Tailored enzyme immobilization: engineering enzymes as application-oriented biocatalysts

Enzymes have been increasingly used as sustainable biocatalysts for a broad range of applications ranging from manufacturing drug intermediates and functional foods to bio-sensing in diagnostics and treating waste water. They have attractive properties including high efficiency, strong specificity, desirable biodegradability and renewability. For their intended applications, there are various additional attributes of enzymes required, including thermal stability, long-term storage stability, and scalable processability. Engineering methods such as enzyme immobilization are routinely used to bring these required attributes. However, application-specific attributes are not always met using generic enzyme immobilization methods. In this work, tailored immobilization strategies have been developed to provide application-specific attributes for enzymes, targeting three representative applications including bio-production, diagnostics and water treatment. Through selection of the suitable immobilization carriers and appropriate immobilization chemistries and conditions, high performance of immobilized enzymes has been achieved for intended applications: i) for bio-production application, the representative enzyme lipase has been immobilized into mesoporous silica yolk/shell nanoparticles and onto solid silica nano-spheres, respectively. The activity, stability, reusability, and selectivity of immobilized lipase have been evaluated in both aqueous solution and organic solvent. The immobilized lipase has shown reasonable activity and reusability in aqueous solution. Due to the efficient protection of the silica shell, the immobilized lipase has demonstrated excellent thermal stability and resistance to degradation by protease in comparison to free lipase. Importantly, the immobilized lipase on silica nano-spheres has enhanced selectivity and stability under high pressure in organic solvent, proving the concept that tailored immobilization can indeed enhance enzyme performance under extreme pressures. ii) for diagnostic application, the mesoporous silica nanorods have been selected to immobilize two representative enzymes, horseradish peroxidase (HRP) and alkaline phosphatase (ALP). The immobilized enzymes have facilitated fabrication of the low-cost sensors in the format of low-cost bio-active papers and printed micro-ring plates. The results have demonstrated that enzyme-based sensors have high storage stability and delivery ability under dry state without refrigeration. Also, micro-ring plate-based sensor has shown dual diagnostic functions in one ring v with attractive semi-quantitative diagnostic ability. iii) For environmental application, the representative enzyme penicillinase has been immobilized on two different yet complementary carriers - magnetic graphene oxide (MGO) and asymmetric polymer membranes with nano-channels, respectively. The selection of MGO provides easy recyclability of immobilized enzymes due to strong magnetic property of MGO. Separately, the optimized dimensions of nano-channels have provided sufficient surface area for immobilization of penicillinase while ensuring there are sufficient reaction times for degrading the substrate (penicillin). For both carriers, the immobilized penicillinase has shown excellent efficiency and high reusability while offering attractive storage stability and thermal stability for degradation of penicillin. The tailored enzyme immobilization methods developed in this work have enabled enzymes to function under different physical states including aqueous solution, organic solvent and dry state as required by their specific applications. The knowledge obtained can guide tailoring immobilized enzymes to develop high-efficient and sustainable processes.