Immobilization of the epoxide hydrolase from Aspergillus niger

Three methods for the immobilization of the epoxide hydrolase from the fungus Aspergillus niger were tested. The highest immobilization yield (90%) and retention of activity (65%) were obtained by adsorption onto DEAE-cellulose compared to adsorption onto hydrophobic porous polypropylene and covalent linkage using Eupergit resin. The enzymatic properties of the immobilized enzyme were similar to those of the free enzyme with respect to the effect of temperature and pH on both activity and stability as well as the effect of solvent (DMF) on activity. The kinetic parameters were affected leading to lower K _M(app) and higher Vm _(app).     

Purification and characterization of a nitrilase from Aspergillus niger K10

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg^-1) at 45°C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of d-sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.     

Culture-Based Strategies for Reduction of Protease Activity in Filtrates from Aspergillus niger NRRL-3

Summary While Aspergillus strains are also being considered as potential hosts for production of extracellular heterologous proteins, the proteases produced by the host are highly problematic in that they typically modify and degrade the recombinant proteins. Culture-based approaches for minimization of protease activity in culture supernatants of Aspergillus niger NRRL-3 included reduction or elimination of peptide nitrogen in the medium, preferential use of a defined salts medium rather than a non-peptide nitrogen medium containing yeast-nitrogen base, supplementation of the medium with carboxymethylcellulose and cultivation at pH 6.5 rather than 7.5. In general, increased proteolytic activity was observed after maximum biomass was observed and biomass was declining suggesting the majority of protease activity was released by cell lysis. Carboxymethylcellulose shifted mycelial morphology from pelleted to filamentous. Mycelium lysis in the centre of pellets, with resultant release of intracellular proteases, would explain why filamentous cultures exhibited much lower proteolytic activity than pelleted cultures.     

Ultrasound effects on invertase from Aspergillus niger

Ultrasound effects on the release and activity of invertase from Aspergillus niger cultivated in a medium containing sucrose and peptone and in another with sugar-cane molasses and peptone were investigated. Irradiation was conducted for periods of 2–10 min. with waves of amplitude 20 and 40 using an ultrasound processor of 20 kHz. Product formation was determined as reducing equivalents formed by time units using 3,5-dinitrosalicylic acid. Total and specific activities of the culture supernatants were compared in the presence and absence of sonication. Both amplitudes promoted a significant increase of total invertase activity in the time periods investigated and the highest values were obtained with an amplitude of 20. Ultrasound irradiation caused cell disruption, thus releasing invertase and, after 4 min, activation of the enzyme also occurred. The best conditions for production, extraction and activation of invertase were in molasses medium containing peptone and irradiation with ultrasound waves at 20 for 8 min. This method showed high efficiency for the extraction and activation of invertase from A. niger as well as a great potential for use in industrial processes.     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of -D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V ₁/K _B , for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of -D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pK _a of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pK _a of His516 in the free reduced enzyme is 6.9.     

Induction,purification and characterisation of arabinases produced by Aspergillus niger

The induction of arabinases in Aspergillus niger N400 was studied on different simple and complex carbon sources. Sugar beet pulp was found to be an inducer of three arabinan degrading enzymes (-l-arabinofuranosidase A, -l-arabinofuranosidase B and endoarabinase). These enzymes were purified from A. niger culture fluid after growth of the fungus in medium employing sugar beet pulp as the carbon source and were characterised both physico-chemically (Mw 83 000, 67 000, 43 000 Da and, pI 3.3, 3.5 and 3.0 for -l-arabinofuranosidases A and B and endo-arabinase, respectively) and kinetically (K _m on p-nitrophenyl--l-arabinofuranoside 0.68 and 0.52 mM for -l-arabinofuranosidases A and B, resp.; K _m on sugar beet arabinan 0.24 and 3.7 g/l for -l-arabinofuranosidase B and endoarabinase, resp.). The amino acid compositions of the three enzymes were determined also. The enzymic properties were compared with those of arabinases purified from a commerical A. niger enzyme preparation. Differences were found though the kinetic data suggest considerable similarity between the enzymes from the different sources. Antibodies raised in mice against the three enzymes were found to be highly specific and no crossreactivity with other proteins present in culture filtrates was observed. A mixture of these antibodies has been used to analyze specific induction of these individual enzymes on simple and complex substrates by Western blotting.Abbreviation PNA p-nitrophenyl--l-arabinofuranoside     

