Aspertaichamide B, a new anti-tumor prenylated indole alkaloid from the fungus Aspergillus japonicus TE-739D.

Prenylated indole alkaloids, which are mainly produced by genera Aspergillus and Penicillium, are a class of structurally intriguing specialized metabolites with remarkable biomedical interests. In this study, chemically guided isolation of the Nicotiana tabacum-derived endophytic fungus Aspergillus japonicus TE-739D yielded eight structurally diverse prenylated indole alkaloids, including an undescribed compound, namely aspertaichamide B (ATB, 1), together with seven previously discovered derivatives (compounds 2 - 8). Their chemical structures as well as the stereochemical features were determined by integrated spectroscopic analyses, including HRESIMS, NMR, NMR calculations with DP4 + probability analysis, and a comparison of the experimental ECD data with computed DFT-based quantum chemical calculations. In vitro cytotoxic effects against the gastric cancer MFC cells revealed that the new compound ATB demonstrated considerable activity. Further studies found that ATB suppressed the viability, colony formation, and migration ability of MFC cells, and induced MFC cells apoptosis in a concentration-dependent way. Moreover, ATB stimulated ROS production in MFC cells and inhibited the tumor growth in the MFC-sourced subcutaneous tumor model while not significantly reducing the weight of mice. The pharmacological results suggested that the newly discovered ATB may be a promising anti-tumor lead compound. KEY POINTS: • Eight structurally diverse prenylated indole alkaloids including a new aspertaichamide B (ATB) were isolated from the fungus Aspergillus japonicus TE-739D. • The structure of ATB was elucidated by HRESIMS, NMR, NMR calculations with DP4 + probability analysis, and ECD calculations. • ATB inhibited cell proliferation, promoted apoptosis, and increased ROS production in gastric cancer cells, and exhibited inhibitory effects on tumor growth in vivo.

Biochemical characterization of an acid-thermostable glucoamylase from Aspergillus japonicus with potential application in the paper bio-deinking.

Aspergillus species have been highlighted in enzyme production looking for industrial applications, notably, amylases are one of the most interesting enzymes. They are capable of hydrolyzing α-glycosidic linkages of starch and widely used in industrial processes to produce ethanol, glucose, and fructose syrup as well as in the textiles, detergents, and paper industries applications. In this context, this work aimed at the biochemical characterization of the glucoamylase from Aspergillus japonicus and its application in the bio-bleaching process of recycled paper. The optimum temperature and pH for the glucoamylase assay were standardized as 50°C and 5.5. After 1 h of incubation, glucoamylase retained 90% of its activity at 30-50°C. It also kept 70% of its activity in the pH range of 4.0-6.5 after an hour of incubation. The enzyme led to an increase of 30% in the relative whiteness of 10 dry grams of sulfite paper and magazine paper when applied along with commercial cellulase and 10 mM MnCl2 . In addition, after the treatments, the glucoamylase recovered activity was 30%-32%, which indicates a prolonged availability of the enzyme and can considerably curtail the redundant downstream process of the recycled paper bio-bleaching. Thus, the glucoamylase from A. japonicus has a significant role in bio-bleaching recycled paper, reducing the necessity of hard chemicals, and improving the industrial process in an interesting economic and ecological mode.

Japonamides A and B, Two New Cyclohexadepsipeptides from the Marine-Sponge-Derived Fungus Aspergillus japonicus and Their Synergistic Antifungal Activities

Two new cyclohexadepsipeptides japonamides A (1) and B (2) were isolated from the ethyl acetate extract of a marine-sponge-derived fungus Aspergillus japonicus based on molecular networking. Their structures were elucidated by comprehensive spectral analysis and their absolute configurations were confirmed by Marfey's method. Compounds 1 and 2 showed no antifungal activities against Candida albicans SC5314 measured by the broth microdilution method but exhibited prominent synergistic antifungal activities in combination with fluconazole, ketoconazole, or rapamycin. The Minimum inhibitory concentrations (MICs) of rapamycin, fluconazole, and ketoconazole were significantly decreased from 0.5 to 0.002 µM, from 0.25 to 0.063 µM, and from 0.016 to 0.002 µM, in the presence of compounds 1 or 2 at 3.125 µM, 12.5 µM, and 6.25 µM, respectively. Surprisingly, the combination of compounds 1 or 2 with rapamycin showed a strong synergistic effect, with fractional inhibitory concentration index (FICI) values of 0.03.

pH-dependent production of himeic acid A and its non-enzymatic conversions to himeic acids B and C.

