Anna Antecka
Lodz University of Technology, Poland
Title: Upstream and downstream processing of fungal laccase
Biography
Biography: Anna Antecka
Abstract
Statement of the Problem: Laccase (EC 1.10.3.2, polyphenol oxidases) belongs to the group of oxidoreductases which is characterized by its specific catalytic properties and the ability to oxidize various organic compounds. Therefore the enzyme is very attractive for a wide range of industrial and environmental purposes. However, due to relatively low effectiveness and the possibility of gradual degradation of bioproducts in the reactor or during the separation and purification stages, there is a need for new approaches and research in this field. Therefore, the purpose of this research was to study and integrate the stages of up- and downstream processing (biosynthesis and purification) of laccase from Cerrena unicolor in order to obtain a highly active enzymatic product.
Methodology: The biosynthesis was performed in a 14-L bioreactor equipped with a set of sensors for process control. Modifications to the medium- addition of microparticles, MPEC, as well as various types of cultivation/growth strategies were examined. The supernatant was concentrated and purified by an aqueous two-phase system (ATPS) consisting of polyethylene glycol and phosphate buffer solutions, and through foam fractionation (FF) at different pH values and with the addition of different detergents. Ultrafiltration and chromatography methods were also investigated. Molecular mass and isoelectric point was determined with the use of electrophoresis.
Findings: Laccase activity increased 3.5-fold after addition of microparticles to the culture media. The fed-batch mode resulted in high laccase activity (up to 4 U/mL) which remains stable during cultivation. The optimal conditions for laccase purification by FF and ATPS were determined with activity partitioning coefficients between foamate and retentate of almost 200 and 2000, respectively, and with yields reaching 50% and 90%, respectively.
Conclusions: Application of MPEC and fed-batch mode proved successful in increasing enzyme production. Both ATPS and FF are successfully applicable for laccase purification.