Discovery and Characteristic of Hyaluronidases from Filamentous Fungi

Background: This is the first report of hyaluronan lyase discovery in filamentous fungi, and the second case of hyaluronan hydrolase production by micromycetes. Hyaluronan (HA) is a glycosaminoglycan composed of Nacetyl- D-glucosamine and D-glucuronic acid. Whereas HA is the major constituent of extracellular matrix and an important regulator of many biological processes in vertebrates, HA degrading enzymes hyaluronidases (HAases) are of particular interest for medicine and pharmacy. Objective: To screen submerged cultures of filamentous fungi for HAase activity, and to characterize the properties of newly discovered HAases. Methods: Fungi were cultivated in bioreactor, enzymes were recovered from the culture broth, and purified by ultrafiltration and chromatography. For enzymes characterization HPLC, LC-ESI-MS, SDS-PAGE, zymography and methods of analytical chemistry were applied. Results: Wood rotting fungus Fistulina hepatica and micromycet Talaromyces stipitatus were identified as producers of extracellular HAases. On the basis of analysis of HA degradation products F. hepatica HAase was identified as HA lyase, while T. stipitatus enzyme was HA hydrolase. The HA lyase had pH optimum close to 4.0 and temperature optimum at around 20°C. T. stipitatus produced acidic HAase with pH optimum at 3.0 and temperature optimum 43°C. T. stipitatus HAase molecular weight was determined, enzyme thermostability, effect of ions on its activity, and substrate specificity were characterized. Conclusion: Filamentous fungi are new and highly promising source of hyaluronidases, which can be used in pharmacy and cosmetics for manufacturing of biologically active HA oligomers.