Endophytic Aspergillus fumigatiaffinis: Novel paclitaxel production and optimization insights.

This study investigated the potential of endophytic fungi to produce paclitaxel (Taxol®), a potent anticancer compound widely employed in chemotherapy. This research aimed to identify, confirm, and characterize endophytic fungi capable of paclitaxel (PTX) production and assess their paclitaxel yield. Additionally, it aimed to investigate factors influencing paclitaxel production. A total of 100 endophytic fungal isolates were collected and identified from the roots of Artemisia judaica. Aspergillus fumigatiaffinis exhibited the highest PTX production (26.373 µg L-1) among the isolated endophytic fungi. The strain was identified as A. fumigatiaffinis (Accession No. PP235788.1). Molecular identification confirmed its novelty, representing the first report of PTX production by A. fumigatiaffinis, an endophyte of Artemisia judaica. Optimization through full factorial design of experiments (DOE) and response surface methodology (RSM) significantly enhanced PTX production to 110.23 µg L-1 from 1 g of dry weight of the fungal culture under optimal conditions of pH 8.0, 150 µg L-1 becozyme supplementation, and 18 days of fermentation in potato dextrose broth. The presence of paclitaxel was confirmed using thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry. These findings maximize the role of endophytic fungus to produce a secondary metabolite that might be able to replace the chemically produced PTX and gives an opportunity to provide a sustainable source of PTX eco-friendly at high concentrations. KEY POINTS: • Endophytic fungi, like A. fumigatiaffinis, show promise for eco-friendly paclitaxel production • Optimization strategies boost paclitaxel yield significantly, reaching 110.23 µg L -1 • Molecular identification confirms novelty, offering a sustainable PTX source.

Metabolic picture of microbial interaction: chemical crosstalk during co-cultivation between three dominant genera of bacteria and fungi in medicinal plants rhizosphere.

INTRODUCTION: Microbial communities affect several aspects of the earth's ecosystem through their metabolic interaction. The dynamics of this interaction emerge from complex multilevel networks of crosstalk. Elucidation of this interaction could help us to maintain the balance for a sustainable future. OBJECTIVES: To investigate the chemical language among highly abundant microbial genera in the rhizospheres of medicinal plants based on the metabolomic analysis at the interaction level. METHODS: Coculturing experiments involving three microbial species: Aspergillus (A), Trichoderma (T), and Bacillus (B), representing fungi (A, T) and bacteria (B), respectively. These experiments encompassed various interaction levels, including dual cultures (AB, AT, TB) and triple cultures (ATB). Metabolic profiling by LC-QTOFMS revealed the effect of interaction level on the productivity and diversity of microbial specialized metabolites. RESULTS: The ATB interaction had the richest profile, while the bacterial profile in the monoculture condition had the lowest. Two native compounds of the Aspergillus genus, aspergillic acid and the dipeptide asperopiperazine B, exhibited decreased levels in the presence of the AT interaction and were undetectable in the presence of bacteria during the interaction. Trichodermarin N and Trichodermatide D isolated from Trichoderma species exclusively detected during coexistence with bacteria (TB and ATB). These findings indicate that the presence of Bacillus activates cryptic biosynthetic gene clusters in Trichoderma. The antibacterial activity of mixed culture extracts was stronger than that of the monoculture extracts. The TB extract exhibited strong antifungal activity compared to the monoculture extract and other mixed culture treatments. CONCLUSION: The elucidation of medicinal plant microbiome interaction chemistry and its effect on the environment will also be of great interest in the context of medicinal plant health Additionally, it sheds light on the content of bioactive constituents, and facilitating the discovery of novel antimicrobials.

Enhancing soil amendment for salt stress using pretreated rice straw and cellulolytic fungi.

