Characterization of Hyaluronan-Degrading Enzymes from Yeasts.

Hyaluronidases (HAases) from yeasts were characterized for the first time. The study elucidated that hyaluronate 4-glycanohydrolase and hyaluronan (HA) lyase can be produced by yeasts. Six yeasts producing HAases were found through express screening of activities. The extracellular HAases from two of the yeast isolates, Pseudozyma aphidis and Cryptococcus laurentii, were characterized among them. P. aphidis HAase hydrolyzed ß-1,4 glycosidic bonds of HA, yielding even-numbered oligosaccharides with N-acetyl-D-glucosamine at the reducing end. C. laurentii produced hyaluronan lyase, which cleaved ß-1,4 glycosidic bonds of HA in ß-elimination reaction, and the products of HA degradation were different-sized even-numbered oligosaccharides. The shortest detected HA oligomer was dimer. The enzymes' pH and temperature optima were pH 3.0 and 37-45 °C (P. aphidis) and pH 6.0 and 37 °C (C. laurentii), respectively. Both HAases showed good thermostability.

Cryptococcus laurentii extracellular biopolymer production for application in wound management.

Cryptococcus laurentii growth and extracellular polysaccharide (EPS) production in bioreactor were studied. Biomass yield 14.3 g/L and EPS synthesis 4.3 g/L in 144 h of submerged cultivation were achieved. EPS synthesis and cell growth had different optima. For EPS formation, pH 3, 25 °C and low aeration (1 % < pO2 < 10 %) were advantageous, while cell growth optimum was at pH 6, 20 °C, and high aeration (pO2 > 30 %). As medium pH changed from pH 3 to pH 6, glucuronic acid (GluAc) content in EPS increased, while galactose, xylose, and glucose decreased. Twenty-five degrees Celsius was optimal for GluAc containing polysaccharide synthesis, while lower temperature (15 °C) increased glucose content in EPS. Aeration intensity and time of cultivation had little effect on EPS composition. Molecular mass distribution of raw C. laurentii EPS was determined by SEC-MALS as 1.352. The row EPS was composed of acidic glucuronoxylomannan for more than 85 %. In the in vivo experiments, EPS significantly improved excisional wound healing in healthy rats. The results suggest that C. laurentii EPS is a promising biotechnological product and an advanced material for application in wound management.

Chitin-glucan complex production by Schizophyllum commune submerged cultivation.

Chitin-glucan complex is a fungal origin copolymer that finds application in medicine and cosmetics. Traditionally, the mycelium of Micromycetes is considered as an industrial chitin-glucan complex source. Basidiomycete Schizophyllum commune submerged cultivation for chitin-glucan complex production was studied. In different S. commune strains chitin-glucan complex composed 15.2 +/- 0.4 to 30.2 +/- 0.2% of mycelium dry weight. Optimized conditions for chitin-glucan complex production (nutrient medium composition in g/l: sucrose - 35, yeast extract - 4, Na2HPO4*12H2O - 2.5, MgSO4*7 H2O - 0.5; medium initial pH 6.5; aeration intensity 21 of air per 11 of medium; 144 hours of cultivation) resulted in 3.5 +/- 0.3 g/l complex yield. Redirection of fungal metabolism from exopolysaccharide synthesis to chitin-glucan complex accumulation was achieved most efficiently by aeration intensity increase. Chitin-glucan complex from S. commune had the structure of microfibers with diameter 1-2 microm, had water-swelling capacity of 18 g/g, and was composed of 16.63% chitin and 83.37% glucan with a degree of chitin deacetylation of 26.9%. S. commune submerged cultivation is a potent alternative to Micromycetes for industrial-scale chitin-glucan complex production.

Synthesis and characterization of new biodegradable hyaluronan alkyl derivatives.

Several new biocompatible and degradable materials were prepared by chemical modification of sodium hyaluronate. The method of activation of hyaluronate by cyanogen bromide was used and subsequent reaction with nucleophile led to the formation of carbamate. This modification of hydroxyl groups of glycosaminoglycans preserves the carboxyl groups and retains properties of polyelectrolyte. This method affords derivatives easily and the reaction condition correlates with degree of substitution. The experimental results show the effect of reaction conditions (reaction time, ratio of reactants) and effect of substitution on biodegradability. The obtained materials were characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy.