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.     

Glucose oxidase ofAspergillus niger

По методу Raper и Fennel (1953) приготовляли гетерока риотический мицелий двух различных по генотипу штаммов Aspergillus niger. Штаммы, изолированные из одного конидиофора в гетерока риотическом валике, не были однородными ни по своим морфологическим свойствам, ни способности к продукции. Повышенная продукция глюкозооксидазы была временной и в третьей генерации спор уже не наблюдалась. Причиной зтого явления было, по-видимомы, биохимическое взаимодействие между ядром и цитоплазмой двуя различных щтаммов, прекращающееся после восстановления равновесия в следущей генерасии спор.     

Arginine and proline genes ofAspergillus niger

Summary Aspergillus niger mutants defective in arginine or proline biosynthesis have been isolated and 12 genetic loci were identified. Mutation was induced by low doses UV, and mutants were isolated after filtration enrichment. The mutants were classified according to their phenotype in growth tests and were further characterized in complementation tests. The arginine auxotrophic mutants represent nine complementation groups. Three additional complementation groups were found for mutants that could grow on proline (two of them on arginine too). Linkage group analysis was done in somatic diploids obtained from a mutant and a master strain with genetic markers on six chromosomes. Thearg genes belong to six different linkage groups and thepro genes to two. Onearg-mutant could be complemented by transformation with theA. nidulans arg B ⁺ gene, and thisA. niger gene thus appeared to be homologous to theA. nidulans arg B. We isolated anA. niger strain with theargB gene tightly linked with thenicA1 marker. This strain is very suitable as acceptor for transformation with anargB-plasmid, because transformants with inserts on the homologous site can be recognized and analyzed genetically using thenicA1 marker gene.     

Inactivation of glucosyltransferase ofAspergillus niger with subtilisin

Summary Treatment of amyloglucosidase ofAspergillus niger with subtilisin, soluble or immobilized, resulted in an almost complete inactivation of glucosyltransferase. Amyloglucosidase preparations treated with subtilisin converted starch to glucose in high yields, thus confirming this procedure to be a simple and efficient way of removing glucosyltransferase from amyloglucosidase.     

Release ofAspergillus niger protoplasts byPenicillium purpurogenum enzymes

Enzymes formed by the fungusPenicillium purpurogenum destroy theAspergillus niger cell wall and if a suitable stabilizing solution is used, protoplasts are released from the hyphae. The rate of release and the quantity and properties of the protoplasts are similar to those of protoplasts obtained by means of snail digestive enzymes. The lytic enzymes also destroy the cell walls of some other filamentous fungi.     

Genetic analysis of the coloured mutants ofAspergillus niger

Seven different classes of coloured mutants were produced in anAspergillus niger strain: brown (B), cinnamon (C), dark brown (DB), fawn (F), green (G), yellow (Y) and white (W). A few of these recurrent colour mutants were allelic and the rest was non-allelic. These were tested by complementation test in heterocaryons. A few colour mutants failed to form heterocaryons. The incompatibility is suggested to be under genetic control. Conidial size of the heterocaryotic heads was measured and found to be significantly larger than that of their parental homocaryons. It is believed that the larger size is due to heterocaryotic vigor manifested in the conidia from heterocaryotic mycelium. It is termed haploid inherited vigor.     

Purification and characterization of pectinesterase produced by a strain ofAspergillus niger

After an 88-fold purification, pectinesterase produced by a strain ofAspergillus niger, isolated from rotten lemons, showed the following main characteristics: maximum activity at 45°C, pH 5; Km, with pectin as substrate, 1.01 mg/L; G^*, 4750 Cal/mol. Polygalacturonic acid and methanol acted as competitive and non-competitive inhibitors, respectively. The activity of the enzyme was impaired by MgCl₂ and stimulated by NaCl.     

Transformation ofAspergillus awamori andA. niger by electroporation

A method is described which allows transformation ofAspergillus awamori andA. niger mediated by electroporation. This procedure gave transformation frequencies similar to those obtained with polyethylene glycol. ForA. niger no differences were observed between the two procedures with respect to the number of integrated plasmid copies or the frequency of homologous integration.     

Mineral nutrition ofAspergillus niger for citric acid production

The mineral requirements of a strain ofAspergillus niger for the production of citric acid in a synthetic medium were studied. It was observed that K₂HPO₄ and MgSO₄.7 H₂O were required at concentrations of 0.1% and 0.02% respectively. The optimum level of each of the trace elements Fe, Mn and Zn was 1.0 μg/ml. NaCl and CaCl₂ at lower concentrations had no effect on citric acid production. Trace elements, Cu, Co and Mo, had an adverse effect on the production of citric acid while Ni and V were without effect.     

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.     