Oxalic acid production from lipids by a mutant of Aspergillus niger at different pH

Crude rapeseed oil and post-refining fatty acids were used as substrates for oxalic acid production by a mutant of Aspergillus niger. Both the final concentration and the yield of the product were highest at pH 4 to 5. With a medium containing 50 g lipids l^–1, production reached a maximum of 68 g oxalic acid l^–1 after 7 d. A high yield of the product (up to 1.4 g oxalic acid g^–1 lipids consumed) was achieved with oil and fatty acids combined.     

Induced reactive oxygen species improve enzyme production from Aspergillus niger cultivation

Intracellular reactive oxygen species (iROS) induction by HOCl was used as a novel strategy to improve enzyme productivities in Aspergillus niger growing in a bioreactor. With induced iROS, the specific intracellular activities of -amylase, protease, catalase, and glucose oxidase were increased by about 170%, 250%, 320%, and 260%, respectively. The optimum specific iROS level for achieving maximum cell concentration and enzyme production was about 15 mmol g cell^–1. The type of iROS inducing the enzyme production was identified to be a derivative of the superoxide radical.     

Decolorization of structurally different textile dyes by Aspergillus niger SA1

The fungal strain, Aspergillus niger SA1, isolated from textile wastewater sludge was screened for its decolorization ability for four different textile dyes. It was initially adapted to higher concentration of dyes (10–1,000 mg l⁻¹) on solid culture medium after repeated sub-culturing. Maximum resistant level (mg l⁻¹) sustained by fungal strain against four dyes was in order of; Acid red 151 (850) > Orange II (650) > Drimarene blue K₂RL (550) > Sulfur black (500). The apparent dye removal for dyes was seen largely due to biosorption/bioadsorption into/onto the fungal biomass. Decolorization of Acid red 151, Orange II, Sulfur black and Drimarine blue K₂RL was 68.64 and 66.72, 43.23 and 44.52, 21.74 and 28.18, 39.45 and 9.33% in two different liquid media under static condition, whereas, it was 67.26, 78.08, 45.83 and 13.74% with 1.40, 1.73, 5.16 and 1.87 mg l⁻¹ of biomass production under shaking conditions respectively in 8 days. The residual amount (mg l⁻¹) of the three products (α-naphthol, sulfanilic acid and aniline) kept quite low i.e., ≤2 in case AR 151 and Or II under shaking conditions. Results clearly elucidated the role of Aspergillus niger SA1 in decolorizing/degrading structurally different dyes into basic constituents.     

Hydroxylation of 10-deoxoartemisinin to 15-hydroxy-10-deoxoartemisinin by Aspergillus niger

The microbial hydroxylation of 10-deoxoartemisinin was investigated with the aim of obtaining preparative yields of hydroxy derivatives. During 14 d at 28°C and pH 6.5 Aspergillus niger transformed 10-deoxoartemisinin (500 mg l^–1) to 15-hydroxy-10-deoxoartemisinin (26%) and 7-hydroxy-10-deoxoartemisinin (69%).     

