Isolation and screening of glucose oxidase producing microorganisms from natural sources.

Among 1486 mould strains isolated from natural sources (screened for extracellular glucose oxidase) only 119 (Aspergillus and Penicillium) showed this enzyme activity. As the best glucose oxidase producer, A. niger 0-1 was isolated from decaying tree. The dynamics of glucose oxidase synthesis in A. niger 0-1 during its culture by submerged method show that the intracellular activity of this enzyme is 10-times higher than its extracellular level. Some properties of the crude glucose oxidase preparation, isolated from the postculture liquids by lyophilization, were examined.

Glucose oxidase biosynthesis using immobilized mycelium of Aspergillus niger.

A simple method is described for the immobilization of Aspergillus niger GIV-10 which produces an extracellular glucose oxidase. A. niger conidia were immobilized on sintered glass Raschig rings, pumice stones or polyurethane foam. Mycella growing out from the spores produced extracellular glucose oxidase: the highest production was with the pumice stone carrlers. This technique facilitates the growth of the filamentous cultures in the spongy structure of a support with continuous accumulation of biomass. After 24 to 36 h, a culture liquid with 2.7 to 3.1 U of glucose oxidase/ml was obtained. This procedure also made possible repeated batch enzyme production and as many as 25 subsequent 24-h batches could be fermented by using the same carrier with only a small loss of glucose oxidase activity.

Screening of microorganisms for improvement of beta-galactosidase production.

The strain of Penicillium notatum 1 most effective for producing beta-galactosidase (see lactase 3.2.1.23), was selected out of 110 moulds belonging to 15 different species, by the test-tube microculture method. The dynamics of beta-galactosidase synthesis was investigated in P. notatum 1 during its culture by submerged method.

Pectinolytic activity of revertants of auxotrophic strains of Aspergillus niger.

From conidia of 4 different auxotrophic A. niger strains 400 spontaneous revertants (100 from each strain) were obtained, and in one case additionally 100 revertants induced by mutagens (UV+NTG). The revertants showed a considerable differentiation with regard to the total pectinolytic activity. Its highest increase occurred in revertants originating from auxotrophs greatly predisposed to synthesize pectinases. In the case of revertants induced by mutagenes an increase in the frequency of their formation was observed, as well as an increased participation of revertants with higher pectinolytic activity compared to both their initial auxotrophic and prototrophic strain.

Selection of amylolytically active Aspergillus niger mutants to 2-deoxy-D-glucose.

As a result of mutagenization and passaging on 2-deoxy-D-glucose containing medium, 10 Aspergillus niger strains resistant to this agent were obtained. These showed (with one exception) an increase in the activity of glucoamylse, the level of which ranged widely in individual cases from several to over 200% in comparison with the parent strain. A weaker rate of glucose accumulation in derepressed strains may account for the fact that the mechanism of their resistance to deoxyglucose is connected with disturbance of the system of glucose transport. However, it is possible that a high activity of acid phosphatase, which the obtained deoxyglucose-resistant cultures showed, may be involved here. Apart from the biochemical character of the catabolic derepression, it seems that it can already be successfully utilized to increase the productivity of industrial mould cultures.

The effect of proteases on the synthesis of glucoamylase by mutants of Aspergillus niger C.

Out of 1230 cultures grown from conidia of the strain Aspergillus niger C, which were previously treated with mutagens, 6 mutants were selected, 5 of which were characterized by considerably lower or lack of proteolytic activity than the parent strain. It was observed that glucoamylase activity and protein synthesis in the mutants examined were directly proportional to the rate of their proteolytic activity. From electrophoretic analysis it was found that in some protease-free mutants or with strongly reduced activity of this enzyme one or two glucoamylase fractions out of four occurring in the parent strain were absent.

Effect of iron on the activity of aconitate hydratase and synthesis of citric acid by Aspergillus niger.

Two strains of A. niger were cultivated on beet molasses medium by the surface method, and the effects of iron on the mycelium formation, the rate of sugar utilization, the activity of aconitate hydratase, and synthesis of citric acid were determined. Among various sources of di- and trivalent iron, ferrous sulphate was chosen for studies, which most effectively limited the synthesis of organic iron, ferrous sulphate was chosen for studies, which most effectively limited the synthesis of organic acids. The molasses solutions were purified with potassium ferrocyanide and FeSO4 at a concentration of 20 mg/100 ml (in terms of Fe2+) was added. It was found in both strains that such an amount of Fe2+, additionally introduced into the molasses medium, only slightly effected the synthesis of the mycelium biomass and the rate and extent of sugar utilization, when at the same time a high increase in the activity of aconitate hydratase occurred. Citric acid production, however, was strongly inhibited by this Fe2+-concentration in case of A. niger 23 (about 94%), whereas A. niger Lcz showed a relatively small decrease (about 20%).

Cellulolytic activity of moulds. III. Enzymatic hydrolysis of cellulose by cellulase preparation from Aspergillus terreus F-413.

The strain Aspergillus terreus F-413 was used to obtain cellulolytic enzymes; it was cultivated on cellucotton by the submerged method in a 101 fermenter. The crude enzymatic preparation obtained from the post-culture liquid, containing small amounts of other enzymes besides active cellulases, was used for hydrolysis of several kinds of cellulose. The highest saccharification percentage was obtained with cellucotton and filter paper, while the lowest with lignocellulosic materials such as straw and sawdust. It was shown that cellulose pretreatment, both mechanical (grinding in a ball mill) and chemical (with alkali) distinctly increased the yield of enzymatic hydrolysis. In the obtained hydrolyzates the presence of glucose as the main product of cellulose decomposition was found.

Cellulolytic activity of moulds. IV. Evaluation of the utility of cellulosic wastes for biosynthesis of cellulases and xylanase by Aspergillus terreus F-413.

The value of various cellulosic wastes for the synthesis of cellulolytic enzymes by the mould Aspergillus terreus F-413 was determined in submerged culture. The best results were obtained by using beet pulp. The dynamics of their synthesis was observed during a 12-day culture on the medium most favourable for the synthesis of cellulases.

Cellulolytic activity of moulds. II. Various methods of precipitating and concentrating enzymes and their influence on the activity of cellulolytic preparation and xylanase of Aspergillus terreus F-413.

Complexes of cellulolytic enzymes and xylanase were precipitated and concentrated by various methods from post-culture liquids of Aspergillus terreus F-143, containing cellucotton as carbon source. The best results in regard to the specific activity of the preparations were obtained by precipitation of enzymes with acetone-denatured ethanol. Besides high cellulolytic and xylanase activity the crude enzyme preparation showed the presence of small amounts of amylase, protease and polygalacturonase.

Cellulolytic activity of moulds. I. Characteristics of the cellulases complex and xylanase of the strain Aspergillus terreus F-413.

Among 79 strains of moulds belonging to 17 different species, the strain Aspergillus terreus F-413 which showed the highest cellulolytic activity was isolated for further studies. Some properties of the complex of cellulases formed by this strain as well as the dynamics of their synthesis under optimal submerged culture conditions were characterized.

Spontaneous segregation of a heterozygous diploid of Aspergillus niger.

A case of spontaneous segregation of a heterozygous diploid was observed with Aspergillus niger, giving rise to a somatic recombinant. Assuming an independence of markers, its genotype is suggested to be brw+lys pab+brw lys+pab.