In order to discover the steroid biotransformation ability of filamentous fungus Aspergillus niger TCCC41650, we studied the fermentation of 4-androstene-3,17-dione with A. niger TCCC41650. The transformation product was purified, crystallized and determined as 16β-hydroxy-androst-4-ene-3,17-dione by X-ray single crystal diffraction method. The best fermentation condition was found to be pH 6.0, ethanol amount 2% with a substrate concentration of 1 per thousand, the transformation rate is 85.81% after 72 h. Based on the best of our knowledge, 16β-hydroxylation rarely occurs in microbial transformations of steroid. This study laid the foundation for the research of 16β-hydroxylation steroids
Androstenedione ; metabolism ; Aspergillus niger ; metabolism ; Biotransformation ; Fermentation ; Hydroxylation ; Industrial Microbiology

OBJECTIVETo study the optimal medium composition for xylanase production by Aspergillus niger XY-1 in solid-state fermentation (SSF).

METHODSStatistical methodology including the Plackett-Burman design (PBD) and the central composite design (CCD) was employed to investigate the individual crucial component of the medium that significantly affected the enzyme yield.

RESULTSFirstly, NaNO(3), yeast extract, urea, Na(2)CO(3), MgSO(4), peptone and (NH(4))(2)SO(4) were screened as the significant factors positively affecting the xylanase production by PBD. Secondly, by valuating the nitrogen sources effect, urea was proved to be the most effective and economic nitrogen source for xylanase production and used for further optimization. Finally, the CCD and response surface methodology (RSM) were applied to determine the optimal concentration of each significant variable, which included urea, Na(2)CO(3) and MgSO(4). Subsequently a second-order polynomial was determined by multiple regression analysis. The optimum values of the critical components for maximum xylanase production were obtained as follows: x(1) (urea)=0.163 (41.63 g/L), x(2) (Na(2)CO(3))=-1.68 (2.64 g/L), x(3) (MgSO(4))=1.338 (10.68 g/L) and the predicted xylanase value was 14374.6 U/g dry substrate. Using the optimized condition, xylanase production by Aspergillus niger XY-1 after 48 h fermentation reached 14637 U/g dry substrate with wheat bran in the shake flask.

CONCLUSIONBy using PBD and CCD, we obtained the optimal composition for xylanase production by Aspergillus niger XY-1 in SSF, and the results of no additional expensive medium and shortened fermentation time for higher xylanase production show the potential for industrial utilization.

In order to investigate the effects of fungal elicitor and macroporous adsorption resin on shikonin accumulation in hairy roots of arnebia euchroma (Royle) Johnst, we used spectrophotometry to determine the total naphthoquinone content of the hairy roots, by adding different volume ratio of Aspergillus niger elicitor, Aspergillus oryzae elicitor, and the macroporous resin into the M-9 liquid medium at different culture time. The results show that the total naphthoquinone content was 2.28 times higher than the control when we added mixed elicitors of Aspergillus niger and Aspergillus oryzae at the ratio of 2.5:50 in the 10th day of hairy roots cultivating. The total naphthoquinone content was 3.71 times higher than that of the control, when we added macroporous adsorption resin NKA-9. Aspergillus niger elicitor exhibited synergistic effect with Aspergillus oryzae elicitor to enhance the naphthoquinone. Also, the total naphthoquinone level was 4.17 times higher than that of the control by adding mixed fungal elicitor and macroporous adsorption resin NKA-9 in the bioreactor. Aspergillus oryzae and mixed elicitor could promote the hairy roots proliferation, and macroporous adsorption resin NKA-9 and mixed elicitor increased the total naphthoquinone content. In summary, the measure developed for Arnebia euchroma (Royle) Johnst hairy roots cultivating in bioreactors may potential for large-scale production of naphthoquinone.
Aspergillus niger ; metabolism ; Boraginaceae ; metabolism ; Fungal Proteins ; pharmacology ; Naphthoquinones ; analysis ; metabolism ; Plant Roots ; chemistry ; metabolism ; Porosity ; Resins, Synthetic ; pharmacology

