Cultivation conditions and properties of extracellular crude lipase from the psychrotrophic fungus Penicillium chrysogenum 9'.

Among 97 fungal strains isolated from soil collected in the arctic tundra (Spitsbergen), Penicillium chrysogenum 9' was found to be the best lipase producer. The maximum lipase activity was 68 units mL(-1) culture medium on the fifth day of incubation at pH 6.0 and 20 degrees C. Therefore, P. chrysogenum 9' was classified as a psychrotrophic microorganism. The non-specific extracellular lipase showed a maximum activity at 30 degrees C and pH 5.0 for natural oils or at pH 7.0 for synthetic substrates. Tributyrin was found to be the best substrate for lipase, among those tested. The Km and Vmax were calculated to be 2.33 mM and 22.1 units mL(-1), respectively, with tributyrin as substrate. The enzyme was inhibited more by EDTA than by phenylmethylsulfonyl fluoride and was reactivated by Ca2+. The P. chrysogenum 9' lipase was very stable in the presence of hexane and 1,4-dioxane at a concentration of 50%, whereas it was unstable in presence of xylene.

Selection of biochemical mutants of Aspergillus niger resistant to some abiotic stresses with increased inulinase production.

AIMS: The present work aimed at evaluating the usefulness of selecting different kinds of biochemical mutants of Aspergillus niger to increase inulinase production in submerged culture. METHODS AND RESULTS: Conidia of A. niger 13/36, an active producer of inulinase, were subjected to mutagenesis with both u.v. and N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and the products were analysed for inulinase activity with our own diffusion plate method. As a result of mutagenization and selection for obtaining biochemical mutants (e.g. surviving conditions of certain abiotic stresses, good growing on basal medium at 15 and 40 degrees C), A. niger strains resistant to these agents were obtained. Studies of the relationship between a criterion of selection and the frequency of mutation showed that the highest frequency of positive mutations in the second selection (86%) was obtained in mutants characteristic of the best growth at the low temperature (15 degrees C), when compared with the parent culture (28%). The most active mutants grown under stress conditions showed significantly higher inulinase activity (about 1.2-4.5-fold), when compared with the parent strain. CONCLUSIONS: The studies presented seem to confirm a high effectiveness of selection in some kinds of biochemical mutants of A. niger with regard to increased inulinase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This screening strategy of mutants can be a contribution to modern commercial enzyme production.

Catalase activity in arctic microfungi grown at different temperatures.

The total of 98 strains of moulds were isolated from soils collected in arctic tundra (Spitzbergen). Among these strains Penicillium cyclopium 1, the most effective for production of catalase, was selected by the method of test-tube microculture. The time course of growth and catalase production by this strain showed the intracellular activity of this enzyme to be about 3-fold higher than its extracellular level Some properties of crude catalase preparation, isolated from postculture liquids by lyophilization, were also examined. Catalase activity showed its maximum at 15 degrees C, indicating adaptation of the enzyme to lower temperatures of the arctic environment.

Production of lignolytic and feed-back type enzymes by Phlebia radiata on different media.

Low molecular-weight compounds, structurally related to lignin, increase the production of laccase, lignin peroxidase, manganese dependent peroxidase, and feed-back type enzymes such as glucose oxidase, cellobioso-quinone oxidoreductase, and glyoxal oxidase in the culture of the white rot fungus Phlebia radiata growing on different carbon sources.

Production of Aspergillus niger catalase under various stress conditions.

The conidia of Aspergillus niger were cultured under various stress conditions. The highest yield of catalase activity was obtained after addition 1.2 M NaCl to 6-h-old culture, which improved the activity of the enzyme by about 2.8-fold in comparison with the control medium without this depressor. A significantly lower increase (1.3- to 1.7-fold) in catalase activity was reached when spores were incubated under oxidative stress (100-200 mM H2O2) or at low pH (2 and 3). Respiratory activities in A. niger culture show important changes with the osmotic stress increase.

Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture.

A novel method for increasing dissolved oxygen concentration in culture media has been developed. It involves adding hydrogen peroxide (H2O2) to the medium which is then decomposed to oxygen and water by catalase. Some factors affecting oxygenation of culture were investigated. Maximal oxygen concentration occurred in 50 ml of the medium containing 0-2 g wet mycelium and 0.2% glucose at pH 5.0. A new apparatus for automated addition of H2O2 to the bioreactor to keep the dissolved oxygen concentration constant over the range 1-100% +/- 2% was tested. A significant increase (over sixfold) of intracellular catalase activity was obtained while the dissolved oxygen concentration remained stable (30% +/- 2%).

