Glucose oxidase biosynthesis in relation to biochemical mutations in Aspergillus niger

The production of extracellular glucose oxidase in a submerged culture by a number of auxotrophic, 2-deoxy-D-glucose resistant and protease-less mutants of Aspergillus niger was evaluated. Among the auxotrophic strains, no evident dependence was found between the kind of the nutritional requirements and the level of the glucose oxidase activity. However, the majority of auxotrophs, requiring serine or niacin, showed a higher enzyme activity (from 16 to 680%) than the parent strain. The dynamics of the glucose oxidase synthesis by the free and immobilized mycelium of the most active niacin^? mutant of A. niger was also investigated.     

Inulinase biosynthesis using immobilized mycelium of Aspergillus niger

Conidia of Aspergillus niger 20 Osm producing extracellular inulinase were immobilized on pumice stones or polyurethane sponge and used in repeated-batch processes. Some factors affecting inulinase biosynthesis by the mycelium A. niger immobilized on pumice stones were investigated. Maximal inulinase production occurred in 50 ml of medium containing 0.5 g of carrier at 30 °C, pH 6.0 and at an agitation speed of 200 rpm. This procedure enabled repeated-batch enzyme production and as many as six subsequent 24 h batches could be fermented by using the same carrier. This is the first report on inulinase biosynthesis by mycelium of A. niger immobilized on polyurethane sponge using unconventional oxygenation of culture which ensures that the dissolved oxygen concentration remains constant.     

Optimization of glucose oxidase production by Aspergillus niger using genetic-and process-engineering techniques

Wild-type Aspergillus niger NRRL-3 was transformed with multiple copies of the glucose oxidase structural gene (god). The gene was placed under the control of the gpd A promoter of A. nidulans. For more efficient secretion the -amylase signal peptide from A oryzae was inserted in front of god. Compared to the wild type, the recombinant strain NRRL-3 (GOD3-18) produced up to four times more extracellular glucose oxidase under identical culture conditions. Addition of yeast extract (2 g l^–1) to a mineral salts medium containing only glucose as carbon source increased volumetric and specific extracellular glucose oxidase activities by 130% and 50% respectively. With the same medium composition and inoculum size, volumetric and specific extracellular glucose oxidase activities increased more than ten times in bioreactor cultivations compared to shake-flask cultures.     

Location of glucose oxidase during production by Aspergillus niger

The production of the enzyme glucose oxidase by Aspergillus niger is well documented. However, its distribution within the fungal culture is less well defined. Since the enzyme location impacts significantly on enzyme recovery, this study quantifies the enzyme distribution between the extracellular fluid, cell wall, cytoplasm and slime mucilage fractions in an A. niger NRRL-3. The culture was separated into the individual fractions and the glucose oxidase activity was determined in each. The extracellular fluid contained 38% of the total activity. The remaining 62% was associated with the mycelia and was distributed between the cell wall, cytoplasm and slime mucilage in the proportions of 34, 12 and 16%, respectively. Intracellular cytoplasmic and cell wall sites were confirmed using immunocytochemical labelling of the mycelia. In the non-viable cell, the mycelial-associated enzyme was distributed between these sites, whereas in the viable cell, it was predominantly associated with the cell wall. The distribution of the enzyme activity indicates that recovery from the solids would result in a 38% loss, whereas recovery from the extracellular fluid would result in a 62% loss. The results also suggest, however, that this 62% loss could be reduced to around 34% by disintegrating the solids prior to separation due to the contribution of the enzyme in the cytoplasm and slime mucilage. This was confirmed by independently establishing the percentage activity in the liquid and solid portions of a disintegrated culture as 62 and 38%, respectively.     

Expression and overproduction of glucose oxidase in Aspergillus niger

Summary This report describes the expression of cloned glucose oxidase gene (god) in glucose-oxidase-deficient mutants (God^–) of Aspergillus niger NRRL-3, the use of this gene for the elevation of glucose oxidase (GOD) productivity in the parental strain, and the further improvement of GOD production by subjecting the transformants to nitrous acid mutagenesis.Correspondence to: F. A. Sharif     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

