antifungal activity and broad specificity toward crystalline chitin degradation.

Chitinases convert chitin into chitin oligomers and are also known antifungal agents. Chitin oligomers have numerous industrial applications. However, chitin's crystalline nature requires pretreatment before breakdown into oligomers. In the study, a novel marine bacterium Bacillus aryabhattai is isolated from the Arabian Sea. Bacterial growth in different crystalline chitin substrates like chitin powder, chitin flakes, and colloidal chitin confirmed the chitinase presence in bacterium could act upon insoluble crystalline chitin with the fractional release of oligomers. The domain architecture analysis of the chitinase confirmed the presence of two N-terminal LysM domains which help enzyme action on crystalline chitin. Statistical optimization of media and Process parameters revealed glycerol, yeast extract, magnesium chloride, and manganese sulfate as significant media components along with colloidal chitin. The optimum process parameters such as pH 7, temperature 40 °C, inoculum size 12.5% (v/v), and inoculum age 20 hours enhanced the specific enzyme activity to ±146.2 U/mL, ±114.9 U/mL and ±175.4 U/mL against chitin powder, chitin flakes and colloidal chitin respectively, which is five to six times higher than basal level activity. The antifungal activity of chitinase against plant pathogenic fungi like Candida albicans and Fusarium oxysporum revealed a zone of inhibition with 14 mm diameter.

Purification, characterization, and antifungal activity of Bacillus cereus strain NK91 chitinase from rhizospheric soil samples of Himachal Pradesh, India.

Newly isolated Bacillus cereus strain NK91 was characterized for extracellular chitinase production. Partially purified chitinase showed a molecular weight of 43.7 kDa in SDS-PAGE analysis. The optimum pH and temperature for the partially purified enzyme were 7.0 and 40°C, respectively. The addition of Mn2+ resulted in a 21% increase in enzyme activity as compared to the control. The Vmax and Km of the enzyme were determined as 76.9 µmol/min and 0.07 mg/mL, respectively. This enzyme exhibited stronger antifungal activity towards Fusarium oxysporum (66.7%), Rhizoctonia solani (64.6%), and Colletotrichum gloeosporioides (63%), and transmission electron microscopy and scanning transmission electron microscopy analysis showed considerable changes in cell wall structure with the treatment of purified chitinase as compared to control. Therefore, this enzyme reveals its biocontrol potential against potent phytopathogens in agriculture that can be helpful in swapping harmful as well as expensive fungicides.

An integrative epi-transcriptomic approach identifies the human cartilage chitinase 3-like protein 2 (CHI3L2) as a potential mediator of B12 deficiency in adipocytes.

Vitamin B12 has multiple biochemical functions including in the one-carbon cycle generating a methyl group for DNA methylation, and metabolism of fatty acids and amino acids to generate energy via the citric acid cycle. The aim of our study was to use a combined epigenomic and transcriptomic approach to identify novel genes mediating the effect of B12 on adipogenesis.Human pre-adipocytes (CHUB-S7) were treated with a range of B12 (0-500 nM) concentrations from the day of cell seeding until harvesting in discovery and validation experiments prior to genome-wide methylation analysis using the Illumina HumanMethylation 450Beadchip. For transcriptomic analysis, RNA-seq libraries were run on the Illumina HiSeq 2500. To further investigate the expression of any genes on human adipogenesis, a second human preadipocyte strain was studied (SGBS) by real-time quantitative PCR (qRT-PCR).A combined epigenetic and transcriptomic approach in differentiated human pre-adipocyte cell line, CHUB-S7, identified that the Human cartilage chitinase 3-like protein 2 (CHI3L2) gene was hypo-methylated and had increased expression in low B12 conditions. Furthermore, there was an approximately 1000-fold increase in CHI3L2 expression in the early days of adipocyte differentiation, which paralleled an increase of lipid droplets in differentiated SGBS cells and an increased expression level of markers of mature adipocytes.In summary, we have identified a potential role of the human cartilage chitinase 3-like protein 2 (CHI3L2) in adipocyte function in the presence of low B12 levels.

Bacterial chitinase biochemical properties, immobilization on zinc oxide (ZnO) nanoparticle and its effect on Sitophilus zeamais as a potential insecticide.

It has been planned to minimize the yield and quality impairment of the seed corn, which is strategically important in the world, by pests under storage conditions with a biological product produced with a biotechnological approach. In this context, the present study aimed to control the maize weevil Sitophilus zeamais, known as a warehouse pest, using a nanoformulation. In the study, the chitinase enzyme from Lactobacillus coryniformis was purified first using ammonium sulfate precipitation and then by using the HiTrap Capto DEAE column, and the molecular mass of the purified enzyme was determined to be ~ 33 kDa, and the optimum pH and the values as pH 6.0 and 65-75 °C, respectively. Five different doses of nanoformulation (2, 4, 6, 8 and 10 mg/L) were applied to corn grains by the spraying method with three repetitions so that the insect can ingest the formulation through feeding. The effects of the applications on the death rate and mean time of death of Sitophilus zeamais were determined. According to these findings, it was concluded that the best practice was nanoformulation with 6 mg/L, considering both the mortality rate (100%) and the average death time (2.4 days). Chitinase from L. coryniformis is a promising candidate for corn lice control and management.