Chitinase Activity Research Articles

Contribution of microbial activity and vegetation cover to the spatial distribution of soil respiration in mountains.

The patterns of change in bioclimatic conditions determine the vegetation cover and soil properties along the altitudinal gradient. Together, these factors control the spatial variability of soil respiration (RS) in mountainous areas. The underlying mechanisms, which are poorly understood, shape the resulting surface CO2 flux in these ecosystems. We aimed to investigate the spatial variability of RS and its drivers on the northeastern slope of the Northwest Caucasus Mountains, Russia (1,260-2,480 m a.s.l.), in mixed, fir, and deciduous forests, as well as subalpine and alpine meadows. RS was measured simultaneously in each ecosystem at 12 randomly distributed points using the closed static chamber technique. After the measurements, topsoil samples (0-10 cm) were collected under each chamber (n = 60). Several soil physicochemical, microbial, and vegetation indices were assessed as potential drivers of RS. We tested two hypotheses: (i) the spatial variability of RS is higher in forests than in grasslands; and (ii) the spatial variability of RS in forests is mainly due to soil microbial activity, whereas in grasslands, it is mainly due to vegetation characteristics. Unexpectedly, RS variability was lower in forests than in grasslands, ranging from 1.3-6.5 versus 3.4-12.7 μmol CO2 m-1 s-1, respectively. Spatial variability of RS in forests was related to microbial functioning through chitinase activity (50% explained variance), whereas in grasslands it was related to vegetation structure, namely graminoid abundance (27% explained variance). Apparently, the chitinase dependence of RS variability in forests may be related to soil N limitation. This was confirmed by low N content and high C:N ratio compared to grassland soils. The greater sensitivity of grassland RS to vegetation structure may be related to the essential root C allocation for some grasses. Thus, the first hypothesis concerning the higher spatial variability of RS in forests than in grasslands was not confirmed, whereas the second hypothesis concerning the crucial role of soil microorganisms in forests and vegetation in grasslands as drivers of RS spatial variability was confirmed.

γ-Cyclodextrin encapsulated thymol for citrus preservation and its possible mechanism against Penicillium digitatum

The volatility of essential oils greatly limits their industrial applications. Here, we successfully prepared γ-cyclodextrin (γ-CD) inclusion compounds (γ-CDTL) containing thymol (TL) for the control of green mold caused by Penicillium digitatum (P. digitatum) in citrus fruit. In vitro experiment showed that the minimum fungicidal concentration (MFC) of γ-CDTL against the hyphae growth of P. digitatum was 2.0 g/L, and 8 × MFC treatment significantly reduced the occurrence of green mold in citrus fruit and had no adverse effect on fruit quality in vivo test compared to prochloraz. Scanning electron microscopy (SEM), x-ray diffraction (XRD), fourier transform-infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), physical properties and sustained release properties were also performed, results indicated that the hydrogen bonds between TL and γ-CD were the basis for the formation of γ-CDTL. We further investigated the inhibition mechanism of γ-CDTL. SEM and TEM experiments showed that γ-CDTL treatment caused severe damage to the hyphal morphology and cells in 30 min and disrupted the permeability of P. digitatum mycelial cell walls by increasing the chitinase activity, thus accelerating the leakage of intracellular lysates. However, the integrity of the cell membrane was obviously damaged only after 60 min of treatment. In conclusion, we prepared a novel inclusion complex γ-CDTL with obvious antifungal effects and preliminarily elucidated its inclusion mechanism and antifungal mechanism. γ-CDTL might be a potent alternative to chemical fungicides for controlling the postharvest decay of citrus.

Conservation tillage increases surface soil organic carbon stock by altering fungal communities and enzyme activity.

