α-(1 → 3)-D-glucans from fruiting bodies of selected macromycetes fungi and the biological activity of their carboxymethylated products.

PURPOSE OF WORK: To show biological activity of carboxymethylated α-(1 → 3)-D-glucans isolated from the selected macromycetes fungi on human tumor and normal cells. Water-insoluble, alkali-soluble polysaccharides (WIP) were isolated from fruiting bodies of four macromycetes fungi: Lentinus edodes, Pleurotus ostreatus, Piptoporus betulinus and Laetiporus sulphureus. The structure of the polysaccharides was determined using composition analysis, methylation analysis, fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The chemical and spectroscopic investigations indicated that the polysaccharides were an α-(1 → 3)-D-glucans. A biological activity analysis of the carboxymethylated (CM) α-(1 → 3)-D-glucans was based on an assessment of their cytotoxic, mitochondrial metabolism-modulating, and free radical scavenging effects. The cytotoxic activity of the CM-glucans was concentration- and cell-type-dependent. The tested CM-glucans, generally, did not have a free radical scavenging effect. The CM-α-(1 → 3)-D-glucans isolated from the selected macromycetes fungi are biologically active and may therefore be used as diet or therapy supplements.

Mutanase from Paenibacillus sp. MP-1 produced inductively by fungal α-1,3-glucan and its potential for the degradation of mutan and Streptococcus mutans biofilm.

Laetiporus sulphureus is a source of α-1,3-glucan that can substitute for the commercially-unavailable streptococcal mutan used to induce microbial mutanases. The water-insoluble fraction of its fruiting bodies from 0.15 to 0.2% (w/v) induced mutanase activity in Paenibacillus sp. MP-1 at 0.35 µ ml(-1). The mutanase extensively hydrolyzed streptococcal mutan, giving 23% of saccharification, and 83% of solubilization of glucan after 6 h. It also degraded α-1,3-polymers of biofilms, formed in vitro by Streptococcus mutans, even after only 3 min of contact.

Paenibacillus strain MP-1: a new source of mutanase.

A mutan-degrading bacterium, closely related to Paenibacillus curdlanolyticus, was isolated from soil. It produced 0.4 U mutanase ml(-1 )in 2 days in shake-flask cultures when bacterial mutan was the sole carbon source. Mutanase activity was optimal at pH 6.2 and 45 degrees C over 1 h and was stable between pH 5.8 and 12 at 4 degrees C for 24 h and up to 40 degrees C for 1 h. Mutan produced by Streptococcus mutans was rapidly hydrolyzed by this enzyme. The hydrolysis of mutan (1 g l(-1)) resulted in 17% saccharification over 2 h and, at the same time, glucan was entirely solubilized.

Enhancement of mutanase production in Trichoderma harzianumby mutagenesis.

Conidia of Trichoderma harzianum F-340, an active producer of fungal mutanase, were mutagenized with physical and chemical mutagens used separately or in combination. After mutagenesis, the drop in conidia viability ranged from 0.004% to 71%. Among the applied mutagens, nitrosoguanidine gave the highest frequency of cultures with enhanced mutanase activity (98%). In total, 400 clones were isolated, and preliminarily evaluated for mutanase activity in flask microcultures. Eight most productive mutants were then quantified for mutanase production in shake flask cultures. The obtained results fully confirmed a great propensity of all the tested mutants to synthesize mutanase, the activity of which increased from 59 to 107% in relation to the parental T. harzianum culture. The best mutanase-overproducing mutant (T. harzianumn F-340-48), obtained with nitrosoguanidine, produced the enzyme activity of 1.36 U/ml (4.5 U/mg protein) after 4 days of incubation in shake flask culture. This productivity was almost twices higher than that achieved by the initial strain F-340, and, at present, is the best reported in the literature. The potential application of mutanase in dentistry is also discussed.

Manganese peroxidase production in submerged cultures by free and immobilized mycelia of Nematoloma frowardii.

