Production and characterization of class II and III Hyaluronidases in eukaryotic and class III prokaryotic systems

Abstract

Hyaluronidases are a class of enzymes belonging to the endoglucanases that cleave chains of the biopolymer Hyaluronic Acid (HA). They are produced by the cosmetic industry, and especially in the medical/pharmaceutical industry, as adjuvants during surgeries and as reversal drugs for aesthetic procedures involving the application of HA. Hyaluronidases, even today, are obtained by extraction from animal tissues, mainly bovine, in an expensive and difficult process to purify for medical application. As an alternative, several hyaluronidases expression systems have been created using bacterial, yeast, mammalian, algal, and insect cells. The hyaluronidases are divided into 3 different classes and act in specific ways as they cleave the HA chains at different sites. Some enzymes cleave the ²-1,3 bonds of AH, while others cleave the ²-1,4 bonds. Expression systems involving the use of bacteria can result in processes with low productivity of Hyaluronidases compared to systems involving the use of yeast. Currently, there is no product or process that has simultaneously united two different classes of hyaluronidases. As a result, this project aims to develop a biotechnological product based on hyaluronidases that is more efficient than the existing ones by combining in the same system the different hyaluronidases, in which classes II and III will be expressed in yeast (Kluyveromyces lactis) and class III expressed in bacteria (Escherichia coli). After selecting the strain of microorganism (K. lactis or E.coli) that provides more favorable characteristics for the production of hyaluronidase, a production process will be developed and optimized in a shaker scale, and then in a 3-liter bioreactor, in batch mode, to collect the kinetic data of cell growth, production and productivity of the enzyme(s). (AU)