Fungal communities play a key role in the decomposition of crop residues and affect soil organic carbon (SOC) dynamics. Conservation tillage enhances SOC sequestration and mitigate global climate change. However, the impact of long-term tillage practices on fungal community diversity and its relation to SOC stock remains unclear. The objectives of this study were to evaluate the relationship between extracellular enzyme activities and fungal community diversity and SOC stock under different tillage practices. A field experiment was conducted with four tillage practices: (i) no-tillage with straw removal (NT0), (ii) no-tillage with straw retention (NTSR, conservation tillage), (iii) plough tillage with straw retention (PTSR), and (iv) rotary tillage with straw retention (RTSR). The results showed that the SOC stock in NTSR was higher than other treatments in the 0-10 cm soil layer. Compared to NT0, NTSR significantly increased soil β-glucosidase, xylosidase, cellobiohydrolase, and chitinase activities at 0-10 cm soil depth (P < 0.05). However, different tillage methods with straw returning had no significant effects on enzyme activity at 0-10 cm soil depth. The observed species and Chao1 index of the fungal communities under NTSR were 22.8% and 32.1% lower than under RTSR in the 0-10 cm soil layer, respectively. The composition, structure, and co-occurrence network of fungal communities differed across tillage practices. A partial least squares path model (PLS-PM) analysis indicated that C-related enzymes were the most influential factors associated with SOC stock. Soil physicochemical properties and fungal communities affected extracellular enzyme activities. Overall, conservation tillage can promote surface SOC stock, which was associated with increased enzyme activity.

Phytopathological and biochemical impacts of Trichoderma harzianum and certain plant resistance inducers on faba bean root rot disease

BackgroundFaba bean attacked by soil-borne pathogens causing root rot disease. This disease has serious damage to both plant stand and produced yield. The present study aimed to evaluate effectiveness of the bioagents; Trichoderma harzianum and some plant resistance inducers as fungicide alternatives against root rot disease incidence at both pre- and post-emergence growth stages.ResultsUnder open greenhouse conditions, the incidence of faba bean root rot in pre- and post-emergence growth phases was considerably reduced by using six inorganic salts and five antioxidants individually or combining with each other or with the bio-stimulator T. harzianum that exceeded the used fungicide, Rhizolex-T. Application of enervit agitated the highest significant defensive impact during pre-emergence stage versus root rot incidence (5.0%), followed by calcium sulfate and [cysteine + T. harzianum] (6.7%). At post-emergence stage, majority of the treatments completely suppressed (100.0%) root rot incidence, except vitamax plus and the fungicide (Rizolex-T) which expressed by 91.7 and 18.8%, respectively. Duplicate irrigations of 23 treatments after faba bean dressing improved the synthesis of different protein contents with the 2nd of which enhanced higher protein contents than the 1st one, except [T. harzianum + vitamin E + vitamin C + enervit + selenium + vitamax plus], [T. harzianum + vitamax plus] and cysteine. Disodium phosphate induced the highest catalase (CAT) activity (1820.8 and 1677.2 U/g FWt) after both irrigations. [T. harzianum + vitamax plus] and vitamin E induced the highest peroxidase (POD) activity 217.4 and 356.9 U/g FWt after 1st and 2nd irrigations, respectively. Disodium phosphate and [T. harzianum + vitamin E + vitamin C + enervit + selenium + vitamax plus] induced the highest chitinase (CHIA) activity 52.8 and 54.4 U/g FWt after 1st and 2nd irrigations, respectively. Application of disodium phosphate, calcium sulfate, potassium metabisulfite, sodium sulfate, cysteine, [cysteine + potash alum], enervit, vitamin E, [vitamin E + vitamin C + enervit + selenium + vitamax plus], [T. harzianum + enervit], [T. harzianum + selenium], [T. harzianum + vitamin E], [T. harzianum + vitamin E + vitamin C + enervit + selenium + vitamax plus] and vitamin C stimulated the formation of new protein bands on SDS-PAGE after the 2nd irrigation treatment.ConclusionsSuch treatments are considered good and environmentally safe alternatives against root diseases for getting rid of the negative effects of fungicides.

Biologic profile to predict overall survival in non-metastatic breast cancer: A latent class analysis on aging biomarkers.