Rice straw breakdown is sluggish, which makes agricultural waste management difficult, however pretreatment procedures and cellulolytic fungi can address this issue. Through ITS sequencing, Chaetomium globosum C1, Aspergillus sp. F2, and Ascomycota sp. SM2 were identified from diverse sources. Ascomycota sp. SM2 exhibited the highest carboxymethyl cellulase (CMCase) activity (0.86 IU/mL) and filter-paper cellulase (FPase) activity (1.054 FPU/mL), while Aspergillus sp. F2 showed the highest CMCase activity (0.185 IU/mL) after various pretreatments of rice straw. These fungi thrived across a wide pH range, with Ascomycota sp. SM2 from pH 4 to 9, Aspergillus sp. F2, and Chaetomium globosum C1 thriving in alkaline conditions (pH 9). FTIR spectroscopy revealed significant structural changes in rice straw after enzymatic hydrolysis and solid-state fermentation, indicating lignin, cellulose, and hemicellulose degradation. Soil amendments with pretreated rice straw, cow manure, biochar, and these fungi increased root growth and soil nutrient availability, even under severe salt stress (up to 9.3 dS/m). The study emphasizes the need for a better understanding of Ascomycota sp. degradation capabilities and proposes that using cellulolytic fungus and pretreatment rice straw into soil amendments could mitigate salt-related difficulties and improve nutrient availability in salty soils.

Unveiling biosynthetic potential of an Arctic marine-derived strain Aspergillus sydowii MNP-2.

BACKGROUND: A growing number of studies have demonstrated that the polar regions have the potential to be a significant repository of microbial resources and a potential source of active ingredients. Genome mining strategy plays a key role in the discovery of bioactive secondary metabolites (SMs) from microorganisms. This work highlighted deciphering the biosynthetic potential of an Arctic marine-derived strain Aspergillus sydowii MNP-2 by a combination of whole genome analysis and antiSMASH as well as feature-based molecular networking (MN) in the Global Natural Products Social Molecular Networking (GNPS). RESULTS: In this study, a high-quality whole genome sequence of an Arctic marine strain MNP-2, with a size of 34.9 Mb was successfully obtained. Its total number of genes predicted by BRAKER software was 13,218, and that of non-coding RNAs (rRNA, sRNA, snRNA, and tRNA) predicted by using INFERNAL software was 204. AntiSMASH results indicated that strain MNP-2 harbors 56 biosynthetic gene clusters (BGCs), including 18 NRPS/NRPS-like gene clusters, 10 PKS/PKS-like gene clusters, 8 terpene synthse gene clusters, 5 indole synthase gene clusters, 10 hybrid gene clusters, and 5 fungal-RiPP gene clusters. Metabolic analyses of strain MNP-2 grown on various media using GNPS networking revealed its great potential for the biosynthesis of bioactive SMs containing a variety of heterocyclic and bridge-ring structures. For example, compound G-8 exhibited a potent anti-HIV effect with an IC50 value of 7.2 nM and an EC50 value of 0.9 nM. Compound G-6 had excellent in vitro cytotoxicities against the K562, MCF-7, Hela, DU145, U1975, SGC-7901, A549, MOLT-4, and HL60 cell lines, with IC50 values ranging from 0.10 to 3.3 µM, and showed significant anti-viral (H1N1 and H3N2) activities with IC50 values of 15.9 and 30.0 µM, respectively. CONCLUSIONS: These findings definitely improve our knowledge about the molecular biology of genus A. sydowii and would effectively unveil the biosynthetic potential of strain MNP-2 using genomics and metabolomics techniques.

Bioprospecting endophytic fungi for bioactive metabolites with seed germination promoting potentials.