Streptomyces S24 has broad spectrum resistance to the Aspergillus in food and feed, such as Aspergillus flavus, Aspergillus niger, Asperegillus alutacells and so on. We studied the adsorption and desorption properties of antifungal substance from Streptomyces S24 on macroporous resins, screened the best elution solution and also investigated some physical and chemical characters of antifungal substance by determining the antifugal activity using oxford plate assay system. According to the analysis results, AB-8 resin offered the best adsorption and desorption capacity for antifungal substance and its saturated absorption capacity was 7.0822 x 10(4) microg/g, the optimal elution solution was 85% acetone and the dynamic desorption rate could reach 93.82%. The antifungal substance was stable to heat and alkali, not sensitive to organic solvents, and sensitive to ultraviolet rays and acid. Based on its ultraviolet spectrometry, the antifungal substance was identified as heptaene macrolide antibiotic.
Adsorption ; Antifungal Agents ; chemistry ; isolation & purification ; pharmacology ; Aspergillus ; drug effects ; Aspergillus flavus ; drug effects ; Aspergillus niger ; drug effects ; Streptomyces ; chemistry ; metabolism

The influence of the physical structure of polyurethane matrix as a support in a solid state culture in tannase production and gallic acid accumulation by Aspergillus niger Aa-20 was evaluated. Three different polyurethane matrices were used as the support: continuous, semi-discontinuous and discontinuous. The highest tannase production at 2479.59 U/L during the first 12 h of culture was obtained using the discontinuous matrix. The gallic acid was accumulated at 7.64 g/L at the discontinuous matrix. The results show that the discontinuous matrix of polyurethane is better for tannase production and gallic acid accumulation in a solid state culture bioprocess than the continuous and semi-discontinuous matrices.
Aspergillus niger ; metabolism ; Bioreactors ; microbiology ; Carboxylic Ester Hydrolases ; metabolism ; Cell Culture Techniques ; methods ; Gallic Acid ; metabolism ; Polyurethanes ; chemistry

Cellulosic material is the most abundant renewable carbon source in the world. Cellulose may be hydrolyzed using cellulase to produce glucose, which can be used for production of ethanol, organic acids, and other chemicals. Cellulase is a complex enzyme containing endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91) and cellobiase (EC 3.2.1.21). The hydrolysis of natural cellulose to glucose depends on the synergism of these three components. The mostly used cellulase produced by Trichoderma reesei has high activity of endoglucanase and exoglucanase, but the activity of cellobiase is relatively low. Therefore, improving the activity of cellobiase in cellulase reaction system is the key to enhance the sacchrification yield of cellulosic resources. Aspergillus niger LORRE 012 was a high productivity strain for cellobiase production. It was found that the spores of this strain were rich in cellobiase. In this work, the cellobiase was immobilized efficiently by simply entrapping the spores into calcium alginate gels instead of immobilizing the pure cellobiase proteins. The immobilized cellobiase was quite stable, and its half-life was 38 days under pH 4.8, 50 degrees C. The thermal stability of the immobilized cellobiase was improved, and it was stable below 70 degrees C. The suitable pH range of the immobilized cellobiase was pH 3.0 - 5.0, with the optimal pH value 4.8. The Km and Vmax value of the immobilized cellobiase were 6.01 mmol/L and 7.06 mmol/min x L, respectively. In repeated batch hydrolysis processes, 50 mL of substrate (10 g/L cellobiose) and 10 mL of immobilized beads containing cellobiase were added into a 150 ml flask. After reacting at pH 4.8, 50 degrees C for several hours, the hydrolysate was harvested for assay, and the immobilized beads were used for the next batch hydrolysis with the fresh substrate. This process was repeated, and the yield of enzymatic hydrolysis kept higher than 97% during 10 batches. The continuous hydrolysis process was carried out in a column reactor (inside diameter 2.8 cm, inside height 40 cm) packed with the immobilized beads. Using 10 g/L cellobiose as substrate, the hydrolysis yield reached 98% under 0.4 h (-1) dilution rate and pH 4.8, 50 degrees C. After corncob was treated by 1% dilute acid, the cellulosic residue (100 g/L) was used as substrate, and hydrolyzed by the cellulase (15 IFPU/g substrate) from Trichoderma reesei, at pH 4.8, 50 degrees C for 48 h. The concentration of reducing sugar in the hydrolysate was only 48.50 g/L (hydrolysis yield 69.5%). When the hydrolysate was further treated by the immobilized cellobiase, the cellobiose was hydrolyzed into glucose, and the feedback inhibition caused by the cellobiose accumulation disappeared sharply. By the synergism of immobilized cellobiase and the cellulase from T. reesei left in the hydrolysate, other oligosaccharides were mostly converted to monosaccharides. At 48 h, the reducing sugar concentration was increased to 58.78 g/L, the hydrolysis yield of the corncob residue was improved to 84.2%, and the ratio of the glucose in the total reducing sugar was increased from 53.6% to 89.5%. The reducing sugars converted from corncob could be used further in the fermentation of valuable industrial products. This research results were meaningful in the conversion and utilization of renewable biomass.
Aspergillus niger ; enzymology ; Biotechnology ; Cellobiose ; metabolism ; Enzyme Stability ; Hydrogen-Ion Concentration ; Kinetics ; Temperature ; beta-Glucosidase ; chemistry ; metabolism

