Integrated extractive fermentation of laccase using aqueous biphasic systems: From production to functional coatings.

Extractive fermentation has increasing attention as intensified bioprocessing approaches by integrating enzyme production, recovery, and primary purification into a single step. This work demonstrates the effective use of aqueous biphasic systems in extractive fermentation, integrating the separation, and concentration of Trametes versicolor laccase expressed in Komagataella phaffii. After assessing K. phaffii growth and laccase activity in various aqueous biphasic phase-forming compounds, two types of aqueous biphasic systems, namely polymer-salt (polyethylene glycol with a molecular weight of 8000 g/mol + K2HPO4/KH2PO4) and polymer-polymer (polyethylene glycol with a molecular weight of 8000 g/mol + dextran with a molecular weight of 40000 g/mol) were selected for laccase separation, and concentration directly from the fermentation broth. The polymer-polymer aqueous biphasic systems (10.0 wt% polyethylene glycol with a molecular weight of 8000 g/mol and 5.0 wt% dextran with a molecular weight of 40000 g/mol) showed the most promising performance, leading to a 19% increase in laccase activity recovered in the dextran-rich phase, a 4.6-fold purification, and recovery of 464.7 U/L, compared to the buffer used as control. The recovered laccase retained its catalytic efficiency, and was successfully applied in the synthesis of polydopamine for functional coating of poly(methyl methacrylate). The coated material was further characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, atomic force microscopy, and optical contact angle measurements. This work shows that aqueous biphasic systems can support a sustainable and integrated platform for enzyme separation and concentration and direct application in biocatalytic surface functionalization.