There is an urgent need for new bioactive molecules with unique mechanisms of action and chemistry to address the issue of incorrect use of chemical fertilizers and pesticides, which hurts both the environment and the health of humans. In light of this, research was done for this work to isolate, identify, and evaluate the germination-promoting potential of various plant species' fungal endophytes. Zea mays L. (maize) seed germination was examined using spore suspension of 75 different endophytic strains that were identified. Three promising strains were identified through screening to possess the ability mentioned above. These strains Alternaria alternate, Aspergilus flavus, and Aspergillus terreus were isolated from the stem of Tecoma stans, Delonix regia, and Ricinus communis, respectively. The ability of the three endophytic fungal strains to produce siderophore and indole acetic acid (IAA) was also examined. Compared to both Aspergillus flavus as well as Aspergillus terreus, Alternaria alternata recorded the greatest rates of IAA, according to the data that was gathered. On CAS agar versus blue media, all three strains failed to produce siderophores. Moreover, the antioxidant and antifungal potentials of extracts from these fungi were tested against different plant pathogens. The obtained results indicated the antioxidant and antifungal activities of the three fungal strains. GC-Mass studies were carried out to determine the principal components in extracts of all three strains of fungi. The three strains' fungus extracts included both well-known and previously unidentified bioactive compounds. These results may aid in the development of novel plant growth promoters by suggesting three different fungal strains as sources of compounds that may improve seed germination. According to the study that has been given, as unexplored sources of bioactive compounds, fungal endophytes have great potential.

Miniaturized Cultivation Profiling (MATRIX)-Facilitated Discovery of Noonazines A-C and Noonaphilone A from an Australian Marine-Derived Fungus, Aspergillus noonimiae CMB-M0339.

Subjecting the Australian marine-derived fungus Aspergillus noonimiae CMB-M0339 to cultivation profiling using an innovative miniaturized 24-well plate format (MATRIX) enabled access to new examples of the rare class of 2,6-diketopiperazines, noonazines A-C (1-3), along with the known analogue coelomycin (4), as well as a new azaphilone, noonaphilone A (5). Structures were assigned to 1-5 on the basis of a detailed spectroscopic analysis, and in the case of 1-2, an X-ray crystallographic analysis. Plausible biosynthetic pathways are proposed for 1-4, involving oxidative Schiff base coupling/dimerization of a putative Phe precursor. Of note, 2 incorporates a rare meta-Tyr motif, typically only reported in a limited array of Streptomyces metabolites. Similarly, a plausible biosynthetic pathway is proposed for 5, highlighting a single point for stereo-divergence that allows for the biosynthesis of alternate antipodes, for example, the 7R noonaphilone A (5) versus the 7S deflectin 1a (6).

Secondary Metabolites with Agricultural Antagonistic Potential from Aspergillus sp. ITBBc1, a Coral-Associated Marine Fungus.

A marine-derived fungal strain, Aspergillus sp. ITBBc1, was isolated from coral collected from the South China Sea in Hainan province. Intensive chemical investigation of the fermentation extract of this strain afforded four new secondary metabolites (1-4), named megastigmanones A-C and prenylterphenyllin H, along with four known compounds (5-8). Their structures were elucidated by extensive spectroscopic analysis including one-and two-dimensional (1D and 2D) NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The modified Mosher's method was undertaken to determine the absolute configurations of new compounds. The phytotoxic activity test showed that compounds 6-8 exhibited significant antagonistic activity against the germination of Triticum aestivum L. and Oryza sativa L. seeds with a dose-dependent relationship.

Mechanism of Fumonisin Self-Resistance: Fusarium verticillioides Contains Four Fumonisin B1-Insensitive-Ceramide Synthases.

Fusarium verticillioides produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B1 (FB1) in fumonisin-producing Fusarium verticillioides than in nonproducing F. graminearum, and likewise between isolates of Aspergillus and Alternaria differing in production of sphinganine-analog toxins. It has been reported that in F. verticillioides, ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of FUM17 and FUM18 by generating a double mutant strain in a fum1 background. Nearly unchanged resistance to added FB1 was observed compared to the parental fum1 strain. A recently developed fumonisin-sensitive baker's yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast LAC1 gene, but not LAG1, increased fumonisin resistance. High-level resistance was conferred by FUM18, but not by FUM17. Likewise, strong resistance to FB1 was caused by overexpression of the presumed F. verticillioides "housekeeping" ceramide synthases CER1, CER2, and CER3, located outside the fumonisin cluster, indicating that F. verticillioides possesses a redundant set of insensitive targets as a self-resistance mechanism.

