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.

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.