Dyestuff industry is responsible for up to 20% of the industrial water pollution, due to dye loss in effluents. Compared to research on treatment of azo dyes (largest category), research of anthraquinone dyes (second largest category) is neglected. Environmental considerations about industrial chemical processes for water treatment have led to a shift towards green chemistry and biocatalysis. Although peroxidases are vastly applied in bioremediation, they cannot be industrially implemented due to low stability, lack of reusability and difficulty in scale-up. Immobilisation offers reusability and can improve the catalytic functions and operational stability of biocatalysts. Novel approaches, include bioinspired supports, synthesised fast and economically, avoiding the environmentally un-friendly methods used in “conventional” immobilisation.
This project focused on understanding the immobilisation of Horseradish Peroxidase (HRP) on bespoke Bioinspired Silicas (BIS), by examining factors affecting the synthesis and performance of the biocatalysts. We immobilised HRP on BIS via in-situ encapsulation and adsorption, and compared the outcomes to that of HRP adsorbed on commercial silicas. We also examined the effect of the controlled presence of amine functionalisation on BIS, of the point of HRP addition during synthesis of the biocatalyst and of increasing HRP concentration, to the immobilisation efficiency and performance of biocatalysts. BIS showed high potential as immobilisation supports, offering high loading (about 20% HRP on BIS-HRP composite) of active enzyme and their ability to protect HRP under exposure to non-optimal conditions. Biocatalysts were characterised for their morphology and porosity before assessing their performance a standard peroxidase assay based on 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate acid oxidation (ABTS assay) and an application assay based on enzymatic degradation of a model anthraquinone dye, Reactive Blue 19 (RB19 assay). Further examination of the best performing BIS-HRP samples, revealed a competitive action of BIS to enzymatic activity, where the support acts as an excellent adsorbent, hindering the diffusion of substrate and product(s) through the pore network. Although free HRP outperforms immobilised HRP (especially via encapsulation), immobilisation results in a highly reusable biocatalyst, for up to 20 times with 60% performance retention towards dye removal, with enhanced storage stability, retaining almost 100% activity over 50 days of storage, compared to 3 days of storage reached with free HRP.
Through this work, we showed the importance of individual factors crucial for enzyme immobilisation, regarding both biocatalyst synthesis and expected performance, as well as the importance of the combination of enzyme, substrate and immobilisation support on biocatalyst performance. This work can be a great base for further optimisation of BIS as enzyme immobilisation support, and its exploration in other applications in the area of water treatment.