Immunochemical relationship between glucoamylases I and II ofAspergillus niger

Rabbit antisera were prepared against the purified glucoamylases I and II ofAspergillus niger. Relationships between the two enzyme forms were investigated by using the antisera in immunodiffusion and immunoinhibition experiments. Both the forms of glucoamylase gave a single continuous precipitin band demonstrating very close structural resemblance. They gave almost identical immunoprecipitation patterns and had the same equivalence points indicating that the two forms ofA. niger gluoamylases were immunologically identical. The enzyme treated with periodate was immunologically identical with the controls and had slightly less enzyme activity but showed greatly reduced stability on storage at 4‡ C.     

Isolation of auxotrophic mutants ofAspergillus niger by ethylmethane sulphonate treatment

The alkylating agent EMS can be employed for the isolation of auxotrophic mutants of theAspergillus niger strain K 10. The maximum number of auxotrophic mutants (2.9–5.1%) corresponded approximately to the number of mutants obtained by EMS treatment of yeasts, but it was by order lower than the number of mutants generally obtained by EMS treatment in bacteria. The majority of isolated mutants grew worse than the parent strain in the liquid medium and also formed lower amount of organic acids. The organic acid which was most frequently accumulated by mutants was citric acid.     

Decolorization of pulp and paper mill effluent by growth ofAspergillus niger

Pulp and paper mill effluent was decolorized by growth ofAspergillus niger. Adding glucose (2.0 g/l) and NH₄H₂PO₄ (1.0 g/l) improved decolorization by the fungus (leaving 19% of original colour) and reduced the BOD₅ (43%) and the COD (41%) of the effluent after 48 h of incubation.

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Résumé L'effluent d'un atelier de pâte à papier a été décoloré par la croissance d'Aspergillus niger. L'ajout de glucose (2.0 g/l) et de NH₄H₂PO₄ (1.0 g/l) a amélioré la décoloration de l'effluent par la molsissure, lalssant 19% de la couleur originale, réduisant la DBO₅ de 43% et la DCO de 41%, après 48 h d'incubation.

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This work was carried out at the Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore-641 003, India.     

Bioenergetic consequences of glucoamylase production in carbon-limited chemostat cultures ofAspergillus niger

Aspergillus niger has been grown in glucose- and maltose-limited continuous cultures to determine the bioenergetic consequences of the production of the extracellular enzyme glucoamylase. Growth yields (g biomass per mol substrate) were high, indicating that growth was very efficient and protein production for biomass was not exceedingly energy consuming. It has been found that the energy costs for the production of this extracellular enzyme is very high. Depending on the efficiency of energy conservation the glucoamylase protein yield on ATP is between 1.3 and 2.6 g protein per mol ATP, which is equal or less than 10% of the theoretical maximum of 25.5. These high energy costs most probably have to be invested in the process of excretion. A comparison between an industrial over-producing strain and the wild typeAspergillus niger showed that this over-producing strain most probably is a regulatory mutant. Two regions of specific growth rates could be determined (one at specific growth rates lower and one at specific growth rates higher than 0.1 h^-1), which are characterized by differences in mycelium morphology and a significant deviation from linearity in the linear equation for substrate utilization. Analysis of the region of specific growth rates higher than 0.1 h^-1 yielded maintenance requirements of virtual zero. It has been concluded that for a good analysis of the growth behaviour of filamentour fungi the linear equation for substrate utilization is not suitable, since it contains no term for the process of differentiation.     

Accumulation of 2-oxo acids in mutants ofAspergillus niger requiring lysine

Abstbact Mutants ofAspergillus niger 194A and 178 requiring lysine differ from the original prototrophic strain K 10 and from each other on the course of accumulation of organic acids. In both mutants less citric acid accumulates during the first phase of cultivation but considerably more 2-oxoglutarate and 2-oxoadipate accumulate than in the original strain. Whereas in the 194A mutant this state remains unchanged also during the second phase of cultivation, in the 178 mutant oxo acids are degraded and citric acid is synthesized intensively. The accumulation of 2-oxoglutarate and 2-oxoadipate in the fermentation medium indicates that inA. niger lysine is synthesizedvia the homocitrate pathway.     

An improved method for characterization of citrate production by conidia ofAspergillus niger

Summary A modification of a method is presented, which enables the characterization of a given conidial material ofAspergillus niger with respect to its capacity to produce citric acid in submerged culture. The procedure can be applied to selection experiments as well as to counteract degeneration during strain maintenance.     

Citric acid production from beet molasses by cell recycle ofAspergillus niger

Summary Production of citric acid from beet molasses at a varying pH profile using cell recycle ofAspergillus niger was investigated. Best results in terms of citric acid concentration, yield, productivity and specific citric acid productivity were obtained with a substrate pH of 3.0.