Comparison of downstream processing methods in purification of highly active laccase

Abstract

Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.

Antimicrobial Activity and Barrier Properties against UV Radiation of Alkaline and Enzymatically Treated Linen Woven Fabrics Coated with Inorganic Hybrid Material

“…The bioreactor was equipped with a set of sensors (pH and oxygen electrodes, pressure and temperature indicators) to control the process parameters. Additionally, a special rotary filter was installed inside the bioreactor and enabled for sterile collection of fermentation broth containing laccase without the biomass [33]. For biosynthesis, the repeated fed-batch mode was applied with a periodic addition of the substrate and simultaneous collection of the culture media with the enzyme.…”

Co-cultivation of filamentous microorganisms in the presence of aluminum oxide microparticles

“…The peak areas of ( +)-geodin, chrysogine, and rimocidin were determined with the use of ESI − quantification traces m/z = 396.989, 173.069, and 766.3990, respectively. The assay of laccase activity in the culture filtrate was performed as previously described (Antecka et al 2019 ).…”

MIR Spectroscopy for Continuous Control and Monitoring of Metabolites

“…These processes are referred to as downstream processes, involve another array of steps such as filtration (amongst others) so that a stable formulation can be produced. These steps involve different types of chromatography, from size exclusion chromatography to ion exchange and affinity chromatography [9]. Chromatography is slow, expensive, and difficult to automate.…”