Abstract
We report the preparation, characterization and application of a novel magnetic four-enzyme nanobiocatalyst prepared by the simultaneous covalent co-immobilization of cellulase (CelDZ1), β-glucosidase (bgl), glucose oxidase (GOx) and horseradish peroxidase (HRP) onto the surface of amino-functionalized magnetic nanoparticles (MNPs). This nanobiocatalyst was characterized by various spectroscopic techniques. The co-immobilization process yielded maximum recovered enzymatic activity (CelDZ1: 42%, bgl: 66%, GOx: 94% and HRP: 78%) at a 10% v/v cross-linker concentration, after 2 h incubation time and at 1:1 mass ratio of MNPs to total enzyme content. The immobilization process leads to an increase of Km and a decrease of Vmax values of co-immobilized enzymes. The thermal stability studies of the co-immobilized enzymes indicated up to 2-fold increase in half-life time constants and up to 1.5-fold increase in their deactivation energies compared to the native enzymes. The enhanced thermodynamic parameters of the four-enzyme co-immobilized MNPs also suggested increment in their thermal stability. Furthermore, the co-immobilized enzymes retained a significant part of their activity (up to 50%) after 5 reaction cycles at 50 °C and remained active even after 24 d of incubation at 5 °C. The nanobiocatalyst was successfully applied in a four-step cascade reaction involving the hydrolysis of cellulose.
Highlights
Enzymes, widely employed as green catalysts, are versatile protein biomolecules with high stereo- and regio-selectivity that catalyze numerous biochemical reactions of industrial importance.They are preferred over conventional chemical catalysts due to their high turnover rates under mild reaction conditions and the reduction of by-product formation [1]
In order to confirm the size of the magnetic nanoparticles, atomic force microscopy (AFM) height images of the γ-Fe2 O3 nanoparticles deposited on a Si-wafer were collected
We have shown for the first time that the simultaneous covalent co-immobilization of four enzymes onto magnetic nanoparticles can lead to the development of robust nanoassemblies able to conduct multi-step cascade reactions
Summary
Widely employed as green catalysts, are versatile protein biomolecules with high stereo- and regio-selectivity that catalyze numerous biochemical reactions of industrial importance. They are preferred over conventional chemical catalysts due to their high turnover rates under mild reaction conditions and the reduction of by-product formation [1]. Its tremendous applications include an expanded range of fields, such as the production of biofuels, pharmaceuticals, foods, textiles and cosmetics [2] Enzymes in their free form are typically unstable and may undergo denaturation under harsh reaction conditions, settling challenges in their handling and separation.
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