12037 Background: The aging process is heterogeneous and the result of different biological mechanisms. Blood biomarkers involved in the aging process, aside from comprehensive geriatric assessment, may help clinicians when facing equivocal therapeutic decisions, for example adjuvant treatment at high risk of toxicities. Methods: This study was conducted on a previously published cohort (PMID 25989735) including 162 patients aged ≥70 years (“Old”) and 82 patients aged ≤60 years (“Young”) with newly diagnosed non-metastatic breast cancer. Serum samples collected from all patients at breast cancer diagnosis, before any treatment were available from the extensive Leuven blood biobank. Overall survival was evaluated with October 7, 2022 as data cutoff. A panel of blood aging biomarkers attributed to inflammation: IL6, MCP1, RANTES, telomere shortening: telomeric DNA ratio T/S, telomeric dysfunction: CRAMP, chitinase activity, and anabolism : IGF1. Considering a high risk of collinearity, we performed a latent class analysis (LCA) on the whole cohort, to delineate the respective roles of inflammaging, replicative senescence and sarcopenia on patient clusters. Results: In univariate analysis were significantly associated with a poorer survival: high MCP1, hazard ratio (HR) = 1.57 (95%CI 1.32-1.87), high chitinase activity, HR = 1.33 (95%CI 1.12-1.57), and low IGF1, HR = 1.76 (95%CI 1.47-2.11). The biomarkers correlation matrix identified a significant correlation of Chitinase activity with MCP1, IGF1, CRAMP; of CRAMP with IL6; and IGF1 with T/S ratio. LCA identified 2 biologic profiles within the population, representing 137 (56.1%, class 1) and 107 (43,9%, class 2) patients. Class 1 was associated with a significantly poorer overall survival: HR = 3.52 (95%CI 2.28-5.45), and age-adjusted HR = 1.77 (95%CI 1.12-2.80). The best predictors for class 1 membership were low IGF1 (89%), high MCP1 (82%), high chitinase activity (79%), high IL6 (69%). Class 1 represented 77% of the “Old” and 15% of the “Young” populations. Conclusions: Our results delineate an unfavorable biologic profile that transcends chronological age and significantly predicts a poorer overall survival, underpinned by markers of both inflammaging, telomeric dysfunction and sarcopenia. The strong correlation between markers argues for a unique underlying biological pathway. Noteworthy, high IGF1, usually considered as a risk factor for breast cancer progression, was associated with a better prognosis. Low IGF1, high chitinase activity and high MCP1 appear as promising predictive biomarkers.

Effects of three essential oil fumigation treatments on the postharvest control of Botrytis cinerea and their efficacy as preservatives of cherry tomatoes.

Cherry tomatoes (Solanum lycopersicum) are becoming increasingly popular due to their nutrition and delicious flavor. However, cherry tomatoes are highly perishable and susceptible to various pathogenic microorganisms after harvest, such as Botrytis cinerea. In the pretest experiment, we screened out 3 kinds of plant essential oils (Torreya grandis oil, Eriobotrya japonica oil, and Citrus medica oil) that have strong fungicidal activity on B. cinerea from cherry tomatoes. To further evaluate the postharvest preservation application prospect of these three oils for cherry tomatoes, these three oils were extracted from different parts of three plants by hydrodistillation, and their chemical constituents were analyzed by Gas Chromatography - Mass Spectrometry (GC-MS). The main representative components of T. grandis oil, E. japonica oil, and C. medica oil were δ-cadinene (11.76%), trans-nerolidol (9.70%), and 5,7-dimethoxycoumarin (23.22%), respectively. These three EOs effectively inhibited the mycelial growth of B. cinerea in vitro, with EC50 values of 81.672, 144.046, and 221.500 μL/L, respectively. Compared with the blank control and other oil treatments, T. grandis oil (at a concentration of 200 µL/L) fumigation treatment was more effective at inhibiting the growth rate of the pathogen. In addition, the phenolic content and phenylalanine ammonia lyase (PAL), β-1,3-glucanase, chitinase, and peroxidase (POD) activities of tomatoes significantly increased on the 7th day due to T. grandis oil treatment. The present study shows that these three oils with high extraction rates have preservation potential for cherry tomatoes. Among these three essential oils (EOs), T. grandis oil can be used to further develop preservative products as a fumigant.

Genome-Wide Identification of the NPR1-like Gene Family in Solanum tuberosum and Functional Characterization of StNPR1 in Resistance to Ralstonia solanacearum.

