In many respects, enzymes offer advantages over traditional chemical processes due to their decreased energy requirements for function and inherent greener processing. However, significant barriers exist for the utilization of enzymes in industrial processes due to their limited stabilities and inability to operate over larger temperature and pH ranges. Immobilization of enzymes onto solid supports has gained attention as an alternative to traditional chemical processes due to enhanced enzymatic performance and stability. This study demonstrates the co-immobilization of glucose oxidase (GOx) and horseradish peroxidase (HRP) as an enzyme system on Metal-Organic Frameworks (MOFs), UiO-66 and UiO-66-NH2, that produces a more effective biocatalyst as shown by the oxidation of pyrogallol. The two MOFs utilized as solid supports for immobilization were chosen to investigate how modifications of the MOF linker affect stability at the enzyme/MOF interface and subsequent activity of the enzyme system. The enzymes work in concert with activation of HRP through the addition of glucose as a substrate for GOx. Enzyme immobilization and leaching studies showed HRP/GOx@UiO-66-NH2 immobilized 6% more than HRP/GOx@UiO-66, and leached only 36% of the immobilized enzymes over three days in the solution. The enzyme/MOF composites also showed increased enzyme activity in comparison with the free enzyme system: the composite HRP/GOx@UiO-66-NH2 displayed 189 U/mg activity and HRP/GOx@UiO-66 showed 143 U/mg while the free enzyme showed 100 U/mg enzyme activity. This increase in stability and activity is due to the amine group of the MOF linker in HRP/GOx@UiO-66-NH2 enhancing electrostatic interactions at the enzyme/MOF interface, thereby producing the most stable biocatalyst material in solution. The HRP/GOx@UiO-66-NH2 also showed long-term stability in the solid state for over a month at room temperature.

A leading biotechnological advancement in the field of biocatalysis is the immobilization of enzymes on solid supports to create more stable and recyclable systems. Metal-organic frameworks (MOFs) are porous materials that have been explored as solid supports for enzyme immobilization. Composed of organic linkers and inorganic nodes, MOFs feature empty void space with large surface areas and have the ability to be modified post-synthesis. Our target enzyme system for immobilization is glucose oxidase (GOx) and chloroperoxidase (CPO). Glucose oxidase catalyzes the oxidation of glucose and is used for many applications in biosensing, biofuel cells, and food production. Chloroperoxidase is a fungal heme enzyme that catalyzes peroxide-dependent halogenation, oxidation, and hydroxylation. These two enzymes work sequentially in this enzyme system by GOx producing peroxide, which activates CPO that reacts with a suitable substrate. This study focuses on using a zirconium-based MOF, UiO-66-NH2, to immobilize the enzyme system via crosslinking with the MOF’s amine group on the surface of the MOF. This study investigates two different crosslinkers: disuccinimidyl glutarate (DSG) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinidimide (NHS), providing stable crosslinking of the MOF to the enzymes. The two crosslinkers are used to covalently bond CPO and GOx onto UiO-66-NH2, and a comparison of the recyclability and enzymatic activity of the single immobilization of CPO and the doubly immobilized CPO and GOx is discussed through assays and characterization analyses. The DSG-crosslinked composites displayed enhanced activity relative to the free enzyme, and all crosslinked enzyme/MOF composites demonstrated recyclability, with at least 30% of the activity being retained after four catalytic cycles. The results of this report will aid researchers in utilizing CPO as a biocatalyst that is more active and has greater recyclability.

Novel biocatalysts that feature enzymes immobilized onto solid supports have recently become a major research focus in an effort to create more sustainable and greener chemistries in catalysis. Many of these novel biocatalyst systems feature enzymes immobilized onto metal−organic frameworks (MOFs), which have been shown to increase enzyme activity, stability, and recyclability in industrial processes. While the strategies used for immobilizing enzymes onto MOFs can vary, the conditions always require a buffer to maintain the functionality of the enzymes during immobilization. This report brings attention to critical buffer effects important to consider when developing enzyme/MOF biocatalysts, specifically for buffering systems containing phosphate ions. A comparative analysis of different enzyme/ MOF biocatalysts featuring horseradish peroxidase and/or glucose oxidase immobilized onto the MOFs UiO-66, UiO-66-NH 2 , and UiO-67 using a noncoordinate buffering system (MOPSO buffer) and a phosphate buffering system (PBS) show that phosphate ions can have an inhibitory effect. Previous studies utilizing phosphate buffers for enzyme immobilization onto MOFs have shown Fourier transform infrared (FT-IR) spectra that have been assigned stretching frequencies associated with enzymes after immobilization. Analyses and characterizations using zeta potential measurements, scanning electron microscopy, Brunauer−Emmett−Teller surface area, powder X-ray diffraction, Energy Dispersive X-ray Spectroscopy, and FT-IR show concerning differences in enzyme loading and activity based on the buffering system used during immobilization.