2-Hydroxy-4-methoxybenzaldehyde inhibits the growth of Aspergillus flavus via damaging cell wall,cell membrane,manipulating respiration thus creating a promising antifungal effect on corn kernels

This study investigated the antifungal activity and the potential antifungal mechanisms of 2-hydroxy-4-methoxybenzaldehyde (HMB) against Aspergillus flavus (A. flavus). The minimum inhibitory concentration (MIC) of HMB in preventing spore germination was 70 μg mL⁻¹. HMB at MIC disrupted cell wall integrity by reducing the number of septa by 86.66% (P < 0.05) in mycelia and increased cell membrane permeability by about 14-fold (P < 0.05) evidenced by propidium iodide (PI) staining. Furthermore, HMB at MIC inhibited respiration by 33.33%. These results revealed that the antifungal activity of HMB against A. flavus could be attributed to the damaged cell wall integrity, cell membrane permeability and respiration metabolism. What’s more, A. flavus was completely restrained in corn kernels due to HMB. Therefore, HMB could be applied as an effective antifungal agent.     

Enzyme Processing of Soy Flour with Minimized Protein Loss

The enzymatic treatment of defatted soy flour (SF) to reduce indigestible carbohydrates can result in undesirable protein loss. Here protein loss was minimized with quantitation of the effects of ionic strength (IS), protease activity, and SF toasting. At the enzyme processing condition (25% w/v SF, 50 °C, pH 4.8, 48 hours), protein loss increased linearly with the IS in enzyme broths (EB); e.g., contacting untoasted SF with water or heat-deactivated EB showed protein loss of 28% in water but up to 40% when IS was increased in the range of 0.04–0.19 M. Protein loss also increased with protease in EB (nondeactivated): after adjusted for IS-related loss, approximately 10% and 25% additional protein loss occurred in EB of 73 and 490–557 U/(g SF) protease, respectively. SDS-PAGE results showed that proteolysis was not extensive, mainly on β-conglycinin α'/α and glycinin acidic 37-kDa subunits; and most of the proteolytic products could be recovered by heat-induced precipitation. SF toasting effects were studied, particularly at 2-hours 160°C, with material balances, protein distributions, and monosaccharide yields in hydrolysates. Overall, protein loss was minimized to 5.2% and the conversion of carbohydrate to monomeric sugars increased to 89.2%.     

Evaluation of microbial toxins,trace elements and sensory properties of a high-theabrownins instant Pu-erh tea produced using Aspergillus tubingensis via submerged fermentation

Theabrownins (TB) are polymeric phenolic compounds associated with the multiple bioactivities of Pu-erh tea, a post-fermented Chinese dark tea. High-TB instant Pu-erh tea was produced via a novel submerged fermentation (SF) using Aspergillus tubingensis and compared with samples produced commercially via the conventional solid-state fermentation (SSF). Viable microorganisms and microbial toxins, especially aflatoxins B₁, G₁, B₂, G₂, cyclopiazonic acid, fumonisins B₁, B₂, B₃ and ochratoxin A, were below the detection limit in all samples. Fewer microbial metabolites were found in SF instant tea compared with the SSF teas. Based on an adult consuming 1 g of instant Pu-erh tea daily, the dietary intake of investigated elements was below the safe limits recommended by various authorities. Tasters viewed the instant tea infusions as very mild, smooth, mellow and full. This suggested that submerged fermentation using A. tubingensis offers a speedy and safe alternative to commercial production of instant Pu-erh tea.     

Analysis of Transglucosylation Products of Aspergillus niger α-Glucosidase that Catalyzes the Formation of α-1,2- and α-1,3-Linked Oligosaccharides

According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger . AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris ; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans . To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0–7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-β-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages.     