The NPR1 (nonexpressor of pathogenesis-related genes 1) gene is an activator of the systemic acquisition of resistance (SAR) in plants and is one of the central factors in their response to pathogenic bacterial infestation, playing an important role in plant disease resistance. Potato (Solanum tuberosum) is a crucial non-grain crop that has been extensively studied. However, the identification and analysis of the NPR1-like gene within potato have not been understood well. In this study, a total of six NPR1-like proteins were identified in potato, and phylogenetic analysis showed that the six NPR1-like proteins in Solanum tuberosum could be divided into three major groups with NPR1-related proteins from Arabidopsis thaliana and other plants. Analysis of the exon-intron patterns and protein domains of the six NPR1-like genes from potato showed that the exon-intron patterns and protein domains of the NPR1-like genes belonging to the same Arabidopsis thaliana subfamily were similar. By performing quantitative real-time PCR (qRT-PCR) analysis, we found that six NPR1-like proteins have different expression patterns in different potato tissues. In addition, the expression of three StNPR1 genes was significantly downregulated after being infected by Ralstonia solanacearum (RS), while the difference in the expression of StNPR2/3 was insignificant. We also established potato StNPR1 overexpression lines that showed a significantly increased resistance to R. solanacearum and elevated activities of chitinase, β-1,3-glucanase, and phenylalanine deaminase. Increased peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities, as well as decreased hydrogen peroxide, regulated the dynamic balance of reactive oxygen species (ROS) in the StNPR1 overexpression lines. The transgenic plants activated the expression of the genes associated with the Salicylic acid (SA) defense response but suppressed the expression of the genes associated with Jasmonic acid (JA) signaling. This resulted in resistance to Ralstonia solanacearum.

Development of a Microbial-Assisted Process for Enhanced Astaxanthin Recovery from Crab Exoskeleton Waste

Astaxanthin is a xanthophyll carotenoid possessing impressive nutraceutical, antioxidant, and bioactive merits. Traditionally, astaxanthin is extracted from crustacean wastes via solvent extraction methods. However, the rigid structure of shells that comprise complex proteins and chitin challenges the extraction process. This investigation addressed an efficient microbial-assisted method to facilitate astaxanthin recovery from crab exoskeleton waste utilizing chitinolytic and proteolytic microorganisms. Herein, we evaluated the effect of pretreatment of the exoskeleton waste with a newly isolated probiotic strain, Bacillus amyloliquefaciens CPFD8, showing remarkable protease and chitinase activity and a proteolytic Saccharomyces cerevisiae 006-001 before solvent extraction, using acetone/hexane, on astaxanthin recovery. Furthermore, the antioxidant and anti-inflammatory activities of the recovered astaxanthin were inspected. Results revealed that both strains boosted the astaxanthin yield from the crab (Callinectes sapidus) exoskeleton compared with solvent extraction using acetone/hexane. Under optimum conditions, astaxanthin yield was 217 and 91 µg/g crab exoskeleton in samples treated with B. amyloliquefaciens CPFD8 and S. cerevisiae 006-001, respectively. Interestingly, pretreatment of crab exoskeleton waste with B. amyloliquefaciens CPFD8 yielded more than 6-fold astaxanthin compared with the solvent extraction method that yielded just 35 µg/g. This increase could be attributed to the proteolytic activity of B. amyloliquefaciens CPFD8 that rendered deproteinized shell chitin accessible to chitinase, facilitating the penetration of solvents and the recovery of astaxanthin. The recovered astaxanthin exhibited excellent antioxidant activity in scavenging DPPH or ABTS free radicals with IC50 values of 50.93 and 17.56 µg/mL, respectively. In addition, the recovered astaxanthin showed a remarkable anti-inflammatory impact on LPS-induced murine macrophage RAW264.7 cells and significantly inhibited the production of nitric oxide, TNF-α, and IL-6 compared with the untreated control. These findings suggest the potential use of the developed microbial-assisted method utilizing chitinolytic and proteolytic B. amyloliquefaciens CPFD8 to maximize the recovery of bioactive astaxanthin from crab (C. sapidus) exoskeleton waste.

Localization of C Cycle Enzymes in Arable and Forest Phaeozems within Levels of Soil Microstructure.

Soil microbial and enzyme activities are closely related to the spatial variability of soil environmental conditions at the microscale (μm-mm). The origin and localization of the enzymes are somewhat neglected when the measured activity is used to evaluate specific soil functions. The activity of four hydrolytic enzymes (β-glucosidase, Cellobiohydrolase, Chitinase, Xylanase) and microbial diversity based on community-level physiological profiling were determined in samples of arable and native Phaeozems with increasing physical impact to soil solids. The level of impact on the soil solids had a significant effect on enzyme activity and depended on both the enzyme type and soil land use. The highest proportion of the activity of Xylanase and Cellobiohydrolase of arable Phaeozem was determined at the dispersion energy in the range of 450-650 J·mL-1 and was associated with the primary soil particles' hierarchy level. The highest proportions of β-glucosidase and Chitinase activities were determined for forest Phaeozem after applying energies lower than 150 J·mL-1 and characterizing the level of soil microaggregates. The increased activity of Xylanase and Cellobiohydrolase in primary soil particles of arable soil compared to those in forest soil might be a reflection of the substrates being unavailable to decomposition, leading to enzyme accumulation on the solid surface. For the Phaeozems, the lower the level of soil microstructure organization, the greater the differences observed between soils of different land use type, i.e., microbial communities, associated with lower microstructure levels, were more specific to land use type.

