Immobilization of defined laccase combinations for enhanced oxidation of phenolic contaminants

Abstract

Immobilization is an important method to increase enzyme stability and allow enzyme reuse. One interesting application in the field of environmental biotechnology is the immobilization of laccase to eliminate phenolic contaminants via oxidation. Fumed silica nanoparticles have interesting potential as support material for laccase immobilization via sorption-assisted immobilization in the perspective of applications such as the elimination of micropollutants in aqueous phases. Based on these facts, the present work aimed to formulate laccase-nanoparticle conjugates with defined laccase combinations in order to obtain nanobiocatalysts, which are active over a broad range of pH values and possess a large substrate spectrum to suitably address pollution by multiple contaminants. A multi-enzymatic approach was investigated by immobilizing five different types of laccases originating from a Thielavia genus, Coriolopsis polyzona, Cerrena unicolor, Pleurotus ostreatus, and Trametes versicolor onto fumed silica nanoparticles, separately and in combinations. The laccases differed concerning their pH optima and substrate affinity. Exploiting their differences allowed the formulation of tailor-made nanobiocatalysts. In particular, the production of a nanobiocatalyst could be achieved that retained a higher percentage of its relative activity over the tested pH range (3-7) compared to the dissolved or separately immobilized enzymes. Furthermore, a nanobiocatalyst could be formulated able to oxidize a broader substrate range than the dissolved or separately immobilized enzymes. Thereby, the potential of the nanobiocatalyst for application in biochemical oxidation applications such as the elimination of multiple target pollutants in biologically treated wastewater has been illustrated.

Insights into laccase producing organisms, fermentation states, purification strategies, and biotechnological applications

“…For example, Hommes et al modified an extracellular flux analyzer (CFA, originally used to assess physiological parameters in cell monolayers) to estimate the oxygen consumption rates of five types of laccases compared with that of the conventional Clark electrode‐based method. The similar assay protocol was applied for estimation of laccase activity in both free and on fumed silica nanoparticles immobilized form in the investigation conducted by Ammann et al In another study performed by Rachinskiy et al, a novel model‐based technique called an enzyme test bench was applied to characterize and optimize a complex and multi‐stage LMS. They used sodium sulfite, catalyzed by cobalt sulfate, to determine the oxygen transfer rate and the respiration activity monitoring system (RAMOS) technique for the determination of laccase activity …”

Fungal Bioremediation of Emerging Micropollutants in Municipal Wastewaters

“…and P. ostreatus laccases led to a wider operative pH range: the recovery of 70% of the activity between pH 3 and 7 was never possible in presence of the single-laccase solutions. As regard the substrate affinity, C. polyzona and T. versicolor laccases worked better together than alone: the multi-enzymes system converted the 5 compounds tested, including benzophenone-2 and gemfibrozil which were never oxidized by C. polyzona and T. versicolor laccase, respectively (Amman et al 2014). The development of this technology could take into consideration also the coimmobilization of different enzymes typology e.g.…”

Laccases to take on the challenge of emerging organic contaminants in wastewater

“…Concerning laccase immobilization for wastewater treatment, mostly covalent binding on solid support materials has been investigated (Cabana et al 2009a;Galliker et al 2010;Zimmermann et al 2011;Demarche et al 2012;Hommes et al 2012;Lloret et al 2012a, c;Catapane et al 2013;Nair et al 2013;Ammann et al 2014) since this approach typically yields more stable biocatalysts compared to the other binding methods. Stability is a crucial factor if the immobilized enzymes are intended for use in tertiary wastewater treatment processes in which the enzymes are exposed to harsh environmental conditions, i.e., high pH values, turbulent flows, and presence of remaining organic, inorganic, or biological denaturing agents.…”