Abstract
This study reports a new strategy for enzyme immobilization based on passive immobilization in neat and magnetically responsive polyamide 4 (PA4) highly porous particles. The microsized particulate supports were synthesized by low-temperature activated anionic ring-opening polymerization. The enzyme of choice was laccase from Trametes versicolor and was immobilized by either adsorption on prefabricated PA4 microparticles (PA4@iL) or by physical in situ entrapment during the PA4 synthesis (PA4@eL). The surface topography of all PA4 particulate supports and laccase conjugates, as well as their chemical and physical structure, were studied by microscopic, spectral, thermal, and synchrotron WAXS/SAXS methods. The laccase content and activity in each conjugate were determined by complementary spectral and enzyme activity measurements. PA4@eL samples displayed >93% enzyme retention after five incubation cycles in an aqueous medium, and the PA4@iL series retained ca. 60% of the laccase. The newly synthesized PA4-laccase complexes were successfully used in dyestuff decolorization aiming at potential applications in effluent remediation. All of them displayed excellent decolorization of positively charged dyestuffs reaching ~100% in 15 min. With negative dyes after 24 h the decolorization reached 55% for PA4@iL and 85% for PA4@eL. A second consecutive decolorization test revealed only a 5–10% decrease in effectiveness indicating the reusability potential of the laccase-PA4 conjugates.
Highlights
Biocatalysts constitute an alternative to traditional catalytic systems, being able to cause transformation of natural or synthetic substrates under mild reaction conditions, with high substance, stereo, and regiospecificity, and lack of toxic byproducts [1,2].With the advances in the production of relatively inexpensive free enzymes, the steady increase of their extensive implementation as effective catalysts in many industrial and biomedical applications is beyond any doubt [3].The wide use of industrial-scale processes with enzymatic catalysts is restricted by the free enzymes’ inactivation by different denaturant agents and by their difficult or even impossible recovery from the reaction medium after use
The fact that activated anionic ring-opening polymerization (AAROP) was possible at 40 ◦ C allowed the entrapment of active laccase into the shell of the forming polyamide 4 (PA4) MP during the polymerization, resulting in three PA4@eL samples
Ai − At where, Ai is the initial absorbance of the dye at the respective wavelength, and At is the absorbance of the test sample. This is the first study reporting on PA4 porous microparticles as effective supports for enzyme immobilization and describes PA4-laccase conjugates with all aspects of their preparation and characterization
Summary
Biocatalysts (extracellular enzymes or whole-cells) constitute an alternative to traditional catalytic systems, being able to cause transformation of natural or synthetic substrates under mild reaction conditions, with high substance-, stereo-, and regiospecificity, and lack of toxic byproducts [1,2]. The wide use of industrial-scale processes with enzymatic catalysts is restricted by the free enzymes’ inactivation by different denaturant agents and by their difficult or even impossible recovery from the reaction medium after use. These problems can be resolved by immobilization (conjugation) of the enzymes to prefabricated matrices of various nature, geometry, and topography. Catalysts 2020, 10, 767 enzymes, apart from their application as reusable heterogeneous biocatalysts, can serve as appropriate platforms for the development of biosensors, biofuel cells, or microscale devices for controlled release of protein drugs [4].
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