The agaric basidiomycete Nematoloma frowardii has been suggested as a good alternative for production of the extracellular ligninolytic enzyme, manganese-dependent peroxidase (MnP). Some cultural and environmental factors influencing the enzymatic activity in shaken flasks and aerated fermenter cultures were evaluated to improve the yields of the process. A low nitrogen medium (1.36 mM N added as ammonium tartrate), containing 16 g/l glucose (C/N ratio=65.3), 2mM Mn2+ and inoculated with immobilized polyurethane foam mycelium, made it possible to obtain a MnP yield of 2304 nkat/l in 8 days. Under these operational conditions, the enzyme productivity in the immobilized cells of N. frowardii was 1.4 times higher than that obtained with the free fungus. In the procedure with the reusable immobilized mycelium (semi-continuous culture) as many as three subsequent 10 day batches could be fermented by using the same carrier with no loss of MnP activity.

Optimization of conditions for the efficient production of mutan in streptococcal cultures and post-culture liquids.

The strain Streptococcus sobrinus CCUG 21020 was found to produce water-insoluble and adhesive mutan. The factors influencing both stages of the mutan production, i.e. streptococcal cultures and glucan synthesis in post-culture supernatants were standardized. The application of optimized process parameters for mutan production on a larger scale made it possible to obtain approximately 2.2 g of water-insoluble glucan per 11 of culture supernate--this productivity was higher than the best reported in the literature. It was shown that some of the tested beet sugars might be successfully utilized as substitutes for pure sucrose in the process of mutan synthesis. Nuclear magnetic resonance analyses confirmed that the insoluble biopolymer synthesized by a mixture of crude glucosyltransferases was a mixed-linkage (1-->3), (1-->6)-alpha-D-glucan (the so-called mutan) with a greater proportion of 1,3 to 1,6 linkages.

Selection of strain and optimization of mutanase production in submerged cultures of Trichoderma harzianum.

Nineteen fungal strains belonging to different genera were tested for extracellular mutanase production in shaken flasks. The optimal enzymatic activity was achieved by Trichoderma harzianum F-470, a strain for which the mutanase productivity has not yet been published. Some of factors affecting the enzyme production in shaken flasks and aerated fermenter cultures have been standardized. Mandels mineral medium with initial pH 5.3, containing 0.25% mutan and inoculated with 10% of the 48-h mycelium, was the best for enzyme production. A slight mutanolytic activity was also found when sucrose, raffinose, lactose and melibiose were carbon sources. Application of optimized medium and cultural conditions, as well as use of a fermenter with automatic pH control set at pH 6.0 enabled to obtain a high mutanase yield (0.33 U/ml, 2.5 U/mg protein) in a short time (2-3 days). The enzyme in crude state was stable over a pH range of 4.5-6.0, and at temperatures up to 35 degrees C; its maximum activity was at 40 degrees C and at pH 5.5.

Insoluble glucans synthesized by cariogenic streptococci: a structural study.

Of the three cariogenic streptococci grown in four various culture media, the strain Streptococcus mutans 20,381 was found to produce large amounts of extracellular glucosyltransferase and water-insoluble, adhesive exopolysaccharide when grown in batch culture on brain-heart infusion broth. Methylation and nuclear magnetic resonance analyses revealed that the insoluble polymers synthesized by the crude glucosyltransferase preparations were mixed-linkage (1-->3), (1-->6)-alpha-D-glucans (so-called mutans) with a greater proportion of 1,3 to 1,6 linkages and major branch points of 3,6-linked glucose. The percentage content of different types of linkages in glucans varied widely and depended on the strain of cariogenic bacteria used to produce glucosyltransferase, and on the kind of medium utilized to cultivate mutans streptococci. The potential application of insoluble glucan produced by mutans streptococci is discussed.

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.

The use of cellulases from a beta-glucosidase-hyperproducing mutant of Trichoderma reesei in simultaneous saccharification and fermentation of wheat straw.