The fungus Aspergillus japonicus MF275 produces himeic acid A (1), containing a 4-pyrone ring, along with its congeners, himeic acids B (2) and C (3). During culture, 1 was gradually converted to 3, the corresponding 4-pyridone derivative. A study of the relationship between the culture pH and the fungal metabolites showed that a decrease from pH 6.5 to pH 2 is essential for production of 1, while a subsequent increase to pH 5 is necessary for production of 3. In addition, we revealed that 1 was non-enzymatically converted to 3 by the incorporation of an ammonium nitrogen atom in a pH 5 buffer, and that 1 was converted to 2 at a conversion ratio of 50% during incubation in MeOH for five days.

Extracellular Secretion of Phytase from Transgenic Wheat Roots Allows Utilization of Phytate for Enhanced Phosphorus Uptake.

A significant portion of organic phosphorus comprises of phytates which are not available to wheat for uptake. Hence for enabling wheat to utilize organic phosphorus in form of phytate, transgenic wheat expressing phytase from Aspergillus japonicus under barley root-specific promoter was developed. Transgenic events were initially screened via selection media containing BASTA, followed by PCR and BASTA leaf paint assay after hardening. Out of 138 successfully regenerated To events, only 12 had complete constructs and thus further analyzed. Positive T1 transgenic plants, grown in sand, exhibited 0.08-1.77, 0.02-0.67 and 0.44-2.14 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, after 4 weeks of phosphorus stress. Based on these results, T2 generation of four best transgenic events was further analyzed which showed up to 1.32, 56.89, and 15.40 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, while in case of real-time PCR, maximum fold increase of 19.8 in gene expression was observed. Transgenic lines showed 0.01-1.18 fold increase in phosphorus efficiency along with higher phosphorus content when supplied phytate or inorganic phosphorus than control plants. Thus, this transgenic wheat may aid in reducing fertilizer utilization and enhancing wheat yield.

Purification and functional properties of a novel glucoamylase activated by manganese and lead produced by Aspergillus japonicus.

Microbial amylases are used to produce ethanol, glucose and can be applied in textiles products, detergents and other industries. This study aimed to determine the best carbon source concentration to induce the amylase production by A. japonicus, and its purification and biochemical characterization. For that, this fungus was cultivated in Khanna medium, pH 5.5, for 4 days, at 25°C, in static condition, supplemented with potato starch and maltose in different concentrations. The fungal crude enzymatic extract was purified in a unique elution in DEAE-cellulose column and the molecular mass was determined as 72kDa. The optimum temperature and pH was 65°C and 5.0, respectively. Amylase remained 75% of its activity after one hour at 50°C and was stable in the pH range 3.0-7.0. The analysis of the end-products by thin layer chromatography showed only glucose formation, which characterizes the purified enzyme as a glucoamylase. Amylopectin was the best substrate for the enzyme assay and Mn+2 and Pb+2 were good glucoamylase activators. This activation, in addition to the biochemical characteristics are important results for future biotechnological applications of this glucoamylase in the recycling and deinking process by the paper industries.

Secondary metabolites from Aspergillus japonicus CAM231, an endophytic fungus associated with Garcinia preussii.

Chemical investigation of Aspergillus japonicus CAM231, isolated from the leaf of Garcina preussii collected in Cameroon, yielded two new compounds; one pyrone derivative, hydroxy neovasinin (1) and one phenol derivative, asperolan (2), together with two known compounds neovasifurarone B (3) and variecolin (4). The structures of the two new compounds were established using intensive NMR spectroscopy and HRMS spectra in comparison with data found in literature. The structure of compound 1 was confirmed by single-crystal X-ray crystallographic analysis in combination with NOESY experiment. The new compounds were screened for their cytotoxic and antibacterial properties; however, the tested compounds displayed no significant activities.

Characteristics and Mechanisms of Biosolubilization of Rock Phosphate by Aspergillus japonicus

ABSTRACT Rock phosphate (RP) is traditionally solubilized by chemical process causing high cost and environmental pollution. To reduce process cost and protect environment, RP solubilization by Aspergillus japonicus was studied and its mechanisms were discussed. Results show that A. japonicus could effectively solubilize RP in NBRIP medium. RP solubilization by A. japonicus included direct and indirect actions of the strain on RP. Cells of A. japonicus attached rapidly to RP surface and the RP surface was seriously corroded by the strain. A. japonicus excreted multiple organic acids, and followed by a significant increase of titratable acidity and decrease of pH in the culture. A positive correlation between content of soluble phosphate and quantity of titratable acidity but a negative correlation between content of soluble phosphate and pH were observed. Results of abiotic solubilization of RP using organic and inorganic acids indicated that the release of soluble phosphate was significantly lower than that of inoculated with A. japonicus. Higher release of soluble phosphate and pH reduction achieved when using ammonium nitrogen rather than nitrate nitrogen.

Potential application of waste from castor bean (Ricinus communis L.) for production for xylanase of interest in the industry.