Using the polymmerse chain reaction (PCR), we amplified the phytase gene phyA from Pichia pastoris GS115-phyA in Aspergillus niger NRRL3135 without the signal peptide sequence and intron sequence,. Then, it was cloned into pINA1297 vector to generate a recombinant vector of pINA1297-phyA. pINA1297-phyA was linearized and transformed into Yarrowia lipolytica po1h by the lithium acetate method. The positive transformants were obtained by YNB(casa) and PPB plates, after induced in YM medium at 28 degrees C for 6 day. The activity of the expressed phytase phyA reached 636.23 U/mL. The molecular weight of the enzyme was 130 kDa measured with SDS-PAGE analysis, whereas its molecular size reduced to 51 kDa after deglycosylation which is correspond with theoretical value. The enzymatic analysis of the recombinant phytase phyA revealed its optimal pH and temperature was 5.5 and 55 degrees C, which had high activity after incubated in pH ranged from 2.0 to 8.0 for 1 h. Moreover, its activity remained 86.08% after exposure to 90 degrees C for 10 min. It also was resistant to pepsin or trypsin treatment.
6-Phytase ; biosynthesis ; genetics ; Aspergillus niger ; genetics ; metabolism ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Yarrowia ; genetics ; metabolism

Carbon-limited continuous culture was used to study the relationship between the growth of Aspergillus niger and the production of glucoamylase. The result showed that when the specific growth rate was lower than 0.068 h(-1), the production of glucoamylase was growth-associated, when the specific growth rate was higher than 0.068 h(-1), the production of glucoamylase was not growth-associated. Based on the result of continuous culture, the Monod dynamics model of glucose consumption of A. niger was constructed, Combining Herbert-Pirt equation of glucose and oxygen consumption with Luedeking-Piret equation of enzyme production, the black-box model of Aspergillus niger for enzyme production was established. The exponential fed-batch culture was designed to control the specific growth rate at 0.05 h(-1) by using this model and the highest yield for glucoamylase production by A. niger reached 0.127 g glucoamylase/g glucose. The black-box model constructed in this study successfully described the glucoamylase production by A. niger and the result of the model fitted the measured value well. The black-box model could guide the design and optimization of glucoamylase production by A. niger.
Aspergillus niger ; metabolism ; Batch Cell Culture Techniques ; Carbon ; Culture Media ; Glucan 1,4-alpha-Glucosidase ; biosynthesis ; Glucose ; Industrial Microbiology ; methods ; Oxygen

The monoamine oxidase mutant A-1 (F210V/L213C) from Aspergillus niger showed some catalytic activity on mexiletine. To futher improve its activity, the mutant was subjected to directed evolution with MegaWHOP PCR (Megaprimer PCR of Whole Plasmid) and selection employing a high-throughput agar plate-based colorimetric screen. This approach led to the identification of a mutant ep-1, which specific activity was 189% of that for A-1. The ep-1 also showed significantly improved enantioselectivity, with the E value increased from 101 to 282; its kinetic k(cat)/K(m) value increased from 0.001 51 mmol/(L x s) to 0.002 89 mmol/(L x s), suggesting that catalytic efficiency of ep-1 had been improved. The mutant showed obviously higher specific activities on 7 of all tested 11 amines substrates, and the others were comparable. Sequence analysis revealed that there was a new mutation T162A on ep-1. The molecular dynamics simulation indicated that T162A may affect the secondary structure of the substrate channel and expand the binding pocket.
Aspergillus niger ; enzymology ; Catalysis ; Kinetics ; Monoamine Oxidase ; genetics ; metabolism ; Mutation ; Polymerase Chain Reaction ; Protein Engineering ; Protein Structure, Secondary ; Substrate Specificity

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