The repertoire and levels of secondary metabolites in microbial cocultures depend on the inoculation ratio: a case study involving Aspergillus terreus and Streptomyces rimosus.

OBJECTIVE: The aim of this study was to determine the influence of the inoculation volume ratio on the production of secondary metabolites in submerged cocultures of Aspergillus terreus and Streptomyces rimosus. RESULTS: The shake flask cocultures were initiated by using 23 inoculum variants that included different volumes of A. terreus and S. rimosus precultures. In addition, the axenic controls were propagated in parallel with the cocultures. UPLC‒MS analysis revealed the presence of 15 secondary metabolites, 12 of which were found both in the "A. terreus vs. S. rimosus" cocultures and axenic cultures of either A. terreus or S. rimosus. The production of the remaining 3 molecules was recorded solely in the cocultures. The repertoire and quantity of secondary metabolites were evidently dependent on the inoculation ratio. It was also noted that detecting filamentous structures resembling typical morphological forms of a given species was insufficient to predict the presence of a given metabolite. CONCLUSIONS: The modification of the inoculation ratio is an effective strategy for awakening and enhancing the production of secondary metabolites that are not biosynthesized under axenic conditions.

Aflatoxins in the rice production chain: A review on prevalence, detection, and decontamination strategies.

Rice (Oryza sativa L.) is one of the most consumed cereals that along with several important nutritional constituents typically provide more than 21% of the caloric requirements of human beings. Aflatoxins (AFs) are toxic secondary metabolites of several Aspergillus species that are prevalent in cereals, including rice. This review provides a comprehensive overview on production factors, prevalence, regulations, detection methods, and decontamination strategies for AFs in the rice production chain. The prevalence of AFs in rice is more prominent in African and Asian than in European countries. Developed nations have more stringent regulations for AFs in rice than in the developing world. The contamination level of AFs in the rice varied at different stages of rice production chain and is affected by production practices, environmental conditions comprising temperature, humidity, moisture, and water activity as well as milling operations such as de-husking, parboiling, and polishing. A range of methods including chromatographic techniques, immunochemical methods, and spectrophotometric methods have been developed, and used for monitoring AFs in rice. Chromatographic methods are the most used methods of AFs detection followed by immunochemical techniques. AFs decontamination strategies adopted worldwide involve various physical, chemical, and biological strategies, and even using plant materials. In conclusion, adopting good agricultural practices, implementing efficient AFs detection methods, and developing innovative aflatoxin decontamination strategies are imperative to ensure the safety and quality of rice for consumers.

Induced production of defensive secondary metabolites from Aspergillus fumigatiaffinis by co-culture with Aspergillus alabamensis.

Seven previously undescribed compounds, including four diketomorpholine alkaloids (1‒4), one indole diketopiperazine alkaloid (9), one chromone (10), and one benzoic acid derivative (13), and nine known compounds (5-8, 11, 12, and 14-16) were isolated from two different fungal sources. Nine of these metabolites (1-9) were obtained from a seagrass-derived Aspergillus alabamensis SYSU-6778, while the others were obtained from a mixed culture of A. alabamensis SYSU-6778 and a co-isolated fungus A. fumigatiaffinis SYSU-6786. The chemical structures of the compounds were deduced via spectroscopic techniques (including HRESIMS, 1D and 2D NMR), chemical reactions, and ECD calculations. It is worth noting that compound 10 was identified as a defensive secondary metabolite of strain SYSU-6786, produced through the induction of compound 8 under co-culture conditions. Compounds 3 and 4 possessed a naturally rare isotryptophan core. Moreover, compounds 1 and 2 exhibited potent inhibitory activities against fish pathogenic bacterium Edwardsiella ictalurid, with minimum inhibitory concentration values of 10.0 µg/mL for both compounds.

Aflatoxin biosynthesis regulators AflR and AflS: DNA binding affinity, stoichiometry, and kinetics.

Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed. The gene cluster responsible for AF biosynthesis encodes biosynthetic enzymes and pathway-specific regulators, notably AflR and AflS. While AflR, a DNA-binding protein, activates gene transcription within the cluster, AflS enhances AF production through mechanisms that are not fully understood. In this study, we developed protocols to purify recombinant AflR and AflS proteins and utilized multiple assays to characterize their interactions with DNA. Our biophysical analysis indicated that AflR and AflS form a complex. AflS exhibited no DNA-binding capability on its own but unexpectedly reduced the DNA-binding affinity of AflR. Additionally, we found that AflR achieves its binding specificity through a mechanism in which either two copies of AflR or its complex with AflS bind to target sites on DNA in a highly cooperative manner. The estimated values of the interaction parameters of AflR, AflS and DNA target sites constitute a fundamental framework against which the function and mechanisms of other AF biosynthesis regulators can be compared.

Material Composition Characteristics of Aspergillus cristatus under High Salt Stress through LC-MS Metabolomics.

Aspergillus cristatus is a crucial edible fungus used in tea fermentation. In the industrial fermentation process, the fungus experiences a low to high osmotic pressure environment. To explore the law of material metabolism changes during osmotic pressure changes, NaCl was used here to construct different osmotic pressure environments. Liquid chromatography-mass spectrometry (LC-MS) combined with multivariate analysis was performed to analyze the distribution and composition of A. cristatus under different salt concentrations. At the same time, the in vitro antioxidant activity was evaluated. The LC-MS metabolomics analysis revealed significant differences between three A. cristatus mycelium samples grown on media with and without NaCl concentrations of 8% and 18%. The contents of gibberellin A3, A124, and prostaglandin A2 related to mycelial growth and those of arabitol and fructose-1,6-diphosphate related to osmotic pressure regulation were significantly reduced at high NaCl concentrations. The biosynthesis of energy-related pantothenol and pantothenic acid and antagonism-related fluvastatin, aflatoxin, and alternariol significantly increased at high NaCl concentrations. Several antioxidant capacities of A. cristatus mycelia were directly related to osmotic pressure and exhibited a significant downward trend with an increase in environmental osmotic pressure. The aforementioned results indicate that A. cristatus adapts to changes in salt concentration by adjusting their metabolite synthesis. At the same time, a unique set of strategies was developed to cope with high salt stress, including growth restriction, osmotic pressure balance, oxidative stress response, antioxidant defense, and survival competition.

Co-production of sugars and aroma compounds from tobacco waste using biomass-degrading enzymes produced by Aspergillus brunneoviolaceus Ab-10.

Biomass-degrading enzymes produced by microorganisms have a great potential in the processing of agricultural wastes. In order to produce suitable biomass-degrading enzymes for releasing sugars and aroma compounds from tobacco scraps, the feasibility of directly using the scraps as a carbon source for enzyme production was investigated in this study. By comparative studies of ten fungal strains isolated from tobacco leaves, Aspergillus brunneoviolaceus Ab-10 was found to produce an efficient enzyme mixture for the saccharification of tobacco scraps. Proteomic analysis identified a set of plant biomass-degrading enzymes in the enzyme mixture, including amylases, hemicellulases, cellulases and pectinases. At a substrate concentration of 100 g/L and enzyme dosage of 4 mg/g, glucose of 17.6 g/L was produced from tobacco scraps using the crude enzyme produced by A. brunneoviolaceus Ab-10. In addition, the contents of 23 volatile molecules, including the aroma compounds 4-ketoisophorone and benzyl alcohol, were significantly increased after the enzymatic treatment. The results provide a strategy for valorization of tobacco waste by integrating the production of biomass-degrading enzymes into the tobacco scrap processing system.

Effects of solid-state fermentation with Aspergillus cristatus (MK346334) on the dynamics changes in the chemical and flavor profile of dark tea by HS-SPME-GC-MS, HS-GC-IMS and electronic nose.