Two Medicago truncatula growth-promoting rhizobacteria capable of limiting in vitro growth of the Fusarium soil-borne pathogens modulate defense genes expression

Main conclusionPGPRs: P. fluorescens Ms9N and S. maltophilia Ll4 inhibit in vitro growth of three legume fungal pathogens from the genus Fusarium. One or both trigger up-regulation of some genes (CHIT, GLU, PAL, MYB, WRKY) in M. truncatula roots and leaves in response to soil inoculation.Pseudomonas fluorescens (referred to as Ms9N; GenBank accession No. MF618323, not showing chitinase activity) and Stenotrophomonas maltophilia (Ll4; GenBank accession No. MF624721, showing chitinase activity), previously identified as promoting growth rhizobacteria of Medicago truncatula, were found, during an in vitro experiment, to exert an inhibitory effect on three soil-borne fungi: Fusarium culmorum Cul-3, F. oxysporum 857 and F. oxysporum f. sp. medicaginis strain CBS 179.29, responsible for serious diseases of most legumes including M. truncatula. S. maltophilia was more active than P. fluorescens in suppressing the mycelium growth of two out of three Fusarium strains. Both bacteria showed β-1,3-glucanase activity which was about 5 times higher in P. fluorescens than in S. maltophilia. Upon soil treatment with a bacterial suspension, both bacteria, but particularly S. maltophilia, brought about up-regulation of plant genes encoding chitinases (MtCHITII, MtCHITIV, MtCHITV), glucanases (MtGLU) and phenylalanine ammonia lyases (MtPAL2, MtPAL4, MtPAL5). Moreover, the bacteria up-regulate some genes from the MYB (MtMYB74, MtMYB102) and WRKY (MtWRKY6, MtWRKY29, MtWRKY53, MtWRKY70) families which encode TFs in M. truncatula roots and leaves playing multiple roles in plants, including a defense response. The effect depended on the bacterium species and the plant organ. This study provides novel information about effects of two M. truncatula growth-promoting rhizobacteria strains and suggests that both have a potential to be candidates for PGPR inoculant products on account of their ability to inhibit in vitro growth of Fusarium directly and indirectly by up-regulation of some defense priming markers such as CHIT, GLU and PAL genes in plants. This is also the first study of the expression of some MYB and WRKY genes in roots and leaves of M. truncatula upon soil treatment with two PGPR suspensions.

Analysis of the Biological Activity and Whole Genome Sequencing of Bacillus cereus CDHWZ7 Isolated from the Rhizosphere of Lycium ruthenicum on the Tibetan Plateau

This study examined the biological activity and genome of Bacillus cereus CDHWZ7 isolated from the root of Lycium ruthenicum in the Dachaidan saline area, Haixi Prefecture, Qinghai Province, China. The results revealed that B. cereus CDHWZ7 exhibited strong inhibition activity against the pathogenic fungi Fusarium graminearum, F. acuminatum, and F. oxysporum. CDHWZ7 also demonstrated cellulose-degrading activity, nitrogen-fixing activity, and the ability to secrete indole-3-acetic acid (IAA) at 55.00 mg∙L−1. The strain CDHWZ7 can grow at a salt concentration of 3–11%, a pH range of 5–11, and a temperature of 4 °C–18 °C, and shows good salt tolerance, acid and alkaline tolerance, and low-temperature fitness. The genome of strain CDHWZ7 was sequenced using Illumina HiSeq + PacBio, revealing a circular structure of 5,648,783 bp in length, containing two intact plasmids with an average GC content of 35.2%, and a total number of 5672 encoded genes. It contained 106 tRNA genes, 42 rRNA genes, and 134 sRNA genes. A total of 137 genes were annotated as carbohydrases, with a total base length of 3,968,396,297 bp. The numbers of coding sequences assigned to the Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of Proteins, and Gene Ontology Databases were 4038, 4133, and 2160, respectively. Further analysis of the genome identified genes encoding chitinase activity, cellulases, secondary metabolites, phytohormone production, volatile compounds, nitrogen and phosphate metabolism, and resistance responses to biotic stresses (glycine betaine transporter protein, catalase, superoxide dismutase, low-affinity potassium transporter protein, cold-shock protein, heat-shock protein), as well as genes related to proliferation, stress response, and resistance to pathogenic fungi. Therefore, this study determined that strain CDHWZ7 has several excellent biological traits, such as antagonism to pathogenic fungi, nitrogen-fixation ability, cellulose-degradation ability, and IAA-production ability. The genome sequence of strain CDHWZ7 and several biodefense functional genes were also analyzed, revealing the potential use of strain CDHWZ7 in the development of biological agents.