Production of amyloglucosidase by Aspergillus niger under different cultivation regimens

Amyloglucosidase (AMG) was produced by Aspergillus niger in solid-state fermentation (SSF), submerged fermentation (SmF) and an aqueous, two-phase system of polyethyleneglycol (PEG) and salt. In SSF, a fed-batch mode of operation gave a yield of 64 U/ml compared with 44 U/ml in batch mode. Similar trends were observed for SmF, where fed-batch cultivation gave a yield of 102 U/ml compared with 66 U/ml in batch. Shorter cultivation times (66 h) were required for SmF than for SSF (96 h). In the aqueous, two-phase cultivation, the productivity and yield of AMG were both twice those in the control fermentation.M. Ramadas is with the Department of Biochemistry, Faculty of Medicine, University of Jaffna, Kokuvil, Sri Lanka. O. Holst and B. Mattiasson are with the Department of Biotechnology, Chemical Center, Lund University, Box 124, S-221 00 Lund, Sweden     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Optimization of enantioselective resolution of racemic ibuprofen by native lipase from Aspergillus niger

Resolution of (R,S)-ibuprofen (2-(4-isobutylphenyl)propionic acid) enantiomers by esterification reaction with 1-propanol in different organic solvents was studied using native Aspergillus niger lipase. The main variables controlling the process (enzyme concentration and 1-propanol:ibuprofen molar ratio) have been optimized using response surface methodology based on a five-level, two-variable central composite rotatable design, in which the selected objective function was enantioselectivity. This enzyme preparation showed preferentially catalyzes the esterification of R(−)-ibuprofen, and under optimum conditions (7% w/v of enzyme and molar ratio of 2.41:1) the enantiomeric excess of active S(+)-ibuprofen and total conversion values were 79.1 and 48.0%, respectively, and the E-value was 32, after 168 h of reaction in isooctane.     

Synthesis of pentylferulate by a feruloyl esterase from Aspergillus niger using water-in-oil microemulsions

Pentylferulate synthesis was achieved at high yields (50–60%) with Aspergillus niger feruloyl esterase using a water-in-oil microemulsion system. The initial rate of synthesis decreased by 15–20% when the water content of the microemulsion was increased from 1.8 to 2.4% (v/v), although a concomitant decrease in conversion was not observed. The enzyme stability was significantly higher in the microemulsion than in an aqueous solution.     

Invertase production by Aspergillus niger in submerged and solid-state fermentation

Three Aspergillus nigerstrains were grown in submerged and solid state fermentation systems with sucrose at 100 g l^–1. Average measurements of all strains, liquid vs solid were: final biomass (g l^–1), 11 ± 0.3 vs 34 ± 5; maximal enzyme titres (U l^–1) 1180 ± 138 vs 3663 ± 732; enzyme productivity (U l^–1h^–1) 20 ± 2 vs 87 ± 33 and enzyme yields (U/gX) 128 ± 24 vs 138 ± 72. Hence, better productivity in solid-state was due to a better mould growth.     

Influence of pectin and glucose on growth and polygalacturonase production by Aspergillus niger in solid-state cultivation

The solid-state production of endo- and exo-polygalacturonases (PG) by Aspergillus niger was studied in a media containing wheat bran, salts, and different citric pectin and/or glucose concentrations. Kinetic analysis of the process indicated that the formation of PG and the growth of A. niger are associated processes. By increasing citric pectin from 0 to 16% (w/w), the maximum A. niger concentration (X _m) was raised from 94 to 121 mg/g dry medium suggesting that pectin can be used by A. niger as a growth substrate besides its role as an inducer. With 16% (w/w) pectin, 281 U exo-PG/gdm and 152 U endo-PG/gdm were obtained. Otherwise, pectin concentrations from 20 to 30% (w/w) hindered both production and growth. A. niger concentrations of 108–113 mg/gdm were achieved in runs with glucose from 5 to 12% (w/w), whereas at 16 and 20% (w/w) glucose, lower X _m values (ca. 100 mg/gdm) were measured. The addition of glucose to the wheat bran medium, up to 10% (w/w) led to maximum endo-PG titers slightly lower than those found in the absence of glucose. Nevertheless, exo-PG formation in these media was strongly increased and activities over 370 U/gdm were achieved. The results suggest that in experiments with pectin concentrations until 16% (w/w), exo-PG production was repressed by pectin-degradation products although these same substances had favored biomass growth. When glucose concentrations over 10% (w/w) were added to the media, the maximum activities of both enzymes decreased drastically, suggesting that glucose at high concentrations also exerts a repressive effect on PG production.     

