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Frontiers in Plant Science 2024Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of...
Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized and root chitinases, and the chitinase of a symbiotic bacterium, 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and , with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that basic endochitinase (Chi19A) and chitinase (Chi19A) are from the GH19 chitinase family. The 1303 chitinase (Ch18A) belongs to the GH18 family. The three enzymes have similar apparent values of (20-40 µM) for the substrate 4-MU-GlcNAc. They vary in their pH and temperature optima with Chi19A activity optimal between pH 5-7 and 30-40°C while Chi19A and Ch18A activities were optimal at pH 7-9 and 50-60°C. Modeling and site-directed mutation of Chi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.
PubMed: 38362446
DOI: 10.3389/fpls.2024.1344142 -
Applied Biochemistry and Biotechnology Feb 2024Aspergillus niger is a species of fungus that is widely found in natural ecosystems and has an important role in various industrial fields and is readily available. To...
Aspergillus niger is a species of fungus that is widely found in natural ecosystems and has an important role in various industrial fields and is readily available. To study the adhesion of microbial cells to solid substrates and to improve their properties, physicochemical characterization of microorganisms is extremely important. For this purpose, in this study, the surface properties of A. niger biomass were determined at low cost and with high accuracy by inverse gas chromatography (IGC), a physicochemical characterization technique. IGC experiments were conducted between 303.2 and 328.2 K at infinite dilution. Among these temperatures, various organic solvent vapors were passed over the A. niger biomass considered as stationary phase and their retention behavior was studied. Using the raw data, net retention volumes were calculated and retention diagrams were drawn. From the linear retention diagrams, the dispersive surface energy was calculated according to Dorris-Gray (48.73-46.09 mJ/m), Donnet-Park (47.12-44.50 mJ/m), Schultz (46.88-42.45 mJ/m), and Hamieh (76.42-64.06 mJ/m) methods. With the IGC method, the acidity-basicity parameters of A. niger biomass were determined and it was found that the surface was basic ( ). In the second part of this study, the butyl acetate isomer series, which are difficult to be separated by conventional methods, were effectively separated by the IGC method using A. niger stationary phase.
PubMed: 38411937
DOI: 10.1007/s12010-024-04881-y -
Journal of Fungi (Basel, Switzerland) Jul 2023Chitin synthases (CHSs) are vital enzymes for the synthesis of chitin and play important and differential roles in fungal development, cell wall integrity, environmental...
Chitin synthases (CHSs) are vital enzymes for the synthesis of chitin and play important and differential roles in fungal development, cell wall integrity, environmental adaptation, virulence, and metabolism in fungi. However, except for ChsC, a class III CHS, little is known about the functions of CHSs in , an important fungus that is widely applied in the fermentation industry and food processing, as well as a spoilage fungus of food and a human pathogen. This study showed the important functions of ChsA, a class II CHS, in using multi-phenotypic and transcriptional analyses under various conditions. The deletion of led to severe defects in conidiation on different media and resulted in the formation of smaller and less compact pellets with less septa in hyphal cells during submerged fermentation. Compared with the WT, the Δ mutants exhibited less chitin content, reduced growth under the stresses of cell wall-disturbing and oxidative agents, more released protoplasts, a thicker conidial wall, decreased production of amylases, pectinases, cellulases, and malic acid, and increased citric acid production. However, Δ mutants displayed insignificant changes in their sensitivity to osmotic agents and infection ability on apple. These findings concurred with the alteration in the transcript levels and enzymatic activities of some phenotype-related genes. Conclusively, ChsA is important for cell wall integrity and mycelial morphology, and acts as a positive regulator of conidiation, cellular responses to oxidative stresses, and the production of malic acid and some enzymes, but negatively regulates the citric acid production in .
PubMed: 37623572
DOI: 10.3390/jof9080801 -
Journal, Genetic Engineering &... Dec 2023Nowadays, farmers are facing a lot of problems for the disposal of banana pseudostem waste after the harvesting of banana. Banana pseudostem is a rich source of fiber,...
BACKGROUND
Nowadays, farmers are facing a lot of problems for the disposal of banana pseudostem waste after the harvesting of banana. Banana pseudostem is a rich source of fiber, which is an alternative source of other natural and synthetic fibers. The banana fibers are biodegradable, and they are expected to be in great demand in the international market. For the textile industry, fibers were extracted using chemical and mechanical methods, but it leads to damage and affects the quality of fibers. So, this study mainly focused on biosoftening of banana pseudostem fiber using crude enzyme produced from Aspergillus niger which is one of the most predominant fungus which can synthesize industrially applicable enzymes and which can soften the surface of banana pseudostem fiber. Through this, biosoftened banana pseudostem fiber can be produced, and the disposal problem of banana pseudostem can be rectified in an eco-friendly manner.
RESULT
The present study was undertaken for the biosoftening of banana pseudostem fiber using crude enzymes isolated from fungal strain. The fungal isolates were subjected to enzyme screening such as cellulase, pectinase, chitinase, peroxidase, and polygalacturonase. The maximum production of enzyme was observed in F2 strain, and it was subjected to crude enzyme production and purification using dialysis and column chromatography. The collected best enzyme fractions were selected for the biosoftening of banana pseudostem fiber. The banana pseudostem fiber was treated with crude enzymes at the time duration of 2, 4, 24, and 48 h. After the treatment, the treated and untreated fibers were evaluated for the mechanical properties and chemical constituent's analysis. The results revealed that the chemical contents were high during 2- and 4-h-treated fibers. After that, chemical constituents were reduced due to the removal of debris by the action of enzymes. The mechanical properties such as breaking load, breaking extension, tenacity, and diameter of fiber were best in the fibers treated for 2 and 4 h. After 4 h due to the removal of chemical constituents the breaking load and tenacity, diameter will be reduced. SEM results proved that the fiber treated at 4th h showed smooth and softened fiber.
CONCLUSION
This study proved that the crude enzymes isolated from the Aspergillus niger can be effectively soften and increase the quality of banana pseudostem fiber.
PubMed: 38108900
DOI: 10.1186/s43141-023-00617-3 -
3 Biotech Nov 2023Inulinases are enzymes of great interest in the food industry, especially due to their application in the synthesis of fructose and fructo-oligosaccharides. Moreover,...
Optimization strategy for inulinase production by URM5741 and its biochemical characterization, kinetic/thermodynamic study, and application on inulin and sucrose hydrolysis.
Inulinases are enzymes of great interest in the food industry, especially due to their application in the synthesis of fructose and fructo-oligosaccharides. Moreover, some inulinases () also present invertase activity (), making them useful for sucrose hydrolysis processes. In the present study, the production of inulinase by URM5741 was evaluated and optimized using two statistical approaches. First, the composition of the cultivation medium was determined through a simplex centroid mixture design, followed by the selection of optimal fermentation conditions using the Box-Behnken design. Based on these experimental designs, the maximum activities of inulinase (16.68 U mL) and invertase (27.80 U mL) were achieved using a mixture of wheat, soy, and oat brans (5 g), along with 2.5% inulin and 40% moisture. The inulinase exhibited optimum temperature and pH of 60 °C and 4.0, respectively, displayed a high affinity for both substrates, as evidenced by very-low Michaelis constant values (1.07-1.54 mM). A relative thermostability was observed at 55-60 °C as indicated by half-lives values ( 169.06-137.27 min; 173.29-141.52 min) and -values ( 561.61-456.00 min; 575.65-470.11 min) which were further confirmed by the high activation energy (123.01 and 143.29 kJ mol). The enzyme demonstrated favorable results in terms of inulin and sucrose hydrolysis, being a maximum release of reducing sugars of 6.04 and 15.80 g L, respectively. These results indicate that the sequential statistical approach proved to be beneficial to produce inulinase by URM5741, with the obtained enzyme considered promising for long-term industrial applications.
PubMed: 37881510
DOI: 10.1007/s13205-023-03790-x -
Journal of Bioscience and Bioengineering Aug 2023CHase catalyzes chlorogenic acid (CGA) hydrolysis to yield equimolar quinic (QA) and caffeic (CA) acids, products of high value and keen industrial interest. We proposed...