Xylanases activity (XY) from Aspergillus japonicus URM5620 produced by Solid-State Fermentation (SSF) of castor press cake (Ricinus communis) on different conditions of production and extraction by PEG/citrate aqueous two-phase system (ATPS) were investigated. XY production was influenced by substrate amount (5-10 g), initial moisture (15-35 %), pH (4.0-6.0) and temperature (25-35 °C), obtaining the maximum activity of 29,085 ± 1808 U g ds-1 using 5.0 g of substrate with initial moisture of 15 % at 25 °C and pH 6.0, after 120 h of fermentation. The influence of PEG molar mass (1000-8000 g mol-1), phase concentrations (PEG 20.0-24.0 % w/w and sodium citrate 15-20 % w/w) and pH (6.0-8.0) on partition coefficient, purification factor, yield and selectivity of XY were determinate. Enzyme partitioning into the PEG rich phase was favored by M PEG 8000 (g mol-1), C PEG 24 % (w/w), C C 20 % (w/w) and pH 8.0, resulting in partition coefficient of 50.78, activity yield of 268 %, 7.20-fold purification factor and selectivity of 293. A. japonicus URM5620 has a potential role in the development of a bioprocess for the XY production using low-cost media. In addition, the present study proved it is feasible to extract xylanase from SSF by adopting the one step ATPS consisting of PEG/citrate.

Biochemical properties of glycosylation and characterization of a histidine acid phosphatase (phytase) expressed in Pichia pastoris.

Phytases catalyze the cleavage of phosphate groups from phytic acid. Here, we have studied the effects of glycosylation on the properties of Aspergillus japonicus C03 phytase expressed in Pichia pastoris. The enzyme ORF of 1338 nucleotides was cloned from genomic DNA, and encoded a secreted mature protein of 446 amino acids, which included the sequence motif RHGXRX and dipeptide HD, classifying the phytase as a histidine acid phosphate. After transformation and 72h of induction, P.pastoris GS115 expressed a 75kDa protein showing 526U/mg phytase activity and 143mg/L of protein. The amino acid sequence showed 8 and 3 potential N- and O-glycosylation sites, respectively. Analysis by ESMS showed two glycoform masses of 75,467 and 72,793, which after deglycosylation decreased to 54,327 and 54,128, respectively, indicating a carbohydrate content of 27-30%. A single GlcNAc was assigned at Asn6, Asn38, Asn84, Asn99, Asn209, Asn218, Asn355 and Asn367. The recombinant phytase showed maximum activity at 50°C, a half-life of 40min, and farUVCD spectroscopy indicated a secondary structure rich in α-helix. Thermal denaturation analyses reveal the melting temperature varied from 50°C at pH 6 to a maximum of 66°C at pH 3 and pH 4.

Increase of the phytase production by Aspergillus japonicus and its biocatalyst potential on chicken feed treatment.

Phytase hydrolyzes phytic acid from the plant components of animal feed, releasing inorganic phosphorus. The phytase production by Aspergillus japonicus was optimized using Plackett-Burman designs (PBD), composite central rotational designs (CCRD), and response surface methodology from standard Czapek medium. The enzyme was applied in broiler chicken and laying hen foods. Analysis from PBD showed that KH2 PO2, MgSO4 · 7H2O, and yeast extract had significant influences on phytase secretion (p < 0.05). The best results from the CCRD experiments were obtained using (A) 0.040% KH2 PO4, (B) 0.050% MgSO4 · 7H2O, and (C) 0.040% yeast extract, enhancing in 49-53 U mg(-1) protein. The determination coefficient (R(2)) was 0.92 and Fcalc was 7.48 times greater than Flisted . Thus, the reduced coded model: Y (U mg-1) = 50.29 + 4.30A - 3.35(A)2 - 4.80(B)2 + 5.62C - 4.26(C)2 was considered predictive and statistically significant (p < 0.05). The optimized culture medium increased the phytase yield in 250%. A. japonicus phytase released high levels of Pi from broiler chicken and laying hen food. A. japonicus is an excellent phytase producer in a culture medium using inexpensive components and agricultural wastes. Therefore, these results provide sound arguments for the formulation of a low cost culture medium for phytase production.

Partial characterization of an inulinase produced by Aspergillus japonicus URM5633

Enzymes obtained by fermentation processes offer a number of advantages and have been widely researched and used throughout the world. This study aimed to partially characterise an inulinase produced from palm and cassava peel. The enzyme was produced via the solid-state fermentation of Aspergillus japonicus URM5633. The optimal temperatures were 50ºC and 55ºC, and the optimal pH values were 5.2 and 3.4 for inulinase fermentatively produced from palm and cassava peel, respectively. The thermostability measurements for inulinase produced in palm showed that the relative activity remained below 100% until 30 minutes of stability for all temperatures, but reached 106.8% at a temperature of 50ºC after 60 minutes. Inulinase from the crude extract of cassava peel was pH stable and only decreased to 55% of the maximal activity over the course of the assay, suggesting that this enzyme can be used in inulinase production and can be utilized in food industries.