Biosorption and solubilization of copper oxychloride fungicide by Aspergillus niger and the influence of calcium

The biosorption of copper oxychloride fungicide particulates(1 m diameter), at concentrations ranging from 25 to 500 ppm active ingredient (ai), by pelleted mycelium of Aspergillus niger grown on Czapek Dox medium was evaluated. The concentration of the fungicide adsorbed to the mycelium, remaining suspended or solubilized in the medium, was determined by analysis of its copper content (CuF)using atomic absorption spectrophotometry (AAS). 2-day-old pellets exhibited highbiosorption efficiency ranging from 97 ± 1.0 to 88 ± 1.2% of the initially added fungicide concentrations, respectively, within 10 min. However, underthe same conditions, amounts of the removed fungicide by 6-day-old mycelial pellets were significantly lower and ranged from 0.5 ± 0.03 to 0.15 ± 0.01%. Scanning electron microscopy studies of 2-day-old pellets supplemented with thefungicide revealed predominant aggregations of clumps and dense particulates on the hyphal tips. The adsorbed CuF of 125 ppm ai fungicide subsequently decreased from 7.5 ± 0.5 to 2.1 ± 0.1 mol Cu (mg dry wt)^-1 after 12 h incubation. Simultaneously, the soluble portion of CuF remaining in the medium increased from 0.9 ± 0.6 to4.9 ± 0.2 mol Cu ml^-1. The presence of 50 mM CaCl₂ resulted in a decrease of the adsorbed CuF to 3.5 ± 0.5 mol Cu (mg dry wt)^-1 and solubilizedcopper in the medium increased to 5.9 ± 0.8 mol Cu ml^-1. Additionally, the cellular copper contents attained after 2 h were 0.08 ± 0.01 and 0.16 ± 0.007 mol Cu (mg dry wt)^-1 in absence and presence of calcium, respectively. The addition of calcium to glucose-starved pellets greatly increased the medium [H⁺] which was conclusively discussed in relation to Ca²⁺/H⁺ exchangecapacity of the fungal cells. These results are of potential environmental,biotechnological and agricultural importance.     

Production of pectinases by Aspergillus niger in solid state fermentation at high initial glucose concentrations

Exopectinase (exo-p) and endopectinase (endo-p) production by Aspergillus niger CH4 in solid state culture was studied at initial glucose concentrations of 100, 250, 350 and 450 g/l. The highest activity of exo-p (35 U/g) was produced at 72 and 120 h in the medium containing 100 and 250 g glucose/l, respectively. The maximum endo-p activity (9 U/g) was produced at 72 h in the medium with 250 g glucose/l. The reduction in pectinase production at 350 and 450 g/l initial glucose concentration was due neither to repression of the synthesis of the enzyme nor to the glucose consumption rate of the strain but due to a drastic drop in pH of the medium.S. Solis-Pereyra, E. Favela-Torres, M. Gutiérrez-Rojas, G. Saucedo-Castañeda and G. Viniegra-González are with the Departamento de Biotecnologia, Universidad Autónoma Metropolitana, A.P. 55-535, C.P. 09340, México D.F., México; S. Roussos is with the Laboratoire de Biotechnologie, ORSTOM, B.P. 5045, 34032, Montpellier Cedex, France, and P. Gunasekaran is with the Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625-021, India.     

Methanol utilization by a strain of Aspergillus niger: influence on the synthesis and activity of pectinases

During the production of pectinases by a strain of Aspergillus niger isolated from rotten lemons, methanol was liberated into the medium due to the cleavage of the pectin molecule used as the carbon source. The methanol was subsequently consumed by the microorganism but neither the synthesis nor the activity of pectinesterase and polygalacturonase was affected. Although not studied in detail, the mechanism involved in the utilization of methanol is similar to that described for methylotrophic yeasts.