Scopoletin is a coumarin compound with various biological activities including detumescence and analgesic, insecticidal, antibacterial and acaricidal effects. However, interference with scopolin and other components often leads to difficulties in purification of scopoletin with low extraction rates from plant resource. In this paper, heterologous expression of the gene encoding β-glucosidase An-bgl3 derived from Aspergillus niger were carried out. The expression product was purified and characterized with further structure-activity relationship between it and β-glucosidase analyzed. Subsequently, its ability for transforming scopolin from plant extract was studied. The results showed that the specific activity of the purified β-glucosidase An-bgl3 was 15.22 IU/mg, the apparent molecular weight was about 120 kDa. The optimum reaction temperature and pH were 55 ℃ and 4.0, respectively. Moreover, 10 mmol/L metal ions Fe²⁺ and Mn²⁺ increased the enzyme activity by 1.74-fold and 1.20-fold, respectively. A 10 mmol/L solution containing Tween-20, Tween-80 and Triton X-100 all inhibited the enzyme activity by 30%. The enzyme showed affinity towards scopolin and tolerated 10% methanol and 10% ethanol solution, respectively. The enzyme specifically hydrolyzed scopolin into scopoletin from the extract of Erycibe obtusifolia Benth with a 47.8% increase of scopoletin. This demonstrated that the β-glucosidase An-bgl3 from A. niger shows specificity on scopolin with good activities, thus providing an alternative method for increasing the extraction efficiency of scopoletin from plant material.
Aspergillus niger/genetics* ; beta-Glucosidase/chemistry* ; Scopoletin ; Polysorbates ; Coumarins

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 Aspergillus niger β-xylosidase An-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 An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The K_i value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The K_m and V_max towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-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 An-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.
Aspergillus niger/genetics* ; Xylose/metabolism* ; Molecular Docking Simulation ; Xylosidases/genetics* ; Cellulases ; Tea ; Hydrogen-Ion Concentration ; Substrate Specificity

Aims: The main aim of the study was to evaluate some methods of application of Aspergillus niger AD1 and Trichoderma hamatum T-113 for enhancing the growth and yield of wheat var. Ibaa99 in pots and field conditions. Methodology and results: Plant growth-promoting fungi (PGPF) loaded with peat moss were used at a rate of 100, 150 and 200 mL pot-1 or m-2 in filed soil; seed treatment (coating) with fungi suspension 19 × 107, soil treatment and combination of all the three methods was employed in the study. Wheat seeds were sown in pots and field plots during 2018-2019, and data regarding various growth and yield attributes were recorded. In both pot and field trials, the results revealed that the best treatments for the desired plant growth and yield attributes were peat moss 150 mL alone or in combination with soil and seed treatments. The soil physicochemical parameters were also improved after inoculation with selected fungal isolates in different application methods compared with un-inoculated control treatment in both pot and field conditions. Conclusion, significance and impact of study The PGPF play a vital role represented phytoremediation, phytostimulation and bio-fertilization. The isolates of PGPF, which were applied with peat moss at 150 mL to the pot and in the field alone or combined with seed treatment and soil application, were significantly the best effective method for improving wheat attributes.
Aspergillus niger ; Trichoderma ; Plant Growth Regulators

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.
Gene Editing ; Aspergillus niger/genetics* ; CRISPR-Cas Systems/genetics* ; Plasmids/genetics*

Aspergillus niger is a vital industrial workhouse widely used for the production of organic acids and industrial enzymes. This fungus is a crucial cell factory due to its innate tolerance to a diverse range of abiotic conditions, high production titres, robust growth during industrial scale fermentation, and status as a generally recognized as safe (GRAS) organism. Rapid development of synthetic biology and systems biology not only offer powerful approaches to unveil the molecular mechanisms of A. niger productivity, but also provide more new strategies to construct and optimize the A. niger cell factory. As a new generation of genome editing technology, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) system brings a revolutionary breakthrough in targeted genome modification for A. niger. In this review, we focus on current advances to the CRISPR/Cas genome editing toolbox, its application on gene modification and gene expression regulation in this fungal. Moreover, the future directions of CRISPR/Cas genome editing in A. niger are highlighted.
Aspergillus niger/genetics* ; CRISPR-Cas Systems/genetics* ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics* ; Gene Editing ; Genome

Aims: This present study focused on purification of fungal β-mannanase produced by Aspergillus niger USM F4 and also physicochemical characterisation of the purified enzyme. Methodology and results: The purified β-mannanase with a molecular mass of ~47.4 kDa was demonstrated on SDSPAGE gel. The enzyme signified a purification degree of 4-fold, with final specific activity of 196.42 U/mg. It reached an optimum catalytic activity at pH 4.0 and 60 °C. The thermal stability of the enzyme was up to 70 °C and maintained the 50% activity after 30 min at 80 °C. Meanwhile, the pH stability was in the range of pH 3.0-9.0 and a 30 min half-life at pH 10.0. All chemical substances manifested an inhibitory effect on purified β-mannanase, with SDS (28.16 ± 0.05% residual activity) as the strongest inhibitor, followed by cupric ion (Cu2+) (49.51 ± 0.09% residual activity). As a whole, the enzyme displayed a substrate specificity in the order of locust bean gum (LBG) > carboxymethylcellulose > soluble starch > xylan from oat spelt > α-cellulose. Its preference for LBG has generated the Km and Vmax values of 0.20 mg/mL and 9.82 U/mL, respectively. Conclusion, significance and impact of study The outcomes of our study offer potential for use at industrial scales, particularly in the oligosaccharides production that involve acid-related activity, wide-ranging temperature and pH stability.
Aspergillus niger ; beta-Mannosidase