Surface characterization and interfacial activity of chitinase chi18-5 against chitosan in langmuir monolayers

One of the challenges for producing active chitinase formulations relies on the gap between the laboratory tests and the biological scenarios where the enzyme will perform its function. In this work, we have employed different Langmuir monolayer arrays to evaluate the interfacial behavior of a recently purified recombinant chitinase, Chi18–5. We have demonstrated that two conformations exist for the chitinase at pH values close to its pI, showing very distinct structural properties at the air/aqueous interface. Enzyme activity was assessed by implementing different kinetic approaches and using a chitosan-1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixed film as organized substrate model membrane. Combining these strategies, we demonstrated that better catalytic efficiencies can be obtained for Chi18–5 at pH 5. Moreover, the chitinase activity at the air/aqueous interface can be tuned by introducing in situ pH modifications over the surrounding milieu. We also studied the changes in the topography at the mesoscale level using Brewster Angle Microscopy (BAM). We found that Chi18–5 segregated onto the chitosan domains of the membrane, showing differences in homogeneity depending on the pH imposed. Alternatively, pure Chi18–5 was tested for immobilization onto a hydrophilic activated solid support using the Langmuir-Blodgett technique. Atomic Force Microscopy (AFM) analyses showed successfully stabilization and preservation of molecular features attributed to the pH at which the enzyme deposition was performed.

Antagonistic potential of rhizobacterial isolates against fungal pathogens causing rhizome rot in turmeric.

The study aims to select potent bacterial antagonists to be used as biocontrol agents against rhizome rot disease in turmeric (Curcuma longa L.). A total of 48 bacterial isolates were isolated from the rhizosphere of turmeric. These isolates were screened for their in vitro antagonism against Fusarium solani FS-01 and Pythium aphanidermatum (ITCC 7908). Production of volatile organic compounds and chitinase activity were also performed. Among the tested isolates, two bacterial isolates (IJ2 and IJ10) showed the highest inhibitory activity against these fungal pathogens. GC/MS analysis of the crude extract produced by Pseudomonas sp. IJ2 and B. subtilis IJ10 was found to contain many bioactive compounds with antifungal and antimicrobial activities. The rhizome treatment with these isolates exhibited the lowest percent disease severity with high biocontrol efficacy against the tested pathogens. These isolates with promising antagonistic potential, therefore, can be used as biocontrol agents against rhizome rot in turmeric.

Cloning, expression, purification and biochemical characterization of the recombinant chitinase enzyme encoded by CTS2 in the budding yeast

Chitin is a polymer of β-1,4-linked N-acetylglucosamine (GlcNAc) and plays a central role in the assembly of the fungal cell wall. Chitinases are hydrolytic enzymes that break down glycosidic bonds in the chitin. Chitinases are classified into three categories, endochitinases, exochitinases and N-acetylglucosaminases, according to the manner in which the enzyme cleaves the chitin polymer. Saccharomyces cerevisiae has two chitinase-encoding genes, CTS1 and CTS2. However, whether Cts2p shows a chitinase activity remains unknown. In this study, we have cloned, expressed and purified the recombinant Cts2p protein from bacterial cells. We have demonstrated that Cts2p has a higher chitobiosidase (exochitinase) activity than endochitinase activity, but no N-acetylglucosaminase activity. The optimal temperature for the chitobiosidase activity of Cts2p is 37 °C.