Optimization of an Aspergillus niger glucose oxidase production process

The purpose of the present study was to ascertain the optimal concentration of dissolved oxygen in order to maximize the intracellular glucose oxidase formation in Aspergillus niger. Cultivations performed in a 3.5 l laboratory reactor showed that a dissolved oxygen concentration at 3% of saturation at a total pressure of 1.2 bar was optimal for maximizing intracellular glucose oxidase activity. Cultivations performed at higher dissolved oxygen concentrations did not produce as much glucose oxidase as those performed at 3%, although the formation rate was high. Experiments revealed that maximal intracellular glucose oxidase formation for the A. niger strain used, is accomplished by limiting the gluconic acid production rate by means of maintaining a low dissolved oxygen concentration. Several attempts to achieve higher intracellular glucose oxidase activity were also made by manipulating the glucose concentration at a 3% dissolved oxygen concentration. However, no enhancement in glucose oxidase activity was observed.     

Effect of continuous feeding of galactose on the production of recombinant glucose oxidase using Saccharomyces cerevisiae

A recombinant strain of Saccharomyces cerevisiae harboring GOD gene originated from Aspergillus niger was used for the production of extracellular glucose oxidase. The effect of continuous galactose feeding on the induction of GAL-10 promoter was examined in a 5 l bioreactor. The highest enzyme production level (164 U cm^х) was achieved at 96 h of cultivation. The production performance was compared with the results of fed-batch cultivations carried out in the same laboratory. Continuous feeding mode was found to be less productive due to excess ethanol formation and plasmid instability.     

Glucose oxidase: natural occurrence, function, properties and industrial applications

Glucose oxidase (GOX) from Aspergillus niger is a well-characterised glycoprotein consisting of two identical 80-kDa subunits with two FAD co-enzymes bound. Both the DNA sequence and protein structure at 1.9 Ǻ have been determined and reported previously. GOX catalyses the oxidation of d-glucose (C₆H₁₂O₆) to d-gluconolactone (C₆H₁₀O₆) and hydrogen peroxide. GOX is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent. GOX is Generally Regarded As Safe, and GOX from A. niger is the basis of many industrial applications. GOX-catalysed reaction removes oxygen and generates hydrogen peroxide, a trait utilised in food preservation. GOX has also been used in baking, dry egg powder production, wine production, gluconic acid production, etc. Its electrochemical activity makes it an important component in glucose sensors and potentially in fuel cell applications. This paper will give a brief background on the natural occurrence, functions as well as the properties of glucose oxidase. A good coverage on the diverse uses of glucose oxidase in the industry is presented with a brief outline on the working principles in the various settings. Furthermore, food grade GOX preparations are relatively affordable and widely available; the readers may be encouraged to explore other potential uses of GOX. One example is that GOX-catalysed reaction generates significant amount of heat (∼200 kJ/mol), and this property has been mostly neglected in the various applications described so far.     

Glucose oxidase overproducing and negative mutants ofAspergillus niger

Summary A colony staining method was used to isolate mutants inAspergillus niger which showed altered glucose oxidase induction. The mutants were isolated under weakly or non-inducing conditions. A stable glucose-oxidase-negative mutant and a series of overproducing mutants were found. Among the overproducing mutants, different phenotypes were found with respect to glucose oxidase induction. The mutants were tested for glucose oxidase production in surface and submerged cultures, indicating a fair correspondence between those methods. From the characteristics of the mutants it can be concuded that oxygen- and carbonsource-dependent induction are mediated by different factors.     

Oxygen supply using modified Aspergillus niger mycelium and hydrogen peroxide

Summary A mycelium of Aspergillus niger was prepared by selective inactivation of glucose oxidase by formaldehyde. Oxygen supplying by hydrogen peroxide decomposed by Aspergillus niger catalase was used for cultivation of Micrococcus luteus, Bacillus amyloliquefaciens, Candida utilis and Kluyveromyces marxianus.     

Glucose oxidase ofAspergillus niger

The authors studied the effect of the various components of synthetic nutrient medium on glucose oxidase production in submerged cultivation ofAspergillus niger. It was found that the optimal glucose concentration was 3.5–6%. The only suitable source of nitrogen was nitrate nitrogen. If the medium contained ammonia nitrogen, glucose oxidase was not formed. The addition of citric acid to the medium very effectively stimulated theQ _{O 2} of the mycelium. Calcium added in the form of calcium nitrate had the same effect. A decrease in the Mg²⁺ ion concentration raised the activity of the enzyme, while inhibiting growth of the mycelium. If the initial pH was less than 4, glucose oxidase production was inhibited and did not start until the pH rose in the course of fermentation. Differences in the initial pH affected not only production of the enzyme, but also the formation of acids and the morphological appearance of the submerged mycelium. On the basis of the findings the synthetic medium for submerged cultivation ofAspergillus niger was modified, resulting in a 50–100% increase in glucose oxidase production as compared with the original medium.     