The L(+)-form of tartaric acid (L(+)-TA) exists extensively in nature, and is widely used in the food, chemical, textile, building, and pharmaceutical industries (Su et al., 2001). The main method for L(+)-TA production is microbial transformation by cis-epoxysuccinate hydrolase (CESH), which can catalyze the asymmetric hydrolysis of cis-epoxysuccinic acid or its salts to TA or tartrate (Bao et al., 2019). Seventeen species containing CESH have been isolated so far. However, most species for L(+)-TA production have been reported from bacteria (Xuan and Feng, 2019). The only fungus isolated from soil by our lab recently, that could be used as catalyst for the process under acidic condition, is Aspergillus niger WH-2 (Bao et al., 2020). In order to find strains with new characteristics, this study attempted to isolate a new CESH source from fungi and investigate its application value.
Aspergillus niger/metabolism* ; Biomass ; Catalysis ; Fermentation ; Hydrogen-Ion Concentration ; Hydrolases/chemistry* ; Hydrolysis ; Industrial Microbiology ; Magnetic Resonance Spectroscopy ; Penicillium/metabolism* ; Phylogeny ; Soil ; Species Specificity ; Stereoisomerism ; Tartrates/chemistry* ; Temperature ; Textiles

In order to elucidate the fungal diversity and community structure in freshwater environments, numerous fungal strains were isolated from freshwater, submerged soils, twigs, dead insects, etc. Among them, the present study has focused specifically on Aspergillus, Penicillium, and Talaromyces species, which produce diverse useful metabolites in general. Twelve strains of Aspergillus isolated were identified as A. japonicus (n = 5), A. tubingensis (3), A. niger (2), and A. flavus (2), 10 strains of which belong to Aspergillus section Nigri, named black Aspergillus. Eight strains of Penicillium were identified as P. brasilianim (n = 3), P. oxalicum (2), P. crustosum (1), P. expansum (1), and P. piscarium (1). Two different strains of Talaromyces were identified as T. pinophilus and T. versatilis. Thus far, Penicillium piscarium and Talaromyces versatilis have been unrecorded in Korea, for which we provide detailed morphological and molecular characteristics.
Aspergillus ; Fresh Water ; Insects ; Korea ; Niger ; Penicillium ; Soil ; Talaromyces

OBJECTIVE: A strain of Aspergillus niger (A. niger), capable of releasing bound phenolic acids from wheat bran, was isolated. This strain was identified by gene sequence identification. The antioxidant and anti-inflammatory capacity of ferulic acid released from wheat bran by this A. niger strain (FA-WB) were evaluated. METHODS: Molecular identification techniques based on PCR analysis of specific genomic sequences were conducted; antioxidant ability was examined using oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA) assays, and erythrocyte hemolysis assays. RAW264.7 cells were used as a model to detect anti-inflammatory activity. RESULTS: The filamentous fungal isolate was identified to be A. niger. ORAC and CAA assay showed that FA-WB had better antioxidant activity than that of the ferulic acid standard. The erythrocyte hemolysis assay results suggested that FA-WB could attenuate AAPH-induced oxidative stress through inhibition of reactive oxy gen species (ROS) generation. FA-WB could significantly restore the AAPH-induced increase in intracellular antioxidant enzyme activities to normal levels as well as inhibit the intracellular malondialdehyde formation. TNF-a, IL-6, and NO levels indicated that FA-WB can inhibit the inflammation induced by lipopolysaccharide (LPS). CONCLUSION Ferulic acid released from wheat bran by a new strain of A. niger had good anti-inflammatory activity and better antioxidant ability than standard ferulic acid.
Animals ; Anti-Inflammatory Agents ; metabolism ; pharmacology ; Antioxidants ; metabolism ; pharmacology ; Aspergillus niger ; genetics ; isolation & purification ; metabolism ; Coumaric Acids ; metabolism ; pharmacology ; DNA, Fungal ; analysis ; Dietary Fiber ; microbiology ; Erythrocytes ; drug effects ; metabolism ; Fermentation ; Hep G2 Cells ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; pharmacology ; Mice ; RAW 264.7 Cells ; Sheep ; Tumor Necrosis Factor-alpha ; metabolism

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.
Animals ; Aspergillus niger ; Calibration ; Cellulase/analysis* ; Chemistry, Clinical/methods* ; Dextranase/analysis* ; Enterocolitis, Necrotizing/diagnosis* ; Equipment Design ; Flavonoids/analysis* ; Glucose/analysis* ; Glucuronic Acid/analysis* ; Glucuronidase/analysis* ; Glycoside Hydrolases/analysis* ; Hydrogen-Ion Concentration ; Linear Models ; Multienzyme Complexes/analysis* ; Plants, Medicinal ; Polygalacturonase/analysis* ; Rats ; Reproducibility of Results ; beta-Galactosidase/analysis* ; beta-Glucosidase/analysis*