Isolation and Characterization of Extracellular Chitinase Produced by Chitinolytic Bacteria Isolated from Soil Samples

Chitin is one of the most common biopolymers found in both marine and terrestrial habitats. Chitinase enzyme has gained popularity due to its diverse biotechnological applications, particularly in agriculture for the biocontrol of phytopathogenic fungi and harmful insects. In this work bacteria producing chitinase enzyme were isolated from soil and water samples. Three bacterial isolates C2, C3, and C4 were found to be Gram-negative coccobacilli while bacterial isolate C9 was found to be Gram-positive cocci. The optimum pH and temperature for all the isolates were studied. The effect of pH, temperature, substrate variation, and incubation period on enzyme activity was studied. The activity of the chitinase enzyme for all the bacterial isolates increased with an increase in temperature up to 37°C. However, a further rise in reaction temperature caused a loss of chitinase activity. Michaelis-Menten plot and Line weaver Burk plot were constructed to calculate the Km and Vmax values. Partial Purification of the chitinase enzyme was carried out and the enzyme activity, protein content, specific activity, and purification fold were estimated. It was observed that the enzyme activity of the crude sample was less than the fractionated sample. The highest enzyme activity was found to be at a 40% fraction. Of the four isolates, C2 was the best in its enzyme activity. The future prospect is the production of chitinases at an industrial scale for various biotechnological applications. Chitinase-producing isolates have the ability to produce chitinase between temperatures 22ºC and 40ºC which is the field temperature for the cultivation of most the crop, so it may be applicable to field conditions against plant pathogenic fungi which is the major problem for agricultural food production. The management of seafood waste businesses may potentially benefit from the usage of this enzyme.

Can you stomach it? Comparative transcriptomics and biochemistry of the stomachs of prickleback fishes (Stichaeidae) consuming different diets

Despite obvious whole animal, tissue level, and biochemical differences among species with different diets, dietary specialization has molecular underpinnings that are not well understood. In this project, we used comparative transcriptomics of stomach tissues to observe how prickleback fishes (Stichaeidae) achieve dietary-driven differences in stomach function. Xiphister mucosus (herbivore), X. atropurpureus (omnivore), Phytichthys chirus (omnivore) and the carnivorous Anoplarchus purpurescens were harvested from the wild, and fed omnivore and/or carnivore diets in the laboratory. Transcriptomes were sequenced for stomach tissues from two individuals per species and diet treatment. The number of shared orthologs varied with phylogenetic relatedness of the species (i.e., sister-taxa X. mucosus and X. atropurpureus shared the most expressed genes among the species), and differential expression and selection analyses are underway, although hydrogen-potassium ATPase appears to be under positive selection in some species. All four fish species strongly expressed pepsinogen and chitinase genes in their stomachs, yet biochemical activity levels (N ≥ 6 per species and diet treatment) of these two digestive enzymes matched natural diet, with the species consuming the most protein ( A. purpurescens) and chitin ( P. chirus) having the highest pepsin and chitinase activities, respectively. These activities were relatively unaltered by the laboratory diets, suggesting that activity level cannot be changed significantly through a dietary shift. Although intestinal carbohydrases typically show support for the Adaptive Modulation Hypothesis—a match among digestive enzyme activities with concentrations of ingested substrate—this may be one of the first investigations to observe such support for gastric enzymes. Thus, beyond being an important vertebrate innovation, the stomach may also provide digestive benefit depending on natural diet. NSF Grant IOS-1355224 and NSF DBI-1852096 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Profiling Plant Proteome and Transcriptome Changes during Grapevine Fanleaf Virus Infection.

Viruses can elicit varying types and severities of symptoms during plant host infection. We investigated changes in the proteome and transcriptome of Nicotiana benthamiana plants infected by grapevine fanleaf virus (GFLV) with an emphasis on vein clearing symptom development. Comparative, time-course liquid chromatography tandem mass spectrometry and 3' ribonucleic acid sequencing analyses of plants infected by two wildtype GFLV strains, one symptomatic and one asymptomatic, and their asymptomatic mutant strains carrying a single amino acid change in the RNA-dependent RNA polymerase (RdRP) were conducted to identify host biochemical pathways involved in viral symptom development. During peak vein clearing symptom display at 7 days post-inoculation (dpi), protein and gene ontologies related to immune response, gene regulation, and secondary metabolite production were overrepresented when contrasting wildtype GFLV strain GHu and mutant GHu-1EK802GPol. Prior to the onset of symptom development at 4 dpi and when symptoms faded away at 12 dpi, protein and gene ontologies related to chitinase activity, hypersensitive response, and transcriptional regulation were identified. This systems biology approach highlighted how a single amino acid of a plant viral RdRP mediates changes to the host proteome (∼1%) and transcriptome (∼8.5%) related to transient vein clearing symptoms and the network of pathways involved in the virus-host arms race.