Growth Characteristics and Glucose Oxidase Production in Mutant Penicillium funiculosum Strains

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th hour of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.     

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.     

High-yield method for immobilization of enzymes

Two types of polyethylenimine-coated glass microbeads (13–44 μm) were synthesized and used for the immobilization of glucose oxidase from Aspergillus niger and catalase from A. niger and beef liver. The two types of beads were distinguishable by differences in their surface topography. Immobilizations were performed by adsorption followed by treatment with glutaraldehyde. The immobilized-enzyme activities per unit support of all of the enzymes tested were compared with and found to be superior to the immobilized activities attainable on aminopropyl-activated glass microbeads. When enzyme was present in less than saturating amounts, the coated beads were able to remove 100% of the glucose oxidase activity initially present in the immobilization solution, with 78–87% of that activity expressed on the support surface. Bound glucose oxidase was more stable to thermal inactivation than native enzyme.     

Control of Aspergillus niger infection in varieties of Arachis hypogeae L. by supplementation of zinc ions during seed germination

Three varieties of Arachis hypogeae, GG 11, GG 20 and GG 24, were compared for resistance against A. niger. GG 20 showed the least disease severity. Infection with A. niger resulted in a rapid increase in NADPH oxidase, Glutathione reductase (GR) and salicylic acid in all the three varieties, indicating hyper increase of reactive oxygen species (ROS) and activation of phenyl propanoid pathway. Ferric reducing antioxidant power value was found to be decreasing due to infection in all the three varieties, confirming the role of ROS in pathogenesis. Since A. niger was found to cause pathogenesis by oxidative stress, the treatment of zinc was given as an antioxidant and its effect was studied. The application of zinc inhibited NADPH oxidase and GR activity in the control as well as in the infected GG 11 and GG 24 varieties but induced in the tolerant variety GG 20. Because zinc treatment could control the ROS in GG 11 and GG 24 varieties, disease severity was reduced but in GG 20 variety, zinc treatment aggravated ROS levels and also the disease severity. The protein profile of GG 20 in comparison to GG 11 and GG 24 varieties revealed one oligomeric protein of 110 kD as one of the responsible factors for its resistance. Total oil and its iodine value were found little higher in GG 20 variety than in other two varieties. It was found that the control of ROS could control the A. niger infection in Arachis hypogeae.     

Aspergillus niger mutants with increased glucose oxidase production

Summary Aspergillus niger NRRL-3, an organism used for the industrial scale production of d-gluconic acid and glucose oxidase (EC 1.1.3.4), was subjected to mutagenesis and selection for acid production on diagnostic media containing methyl red. The plates contained 0.1 M d-glucose, a concentration that does not produce a color change in the medium surrounding mycelia of the parental strain under the conditions employed. Mutagenized spores yielded occasional colonies which were able to grow rapidly and were surrounded by a reddish zone. A number of such presumptive mutants were selected and isolated. Twenty-six such strains were grown in shaken cultures with liquid media containing 0.01, 0.1 or 0.5 M d-glucose, harvested, disrupted and the specific activity of d-glucose oxidase determined. Seven of the mutant strains had glucose oxidase specific activities markedly higher than the parental strain.Paper No. 8393, Nebraska Agricultural Research Division.     

Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger

A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l⁻¹ for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l⁻¹.     

The role of aeration and agitation in the production of glucose oxidase in submerged culture. II

The main purpose of the work reported here was to establish the effectiveness of aeration and agitation, and to determine the best conditions of aeration for the growth and production of glucose oxidase of Aspergillus niger, on a semi-industrial scale. Concentration of dissolved O₂, O₂ consumption and CO₂ production were measured. It was found that the rate of growth and the activity of glucose oxidase per gram mycelium increased with the increase of speed of agitation. The concentration of dissolved oxygen of the fermentation broth, as well as the rate of respiration (O₂ consumption and CO₂ production) increased in direct proportion to the increase of speed of agitation, while assimilation of sugars was accelerated. The values of the respiratory ratio showed a fluctuation according to the presence or absence of sugar in the medium.