Strategies based on aqueous two-phase systems for the separation of laccase from protease produced by Pleurotus ostreatus

Laccases are currently employed in several biotechnological applications such as pharmaceutical, environmental, textile and petrochemical. Their production in submerged bioreactors has been extensively reported using basidiomycetes, however, this approach exhibits a major drawback: the concomitant production of proteases, which reduces laccase activity in broths. In this work, aqueous two-phase systems (ATPS) composed by polyethylene glycol (PEG)-salt, UCON-salt and PEG-dextran were tested to separate laccase from proteases in culture media. Results showed that PEG of low molecular weight (400 g mol-1), volume ratio of 0.33 and tie line length of 45% (w/w) in a PEG-salt system allowed the separation of both enzymes. The extraction efficiency decreased when sodium chloride was added into the system, but increased with a second ATPS stage. A PEG 4600-dextran 500 000 system was also suitable to separate laccase from proteases with an enzymatic activity recovery of 95%. The UCON-salt systems tested were not adequate for the separation of both enzymes since they partitioned to the same phase. Therefore, two options for laccase separation from proteases are proposed: a two step PEG 400-salt system for laccase separation after the fermentation process and PEG 4600-dextran 500 000 for in situ recovery of laccase due to its lower salt content which is suitable for fungal growth. To our knowledge, this is the first time that conditions for the separation of laccase and proteases on ATPS different phases are reported. These findings open the opportunity to establish optimum process conditions for the efficient production and recovery of laccases produced by Pleurotus ostreatus. (C) 2019 Elsevier B.V. All rights reserved.