Aspergillus cristatus, the predominant microbe of Fuzhuan brick tea (FBT), is responsible for the creation of distinctive golden flower and unique floral aroma of FBT. The present study examined the alterations in chemical and aromatic components of raw dark tea by solid-state fermentation using A. cristatus (MK346334), the strain isolated from FBT. As results, catechins, total ployphenols, total flavonoids, theaflavins, thearubigins and antioxidant activity were significantly reduced after fermentation. Moreover, 112 and 76 volatile substances were identified by HS-SPME-GC-MS and HS-GC-IMS, respectively, primarily composed of alcohols, ketones, esters and aldehydes. Furthermore, the calculation of odor activity values revealed that 19 volatile chemicals, including hexanal, heptanal, linalool and methyl salicylate, were the main contributors to the floral, fungal, woody and minty aroma of dark tea. The present research highlights the pivotal role played by the fermentation with A. cristatus in the chemical composition, antioxidant property and distinctive flavor of dark tea.

Galf-Specific Neolectins: Towards Promising Diagnostic Tools.

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.

Determination of soil phenanthrene degradation through a fungal-bacterial consortium.

Fungal-bacterial consortia enhance organic pollutant removal, but the underlying mechanisms are unclear. We used stable isotope probing (SIP) to explore the mechanism of bioaugmentation involved in polycyclic aromatic hydrocarbon (PAH) biodegradation in petroleum-contaminated soil by introducing the indigenous fungal strain Aspergillus sp. LJD-29 and the bacterial strain Pseudomonas XH-1. While each strain alone increased phenanthrene (PHE) degradation, the simultaneous addition of both strains showed no significant enhancement compared to treatment with XH-1 alone. Nonetheless, the assimilation effect of microorganisms on PHE was significantly enhanced. SIP revealed a role of XH-1 in PHE degradation, while the absence of LJD-29 in 13C-DNA indicated a supporting role. The correlations between fungal abundance, degradation efficiency, and soil extracellular enzyme activity indicated that LJD-29, while not directly involved in PHE assimilation, played a crucial role in the breakdown of PHE through extracellular enzymes, facilitating the assimilation of metabolites by bacteria. This observation was substantiated by the results of metabolite analysis. Furthermore, the combination of fungus and bacterium significantly influenced the diversity of PHE degraders. Taken together, this study highlighted the synergistic effects of fungi and bacteria in PAH degradation, revealed a new fungal-bacterial bioaugmentation mechanism and diversity of PAH-degrading microorganisms, and provided insights for in situ bioremediation of PAH-contaminated soil.IMPORTANCEThis study was performed to explore the mechanism of bioaugmentation by a fungal-bacterial consortium for phenanthrene (PHE) degradation in petroleum-contaminated soil. Using the indigenous fungal strain Aspergillus sp. LJD-29 and bacterial strain Pseudomonas XH-1, we performed stable isotope probing (SIP) to trace active PHE-degrading microorganisms. While inoculation of either organism alone significantly enhanced PHE degradation, the simultaneous addition of both strains revealed complex interactions. The efficiency plateaued, highlighting the nuanced microbial interactions. SIP identified XH-1 as the primary contributor to in situ PHE degradation, in contrast to the limited role of LJD-29. Correlations between fungal abundance, degradation efficiency, and extracellular enzyme activity underscored the pivotal role of LJD-29 in enzymatically facilitating PHE breakdown and enriching bacterial assimilation. Metabolite analysis validated this synergy, unveiling distinct biodegradation mechanisms. Furthermore, this fungal-bacterial alliance significantly impacted PHE-degrading microorganism diversity. These findings advance our understanding of fungal-bacterial bioaugmentation and microorganism diversity in polycyclic aromatic hydrocarbon (PAH) degradation as well as providing insights for theoretical guidance in the in situ bioremediation of PAH-contaminated soil.

Biochemical characterization of purified phytase produced from Aspergillus awamori AFE1 associated with the gastrointestinal tract of longhorn beetle (Cerambycidae latreille).

