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 β-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 β-1,4 glycosidic bonds of HA in β-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 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.