CHase catalyzes chlorogenic acid (CGA) hydrolysis to yield equimolar quinic (QA) and caffeic (CA) acids, products of high value and keen industrial interest. We proposed the preparation and characterization of the nonviable mycelium of Aspergillus niger AKU 3302 containing a cell-associated CHase (CHase biocatalyst) for application in hydrolyzing the CGA from yerba mate residues to produce QA and CA. When the vegetative mycelium was heated at 55 °C for 30 min, no loss of CHase activity occurred, but vegetative mycelial growth and spore germination ended. The CHase biocatalyst did not limit mass transfer above 100 strokes min. The reaction rate increased with catalyst loading and was kinetically controlled. The CHase biocatalyst exhibited suitable biochemical properties (optimum pH 6.5 at 50 °C) and high thermal stability (remaining stable at up to 50 °C for 8 h). The cations in yerba mate extracts did not affect CHase activity. We observed no apparent loss in the activity of the CHase biocatalyst after even 11 batch cycles of continuous use. The biocatalyst retained 85% of its initial activity after 25 days of storage at pH 6.5 and 5 °C. When a yerba mate extract was passed through a glass column packed with the biocatalyst, an effective bioconversion of CGA into CA and QA occurred. CHase activity produced a natural biocatalysis with considerable operational and storage stability; which capability, being a novel biotechnological process, can be used in the bioconversion of CGA from yerba mate residues into CA and QA at a substantially reduced cost.
Topics: Aspergillus niger; Hydrolases; Hydrolysis; Ilex paraguariensis
PubMed: 37311683
DOI: 10.1016/j.jbiosc.2023.05.007 -
Sheng Wu Gong Cheng Xue Bao = Chinese... Nov 2023The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited...
The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of β-xylosidase -xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of -xyl to xylose was lower than that of β-xylosidase with poor xylose tolerance. The value of xylose inhibition constant of recombinant-expressed -xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The and towards NPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of -xyl was 65 ℃, the optimum pH was 4.0, 61% of the -xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the -xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by -xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the -xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.
Topics: Aspergillus niger; Xylose; Molecular Docking Simulation; Xylosidases; Cellulases; Tea; Hydrogen-Ion Concentration; Substrate Specificity
PubMed: 38013186
DOI: 10.13345/j.cjb.230206 -
Medical Mycology Sep 2023Aspergillus species is a widespread environmental mould that can cause aspergillosis. The purpose of this study was to investigate the antifungal susceptibility profile...
In vitro antifungal susceptibility profile and genotypic characterization of clinical Aspergillus isolates in Eastern China on behalf of Eastern China Invasive Fungi Infection Group.
Aspergillus species is a widespread environmental mould that can cause aspergillosis. The purpose of this study was to investigate the antifungal susceptibility profile and genotypic characterization of clinical Aspergillus isolates from different provinces in Eastern China. The data included the antifungal susceptibility distributions with eight common antifungal drugs, cyp51A gene mutations of triazole-resistant Aspergillus fumigatus sensu stricto, and the genotypic relationships among the A. fumigatus sensu stricto isolates based on microsatellite typing. A. fumigatus sensu lato was the most common clinical Aspergillus species (n = 252), followed by A. flavus (n = 169), A. terreus (n = 37), A. niger (n = 29), and A. nidulans (n = 4). The modal minimum effective concentration values of micafungin and anidulafungin were lower than those of caspofungin for all Aspergillus species. The in vitro efficacy of isavuconazole was similar to that of voriconazole against most Aspergillus species. Sequencing revealed cyp51A gene mutations TR34/L98H, TR34/L98H/S297T/F495I, and TR46/Y121F/T289A in four triazole-resistant A. fumigatus sensu stricto. Phylogenetic analyses using microsatellite markers of A. fumigatus sensu stricto revealed that 211 unique genotypes clustered into two clades. The data demonstrate the diversity of clinically relevant Aspergillus species in Eastern China. Routine antifungal susceptibility testing should be performed to monitor the antifungal resistance and guide clinical therapy.