Purification of extracellular catalase from Aspergillus niger.

The extracellular catalase (EC 1.11.1.6) produced by Aspergillus niger culture in 5-liter fermentor was isolated and purified by ion-exchange chromatography on DEAE Sepharose (fast flow) column, hydrophobic interaction on phenyl-Sepharose column and chromatofocusing on PBE 94 column. Some physico-chemical properties of two purified catalase forms were also determined (molecular weight, isolelectric point, polysaccharides contents, Km, and Ea).

Effect of osmotic stress on glucose oxidase production and secretion by Aspergillus niger.

A mycelium of Aspergillus niger was cultured under various osmotic stresses. When fungus was cultured under osmotic stress, total catalase activity increased with increasing concentration of NaCl, up 0.4 M, but glucose oxidase under this condition significantly decreased. Mycelial growth was repressed with increased media osmolarity. To release periplasmic glucose oxidase 72-h old mycelium was suspended on a concentrated solution of NaCl (0.4-2.8 M/l). The highest yield of GOD activity was obtained at a NaCl concentration of 1.2 M at pH 6.0, which improved the activity of this enzyme by about 2.1-fold in comparison with the control medium without this depressor.

Simultaneous production of catalase, glucose oxidase and gluconic acid by Aspergillus niger mutant.

The production of gluconic acid, extracellular glucose oxidase and catalase in submerged culture by a number of biochemical mutants has been evaluated. Optimization of stirrer speed, time cultivation and buffering action of some chemicals on glucose oxidase, catalase and gluconic acid production by the most active mutant, AM-11, grown in a 3-L glass bioreactor was investigated. Three hundred rpm appeared to be optimum to ensure good growth and best glucose oxidase production, but gluconic acid or catalase activity obtained maximal value at 500 or 900 rpm, respectively. Significant increase of dissolved oxygen concentration in culture (16-21%) and extracellular catalase activity were obtained when the traditional aeration was employed together with automatic dosed hydrogen peroxide.

Selective isolation of Aspergillus niger mutants with enhanced glucose oxidase production.

After mutagenization and selection, mutant Aspergillus niger strains resistant to certain agents were obtained. Seven of the mutants showed increased extracellular glucose oxidase (GOD), the level for individual cases ranged widely from 8.8 to over 138.5% in comparison with the parental strain. Studies of the relationship between method of selection and frequency of mutation showed that the highest frequency of positive mutations (15.8% and 17.3%) was obtained from mutants resistant to ethidium bromide (1 mmol l-1 and sodium gluconate (45%), respectively. The time course of growth and enzyme production by the most active mutant AM-11 showed intra- and extracellular GOD activities to have increased about 2.2- and 2.4-fold, respectively, compared with the parental strain.

Selection of biochemical mutants of Aspergillus niger with enhanced catalase production.

The production of extracellular catalase in a submerged culture by a number of biochemical mutants has been evaluated. Eight of these mutants showed increased extracellular catalase, the level of which ranged widely in individual cases from 44% to over 94% in comparison with the parental strain. Studies of the relationship between a criterion of selection and the frequency of mutation showed that the highest frequency of positive mutations (15.8% and 24.2%) was obtained with respect to mutants resistant to ethidium bromide (1 mmol/l) and sodium gluconate (45%) respectively. The time course of growth and enzyme production by the most active mutant, AM-20, showed extra- and intracellular catalase activities increasing about 2- and 2.6-fold respectively, compared with the parental strain.

Optimization of catalase biosynthesis in submerged cultures of Aspergillus niger mutant.

The effect of some medium components, viscous substances and metabolic inhibitors, on catalase production by mutant Aspergillus niger has been studied in shake culture. Altering the composition of the basal medium, particularly substituting NaNO3 for KNO3, and peptone for yeast extract brought an increase in extra- and intracellular catalase activity by 1.5- and 3-fold, respectively. The addition of 2.0-6.0 mg sodium alginate or pectin/ml as viscous additive to the medium, containing glucose as carbon source, increased the medium viscosity and catalase production in shake culture by about 2.8- to 3.0-fold. The highest yield of extracellular catalase activity of A. niger was obtained in the presence of sodium orthovanadate and Triton X-100, which improved the activity of this enzyme by about 1.5-2.2-fold. A significant increase in intracellular catalase activity was observed in the presence of hematin, Tween 80 and sodium orthovanadate (1.7-, 1.6- and 1.4-fold respectively). The time course of growth and enzyme production by A. niger in the optimized medium is also reported.