N-Heterocyclization in Gliotoxin Biosynthesis is Catalyzed by a Distinct Cytochrome P450 Monooxygenase

Gliotoxin and related epidithiodiketopiperazines (ETP) from diverse fungi feature highly functionalized hydroindole scaffolds with an array of medicinally and ecologically relevant activities. Mutation analysis, heterologous reconstitution, and biotransformation experiments revealed that a cytochrome P450 monooxygenase (GliF) from the human-pathogenic fungus Aspergillus fumigatus plays a key role in the formation of the complex heterocycle. In vitro assays using a biosynthetic precursor from a blocked mutant showed that GliF is specific to ETPs and catalyzes an unprecedented heterocyclization reaction that cannot be emulated with current synthetic methods. In silico analyses indicate that this rare biotransformation takes place in related ETP biosynthetic pathways.     

Mycoremediation of chlorpyrifos and lambda-cyhalothrin by two species of filamentous fungi

Chlorpyrifos and lambda-cyhalothrin are two of the most commonly used insecticides in agriculture. Their residues reach water courses, affecting aquatic fauna and subsequently human beings. Aspergillus viridinutans and Penicillium implicatum isolated from water drains at El-Giza and El-Ismailia governorates were grown in potato dextrose broth media supplemented with different concentrations of chlorpyrifos and lambda-cyhalothrin. Samples, taken at 0, 7 and 14 days, were analysed for pesticide residues by gas chromatography. The fungal species showed biodegradation potentials of 2.5, 5 and 20 ppm of chlorpyrifos and lambda-cyhalothrin. Degradation percentage reached 100% after 14 days of incubation of both pesticides (2.5 ppm) individually with the two tested fungal species. The values of degradation rate, besides the weights of dry fungal biomasses ensured that the optimum chlorpyrifosconcentration for A. viridinutans and P. implicatum was 5 ppm, while the optimum lambda-cyhalothrin concentration for A. viridinutanswas 5 ppm and 2.5 ppm for P. implicatum.     

Response of Aspergillus flavus,Aspergillus niger and Penicillium citrinum to oxidative stress induced by Fenton’s reagent

The presence of fungi in water systems represents a threat to human health. Hydrogen peroxide is known for its disinfecting properties and easy decomposition to water and oxygen. Its activity can be enhanced by the addition of iron as a catalyst, a reagent known as Fenton?s reagent. In the present study, different Fenton concentrations were investigated on the spores of Aspergillus flavus, Aspergillus niger and Penicillium citrinum at different time intervals. The results indicate that complete inactivation of spores was noticed after 60 min of exposure to both 2 and 3% H₂O₂ catalyzed by 0.025 g Fe²⁺/100 ml for A. niger, and 3% H₂O₂ catalysed by 0.075 g Fe²⁺/100 ml for P. citrinum. The activity of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), was determined in mycelial mat harvested after 7 days. Their activities were either highly increased or reached their minimum values prior to inactivation of spores.     

AstPT Catalyses both Reverse N1‐ and Regular C2 Prenylation of a Methylated Bisindolyl Benzoquinone

Prenylated bisindolyl benzoquinones exhibit interesting biological activities, such as antidiabetic or anti‐HIV activities. A number of these compounds, including asterriquinones, have been isolated from Aspergillus terreus. In this study, we identified two putative genes by genome mining, ATEG_09980 and ATEG_00702, which share high sequence similarity with the known bisindolyl benzoquinone prenyltransferase TdiB from Aspergillus nidulans. The coding sequences were cloned and overexpressed in E. coli. The overproduced recombinant proteins were purified to near homogeneity and used for enzyme assays with asterriquinone D in the presence of dimethylallyl diphosphate. HPLC analysis showed that product formation was only detected in enzyme assays with EAU29429 encoded by ATEG_09980, not in those with EAU39348 encoded by ATEG_00702. Product isolation and structure elucidation by NMR and MS analyses led to identification of N1‐reversely and C2‐regularly monoprenylated derivatives, as well as N1′,N1′′reversely, N1′‐reversely, C2′′‐regularly diprenylated derivatives. This proved that EAU29429 functions as an asterriquinone prenyltransferase (AstPT) and indicated the involvement of EAU29429 rather than EAU39348 in the biosynthesis of methylated asterriquinones. Furthermore, incubation of monoprenylated enzyme products with AstPT resulted in the formation of the diprenylated derivatives.     

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.