Conidia of the cellulolytic strain Trichoderma reesei F522 were mutagenized with UV irradiation and N-methyl|-N'-nitro-N-nitrosoguanidine (NTG). A visual agar plate detection system was developed, using esculin and ferric ions, to identify mutants of T. reesei with increased beta-glucosidase activity. Selected mutants were tested for production of extracellular cellulases in shake flasks on autohydrolyzed wheat straw as carbon source. The most active mutant V-7 showed about 6-times higher activity of beta-glucosidase than the parent strain F-522, whereas the filter paper degrading and endo-1,4-beta-D-glucanase activities increased by 45% and by almost 31%, respectively. Cellulase preparations obtained from the parent and mutant strains were then used along with Kluyveromyces fragilis cells for ethanol production from ethanol-alkali pulped straw in the simultaneous saccharification and fermentation (SSF) process. From 10% (w/v) of straw pulp (dry matter), 2.5% (w/v) ethanol was obtained at 43 degrees C after 48 h using cellulase derived from the parent strain of T. reesei. When the beta-glucosidase-hyperproducing mutant V-7 was employed, the ethanol yield in the SSF process increased to 3.4% (w/v), the reaction time was shortened to 24 h and no cellobiose was detected in straw hydrolyzates.

The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol.

Autohydrolysis and ethanol-alkali pulping were used as pretreatment methods of wheat straw for its subsequent saccharification by Trichoderma reesei cellulase. The basic hydrolysis parameters, i.e., reaction time, pH, temperature, and enzyme and substrate concentration, were optimized to maximize sugar yields from ethanol-alkali modified straw. Thus, a 93% conversion of 2.5% straw material to sugar syrup containing 73% glucose was reached in 48 h using 40 filter paper units/g hydrolyzed substrate. The pretreated wheat straw was then fermented to ethanol at 43 degrees C in the simultaneous saccharification and fermentation (SSF) process using T. reesei cellulase and Kluyveromyces fragilis cells. From 10% (w/v) of chemically treated straw (dry matter), 2.4% (w/v) ethanol was obtained after 48 h. When the T. reesei cellulase system was supplemented with beta-glucosidase from Aspergillus niger, the ethanol yield in the SSF process increased to 3% (w/v) and the reaction time was shortened to 24 h.

Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose.

A total of 58 yeast strains from 12 genera were assayed for their ability to grow and ferment carbohydrates in standard Durham tube test at 40, 43, and 46 degrees C. Based on the kinetic parameters for glucose fermentation in shaken flask cultures, the strain Fabospora fragilis CCY51-1-1 was chosen for further studies. It reached about 56.0 and 35.0 g ethanol/L from approximately 140 g glucose/L at 43 and 46 degrees C in less than 48 h, respectively. Trichoderma reesei cellulase preparation (400 FPU/L) had not distinct effect on the ethanol yield and biomass production by the selected strain in the first 12 h fermentation at 46 degrees C. Later a negligible decrease in both yields was observed. It was found that Fabospora fragilis did not grow or produce ethanol at 46 degrees C as tho initial ethanol concentration overcame 40 g/L.

Intensification of oak sawdust enzymatic hydrolysis by chemical or hydrothermal pretreatment.

The activities of cellulases and xylanase were determined in laboratory cultures of Aspergillus terreus F-413 performed on natural and chemically or hydrothermally pretreated oak sawdust. The best stimulation effects were obtained in the cultures containing sawdust treated with dioxane, sodium hydroxide, or phosphoric acid. Moreover, the sawdust pretreatment distinctly affected its enzymatic hydrolysis, especially when the preparation of hydrolase complex was isolated from the culture of A. terreus F-413 growing on the modified sawdust as a sole carbon source. The highest saccharification effect was observed when the sawdust was treated with dioxane, sodium hydroxide, or phosphoric acid. Glucose was the main product of sawdust decomposition found in the hydrolyzates.

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.

[Characterization of mitochondria of Aspergillus niger from mycelium growing in surface and submerged culture conditions (author's transl)]. / Charakteristik der Mitochondrien der Myzelien von Aspergillus niger aus Oberflächen- und Submerskulturen.

This paper deals with studies on tightly coupled mitochondria present in the active citric acid producing mycelium of Aspergillus niger growing in surface and submerged culture. A special homogenizer had been used for rapid extraction of mitochondria. Observation in the electron microscope indicated that some of the isolated mitochondria were probably damaged during preparation. Nevertheless, the crude mitochondrial fraction was capable of coupling phosphorylation to the oxidation of three different substrates tested viz., succinate, citrate and NADH. It was found that yield of mitochondria was greater in submerged mycelium than in the surface mycelium of A. niger.