The need for industrially and biotechnologically significant enzymes, such as phytase, is expanding daily as a result of the increased use of these enzymes in a variety of operations, including the manufacture of food, animal feed, and poultry feed. This study sought to characterize purified phytase from A. awamori AFE1 isolated from longhorn beetle for its prospect in industrial applications. Ammonium sulfate precipitation, ion-exchange chromatography, and gel-filtration chromatography were used to purify the crude enzyme obtained from submerged fermentation using phytase-producing media, and its physicochemical characteristics were examined. The homogenous 46.8-kDa phytase showed an 8.1-fold purification and 40.7% recovery. At 70 C and pH 7, the optimum phytase activity was noted. At acidic pH 4-6 and alkaline pH 8-10, it likewise demonstrated relative activity of 88-95% and 67-88%, respectively. It showed 67-70% residual activity between 30 and 70 C after 40 min, and 68-94% residual activity between pH 2 and 12 after 2 h. The presence of Hg+, Mg2+, and Al3+ significantly decreased the enzymatic activity, whereas Ca2+ and Cu2+ enhanced it. Ascorbic acid increased the activity of the purified enzyme, whereas ethylenediaminetetraacetic acid (EDTA) and mercaptoethanol inhibited it. The calculated values for Km and Vmax were 55.4 mM and1.99 µmol/min/mL respectively. A. awamori phytase, which was isolated from a new source, showed unique and remarkable qualities that may find use in industrial operations such as feed pelleting and food processing.

Lignocellulolytic Enzymes Production by Four Wild Filamentous Fungi for Olive Stones Valorization: Comparing Three Fermentation Regimens.

Lignocellulolytic enzymes play a crucial role in efficiently converting lignocellulose into valuable platform molecules in various industries. However, they are limited by their production yields, costs, and stability. Consequently, their production by producers adapted to local environments and the choice of low-cost raw materials can address these limitations. Due to the large amounts of olive stones (OS) generated in Morocco which are still undervalued, Penicillium crustosum, Fusarium nygamai, Trichoderma capillare, and Aspergillus calidoustus, are cultivated under different fermentation techniques using this by-product as a local lignocellulosic substrate. Based on a multilevel factorial design, their potential to produce lignocellulolytic enzymes during 15 days of dark incubation was evaluated. The results revealed that P. crustosum expressed a maximum total cellulase activity of 10.9 IU/ml under sequential fermentation (SF) and 3.6 IU/ml of ß-glucosidase activity under submerged fermentation (SmF). F. nygamai recorded the best laccase activity of 9 IU/ml under solid-state fermentation (SSF). Unlike T. capillare, SF was the inducive culture for the former activity with 7.6 IU/ml. A. calidoustus produced, respectively, 1,009 µg/ml of proteins and 11.5 IU/ml of endoglucanase activity as the best results achieved. Optimum cellulase production took place after the 5th day under SF, while ligninases occurred between the 9th and the 11th days under SSF. This study reports for the first time the lignocellulolytic activities of F. nygamai and A. calidoustus. Furthermore, it underlines the potential of the four fungi as biomass decomposers for environmentally-friendly applications, emphasizing the efficiency of OS as an inducing substrate for enzyme production.

Analysis of the molecular basis for the non-amylolytic and non-proteolytic nature of Aspergillus vadensis CBS 113365.

Aspergillus vadensis CBS 113365, a close relative of A. niger, has been suggested as a more favourable alternative for recombinant protein production as it does not acidify the culture medium and produces very low levels of extracellular proteases. The aim of this study was to investigate the underlying cause of the non-amylolytic and non-proteolytic phenotype of A. vadensis CBS 113365. Our results demonstrate that the non-functionality of the amylolytic transcription factor AmyR in A. vadensis CBS 113365 is primarily attributed to the lack of functionality of its gene's promoter sequence. In contrast, a different mechanism is likely causing the lack of PrtT activity, which is the main transcriptional regulator of protease production. The findings presented here not only expand our understanding of the genetic basis behind the distinct characteristics of A. vadensis CBS 113365, but also underscore its potential as a favourable alternative for recombinant protein production.