Effect of medium components and metabolic inhibitors on beta-galactosidase production and secretion by Penicillium notatum 1.

Factors affecting the beta-galactosidase production by Penicillium notatum 1 were studied using fermentation media of different chemical composition. The medium containing lactose, salts, peptone and yeast extract with initial pH 2.5 was selected as the best for enzyme production. Monobasic ammonium phosphate (0.9%) was found to be the best inorganic nitrogen source for lactase production. Various extraction media and metabolic inhibitors were examined for effective releasing of beta-galactosidase from the fungal cells. Using a simple method of mycelium extraction with 0.1 Triton X-100, it was possible to obtain about 4-fold higher amounts of enzyme in the cell free extracts, than those excreted into the post-culture liquid.

Influence of medium components and metabolic inhibitors on glucose oxidase production by Aspergillus niger mycelium.

The effect of some medium components and metabolic inhibitors on glucose oxidase (GOD) production by mutant Aspergillus niger was studied in shaken culture. Altering the composition of the basal medium, particularly substitution of (NH4)2HPO4 for NaNO3, and lack of Mg2+ ions caused an increase in GOD activity by 269.6%. The highest yield of extracellular GOD activity of A. niger mycelium was obtained in the presence of hematin (1 mM), choline (40 mM) and Tween 80 (0.1%), which improved the activity of this enzyme by about 31.4-53.9%. A significant increase (68.3%) in intracellular GOD activity was found in the presence of sodium orthovanadate (1 mM). The time course of growth and enzyme production by A. niger in the optimized medium is also reported.

Seeds as natural matrices for immobilization of Aspergillus niger mycelium producing pectinases.

A simple method for the immobilization of Aspergillus niger mycelium producing polygalacturonase (PG) and pectinesterase (PE) is described. Fungal conidia were immobilized on wheat, rye, barley, peas, buckwheat and mustards seeds. Spongy mycelia overgrowing the seed surfaces on mineral medium with pectin produced extracellular PG and PE; the highest production was reached on the wheat carrier. Some of the variables influencing the enzymatic activity have been optimized. After every 24 h, a culture liquid with 6.8-7.8 U of PG ml-1 and 7.0-10.1 U of PE ml-1 was obtained. This procedure also made possible repeated batch enzyme production and, as many as eight subsequent 24-h batches could be fermented by using the same carrier without any loss of PG activity. The addition of sodium orthovanadate (1 mmol) into the medium with pectin caused a significant increase in PG and PE activity produced by free cells of A. niger (by 1.59-fold and 1.67-fold respectively), and only 0.47-fold of PG activity in case of the immobilized mycelium.

Selection of strain, culture conditions and extraction procedures for optimum production of beta-galactosidase from Kluyveromyces fragilis.

Among 47 tested yeast strains, belonging to different genera, only two cultures of Kluyveromyces fragilis and Fabospora fragilis showed beta-galactosidase activity in shaken flasks. Three types of extraction were used to release the enzyme from K. fragilis cells: solvent and detergent extraction, freezing and thawing extraction, and mechanical disintegration prior to extraction, using Triton X-100. The results indicate that the highest yield could be obtained by mechanical disintegration of cells. Factors affecting the enzyme production were studied using fermentation media of different chemical composition. The medium containing lactose, salts and yeast extract with initial pH 7.0 was selected as the best for enzyme production. Monobasic ammonium phosphate (2.0 g/dm3) was found to be the best inorganic nitrogen source for enzyme production. The effect of phosphorus level was also studied.

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 synthesis by Aspergillus niger GIV-10 on starch.

The effect of various kinds of starch, as the sole source of organic carbon, on the biosynthesis of glucose oxidase by A. niger GIV-10 was examined. A. niger grown on 6% wheat starch medium provided extracellular and intracellular glucose oxidase with the highest enzymatic activities. A new method of intracellular glucose oxidase extraction (without disruption of mycelium), developed and discussed in this paper, increased 2 to 3.8-times glucose oxidase yield, as compared to that described earlier.

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.