Topics: Animals; Antifungal Agents; Aspergillus fumigatus; Phylogeny; Fungal Proteins; Azoles; Drug Resistance, Fungal; Aspergillus; Triazoles; Genotype; Invasive Fungal Infections; Microbial Sensitivity Tests
PubMed: 37580143
DOI: 10.1093/mmy/myad082 -
Occurrence of fungi and mycotoxins in herbal medicines and rapid detection of toxin-producing fungi.Environmental Pollution (Barking, Essex... Sep 2023Contamination from external hazardous materials may greatly influence the safety and efficacy of herbal medicines. This paper aimed to evaluate the levels of...
Contamination from external hazardous materials may greatly influence the safety and efficacy of herbal medicines. This paper aimed to evaluate the levels of contamination by mycotoxins and toxigenic fungi in herbal medicines and establish a rapid method for detecting toxin-producing fungi. Herein, 62.92%, 36.25%, and 64.17% of herbal medicines were contaminated by aflatoxins (AFs), ochratoxins, and fumonisins, respectively. Aspergillus (43.77%), Fusarium (5.17%), and Cladosporium (4.46%) were the three predominant genera. Spearman's correlation results showed that Aspergillus and Fusarium were significantly and positively correlated with mycotoxin content (R > 0.5, P < 0.05). In addition, 323 fungal strains were isolated from herbal medicines, and 20 species were identified, mainly belonging to Aspergillus and Penicillium. Analysis of potential mycotoxin-producing fungi showed that Aspergillus flavus can produce AFs, and Aspergillus ochraceus and Aspergillus niger can produce ochratoxin A (OTA). Multiplex real-time polymerase chain reaction showed that A. flavus harbored AF synthesis genes (aflR), and A. ochraceus and A. niger harbored OTA synthesis genes (aoksl). With these synthesis genes, 67.07% and 37.20% of 164 herbal medicines were positive for toxigenic genes. Furthermore, an excellent correlation was found between the above gene copies and mycotoxin content (R = 0.99). Our results confirmed the high detection rate of mycotoxins in herbal medicines and identified pivotal AF- and OTA-producing fungi. In conclusion, this paper provided the contamination status of fungi and mycotoxins in herbal medicines and established a rapid method for detecting toxigenic fungi.
Topics: Mycotoxins; Fungi; Aflatoxins; Fumonisins; Plant Extracts; Food Contamination
PubMed: 37343918
DOI: 10.1016/j.envpol.2023.122082 -
Journal of Food Science Oct 2023Tank fermentation is a novel approach to fermenting teas; however, the species of microorganisms present remain unclear. The microbial community composition of Liupao...
Tank fermentation is a novel approach to fermenting teas; however, the species of microorganisms present remain unclear. The microbial community composition of Liupao tea at various stages of tank fermentation was analyzed using high-throughput sequencing. Sphingomonas, Aquabacterium, Pelomonas, Acinetobacter, Blastobotrys, Aspergillus, Debaryomyces, and Aureobasidium were the predominant genera, which is different from pile fermentation. Fifteen genera (including Lactobacillus, Debaryomyces, Candida, Allobaculum, Flavobacterium, Caulobacter, Blastobotrys, Aspergillus, and Rasamsonia) were identified as biomarkers. PICRUSt analysis predicted that the most abundant functional genes were related to metabolism of carbohydrates, amino acids, cofactors, vitamins, and other secondary metabolites. Using the pure culture method, 283 strains were isolated at various stages of fermentation, representing 20 genera and 29 species of bacteria, and 11 genera and 18 species of fungi. Most of the dominant Sphingomonas, Staphylococcus, Aspergillus, and Blastobotrys identified by sequencing were also isolated. Of these, Sphingomonas olei, Aspergillus luchuensis, Aspergillus niger, Aspergillus aculeatus, Aspergillus amstelodami, Blastobotrys adeninivorans, Candida metapsilosis, and Candida blankii were beneficial strains that might be used to ferment Liupao tea. This study provides a basis for the development of processing technologies and utilization of microbial strains in the production of dark teas. PRACTICAL APPLICATION: Microbial diversity in tank-fermented Liupao tea was reported for the first time. 8 microorganisms were the predominant genera. The species, functions and potential risks of microorganisms was revealed. We clarified the differences between tank and pile fermentation.
PubMed: 37623914
DOI: 10.1111/1750-3841.16748