Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris

We proposed a yeast transformant cell incorporating the Aspergillus niger glucose oxidase gene (GOX gene), which is capable of constitutively as well as secretory expression. The GOX gene has been cloned in this study. This conclusion is based on the following: first, the ligated DNA determined by electrophoresis, was a 1489-1882bp fragment, close to the size of glucose oxidase (GOD), which is 1818bp. Secondly, the single open reading frame encoded a protein of 605 amino acids. Thirdly, secreted GOD recombinant proteins in the culture supernatants of the GOX gene transformant migrated as a single band in SDS-PAGE with an apparent molecular mass of between 75,000 and 100,000 Da, which is glycosylated GOD by the Pichia pastoris X-33 host machinery during the secretion process. Finally, the clones were cultured and secreted a protein, which possessed the GOD activity of catalyzing beta-d-glucose oxidation. With regard to the pH characteristics, the activity was more than 80% of the maximum activity in the range between pH 5 and pH 7. As for the temperature characteristics, the activity was not less than 92% of the maximum in the temperature range between 10 and 45 degrees C. The GOX gene transformant was able to maintain the GOD enzyme activity and produce recombinant GOD continuously for at least 2 weeks.     

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 beta-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, V1/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 beta-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 pKa of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pKa of His516 in the free reduced enzyme is 6.9.     

Co-immobilization of manganese peroxidase from Phlebia radiata and glucose oxidase from Aspergillus niger on porous silica beads

Manganese peroxidase (MnP) from Phlebia radiata and glucose oxidase from Aspergillus niger were co-immobilized on porous silica beads. Immobilization of both enzymes on the same carrier provided an integrated system in which H₂O₂ required by MnP was produced by glucose oxidase. The immobilization process resulted in a decrease of both enzymatic activities and substrate affinities. However, immobilization improved the stability of MnP against H₂O₂ or high pH, as well as the storage stability of this enzyme.     

Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production

Various strains of Aspergillus niger were screened for extracellular glucose oxidase (GOD) activity. The most effective producer, strain FS-3 (15.9 U mL^–1), was mutagenized using UV-irradiation or ethyl methane sulfonate. Of the 400 mutants obtained, 32 were found to be resistant to 2-deoxy d-glucose, and 17 of these exhibited higher GOD activities (from 114.5 to 332.1%) than the original FS-3 strain. Following determination of antifungal resistance of the highest producing mutants, four mutants were selected and used in protoplast fusions in three different intraspecific crosses. All fusants showed higher activities (from 285.5 to 394.2%) than the original strain. Moreover, of the 30 fusants isolated, 19 showed higher GOD activity than their corresponding higher-producing parent strain.     

Assay for glucose oxidase from Aspergillus niger and Penicillium amagasakiense by Fourier transform infrared spectroscopy

A simple and direct assay method for glucose oxidase (EC 1.1.3.4) from Aspergillus niger and Penicillium amagasakiense was investigated by Fourier transform infrared spectroscopy. This enzyme catalyzed the oxidation of d-glucose at carbon 1 into d-glucono-1,5-lactone and hydrogen peroxide in phosphate buffer in deuterium oxide ((2)H(2)O). The intensity of the d-glucono-1,5-lactone band maximum at 1212 cm(-1) due to CO stretching vibration was measured as a function of time to study the kinetics of d-glucose oxidation. The extinction coefficient epsilon of d-glucono-1,5-lactone was determined to be 1.28 mM(-1)cm(-1). The initial velocity is proportional to the enzyme concentration by using glucose oxidase from both A. niger and P. amagasakiense either as cell-free extracts or as purified enzyme preparations. The kinetic constants (V(max), K(m), k(cat), and k(cat)/K(m)) determined by Lineweaver-Burk plot were 433.78+/-59.87U mg(-1) protein, 10.07+/-1.75 mM, 1095.07+/-151.19s(-1), and 108.74 s(-1)mM(-1), respectively. These data are in agreement with the results obtained by a spectrophotometric method using a linked assay based on horseradish peroxidase in aqueous media: 470.36+/-42.83U mg(-1) protein, 6.47+/-0.85 mM, 1187.77+/-108.16s(-1), and 183.58 s(-1)mM(-1) for V(max), K(m), k(cat), and k(cat)/K(m), respectively. Therefore, this spectroscopic method is highly suited to assay for glucose oxidase activity and its kinetic parameters by using either cell-free extracts or purified enzyme preparations with an additional advantage of performing a real-time measurement of glucose oxidase activity.     

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).     

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.     

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.     

Involvement of NADPH oxidase in hydrogen peroxide accumulation by Aspergillus niger elicitor-induced Taxus chinensis cell cultures

After determining that hydrogen peroxide (H2O2) accumulation induced by a fungal elicitor from Aspergillus niger was from the superoxide dismutase-catalyzed dismutation of superoxide radical, the site of H2O2 generation in cell suspension cultures of Taxus chinensis was studied. The results showed that 90% and 10% of the elicitor-induced H2O2 accumulation respectively appeared in intracellular and extracellular fractions of cells, and that the elicitor-induced H2O2 accumulation in protoplasts and plasma membranes was similar to that in intact cells, indicating that the site of H2O2 accumulation was plasma membranes but not in extracellular fraction of Taxus cells. The H2O2 forming enzyme was also investigated. The elicitor-induced H2O2 accumulation in intact cells was not changed by loss of apoplastic peroxidase (POD) by the washing, and the H2O2 accumulation in plasma membranes was inhibited by the mammalian neutrophil NAD(P)H oxidase inhibitor diphenylene iodonium (DPI), but was slightly affected by exogenous POD and its inhibitor. Furthermore, in plasma membranes, the H2O2 accumulation was more significantly enhanced by NADPH than by NADH, and the former was more obviously decreased by DPI than the latter. The present results show that NADPH oxidase in plasma membranes is involved in H2O2 accumulation in fungal elicitor-induced Taxus chinensis cell cultures.     

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.     

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.     

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.     

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.     

Expression of recombinant Aspergillus niger xylanase A in Pichia pastoris and its action on xylan

The mature peptide of Aspergillus niger xylanase A (AnxA) was successfully expressed in Pichia pastoris at high levels under the control of AOX1 promoter. The recombinant AnxA (reAnxA) was secreted into culture medium. After 96-h 0.25% methanol induction, the activity of reAnxA in the culture supernatant reached the peak, 175 U/mg, which was 1.9 times as high as that of the native AnxA (92 U/mg). Studies on enzymatic properties showed that the optimum temperature and optimum pH of reAnxA were 50 degrees C and 5.0, respectively. The reAnxA was very stable in a wide pH range of 3.0-8.0. After incubation at the pH 3.0-8.0, 25 degrees C for 1h, all the residual activities of reAnxA were over 80%. The K(m) and k(cat) values for reAnxA were 4.8 mg/ml and 123.2s(-1), respectively. HPLC analysis showed that xylotriose was the main hydrolysis product of birchwood xylan and bran insoluble xylan by reAnxA.     

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.     

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.