Chitinase from Streptomyces mutabilis as an Effective Eco-friendly Biocontrol Agent.

Blood sucking parasites not only cause economic loss but also transmit numerous diseases. Dermanyssus gallinae, an obligatory blood feeding ectoparasite causes huge production loss to the poultry industry. Mosquitoes act as vector for transmitting several viral and parasitic diseases in humans. Acaricide resistance limits the control of these parasites. The present study was aimed to control the parasites using chitinase that have selective degradation of chitin, an important component in exoskeleton development. Chitinase was induced in Streptomyces mutabilis IMA8 with chitin extracted from Charybdis smithii. The enzyme showed more than 50% activity at 30-50°C and the optimum activity at 45°C. The enzyme activity of chitinase was highest at pH 7.0. The kinetic parameters Km and Vmax values of chitinase were determined by non-linear regression using Michaelis-Menten equation and its derivative Hanes-Wolf plot. The larvicidal effect of different concentrations of chitinase was evaluated against all instar larvae (I-IV) and pupae of An. stephensi and Ae. aegypti after 24h of exposure. The percentage of mortality was directly proportional to the chitinase concentration. Bioassay for miticidal activity showed that chitinase had excellent miticidal activity (LC50 = 24.2ppm) against D. gallinae. The present study suggested the usage of Streptomyces mutabilis for preparation of chitinase in mosquito and mite control.

E3 ubiquitin ligase PbrATL18 is a positive factor in pear resistance to drought and Colletotrichum fructicola infection

The Arabidopsis Tóxicos en Levadura (ATL) protein is a subfamily of the E3 ubiquitin ligases, which exists widely in plants and is extensively involved in plant growth and development. Although the ATL family has been identified in other species, such as Arabidopsis, Oryza sativa, and grapevine, few reports on pear ATL gene families have been reported. In this study, 92 PbrATL genes were identified and analyzed from the Pyrus breschneideri genome. Motif analysis and phylogenetic tree generation divided them into nine subgroups, and chromosome localization analysis showed that the 92 PbrATL genes were distributed in 16 of 17 pear chromosomes. Transcriptome data and quantitative real-time polymerase chain reaction (qRT-PCR) experiments demonstrated that PbrATL18, PbrATL41, and PbrATL88 were involved in both pear drought resistance and Colletotrichum fructicola infection. In addition, Arabidopsis thaliana overexpressing PbrATL18 showed greater resistance to drought stress than the wild type (WT), and PbrATL18-silenced pear seedlings showed greater sensitivity to drought and C. fructicola infection than the controls. PbrATL18 regulated plant resistance by regulating chitinase (CHI), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities. This study provided a reference for further exploring the functions of the PbrATL gene in drought resistance and C. fructicola infection.

Disruption of peritrophic matrix chitin metabolism and gut immune by chlorantraniliprole results in pathogenic bacterial infection in Bombyx mori

Chlorantraniliprole (CAP) is widely used in pest control, and its environmental residues affect the disease resistance of non-target insect silkworms. Studies have demonstrated that changes in gut microbial communities of insects are associated with susceptibility to pathogens. In the present study, we examined the effects of CAP exposure on the immune system and gut microbial community structure of silkworms. The results showed that after 96 h of exposure to low-concentration CAP, the peritrophic matrix (PM) of silkworm larvae was disrupted, and pathogenic bacteria invaded hemolymph. The trehalase activity in the midgut was significantly decreased, while the activities of chitinase, β-N-acetylglucosaminidase, and chitin deacetylase were increased considerably, resulting in decreased chitin content in PM. In addition, exposure to CAP reduced the expressions of key genes in the Toll, IMD, and JAK/STAT pathways, ultimately leading to the downregulation of antimicrobial peptides (AMPs) genes and alterations in the structure of the gut microbial community. Therefore, after infection with the conditional pathogen Enterobacter cloacae (E. cloacae), CAP-exposed individuals exhibited significantly lower body weight and higher mortality. These findings showed that exposure to low-concentration CAP impacted the biological defense system of silkworms, changed the gut microbial community structure, and increased silkworms' susceptibility to bacterial diseases. Collectively, these findings provided a new perspective for the safety evaluation of low-concentration CAP exposure in sericulture.