A chemo-biocatalyst based on glutamate oxidase-integrated biomimetic trimanganese tetraoxide as cascade composite nano-catalyst for synthesis of α­Ketoglutaric acid.

The combination of chemo- and biocatalysts to perform one-pot synthetic route has presented great challenges for decades. Herein, glutamate oxidase (GLOX) and trimanganese tetraoxide (Mn3O4) nanocrystals were combined for the first time by one-step biomineralization to construct a mimic multi-enzyme system (GLOX@Mn3O4) for chemoenzymatic synthesis of α­ketoglutaric acid (α­KG). Mn3O4 not only served as a support for the enzyme immobilization, but also contributed its catalytic activity to co-operate with natural enzymes for the cascade reactions. The as-synthesized chemo-enzyme catalysts with directly contacted catalytic sites of the enzyme and inorganic catalyst maximizes the substrate channeling effffects for in situ rapid decomposition of the oxidative intermediate, H2O2, during the enzymatic oxidation of sodium glutamate, thus relieving the inhibition of H2O2 accumulation for GLOX. Benefiting from the excellent stability and reusability of GLOX@Mn3O4, a nearly 100% conversion (99.7%) of l-glutamate to α-KG was achieved, over 4.7 times higher than that of the free GLOX system (21.2%). This work provides a feasibility for constructing a high-performance chemo-enzyme catalyst for cascade catalysis, especially for those reactions with toxic intermediates.

Glutamate Oxidase-Integrated Biomimetic Metal-Organic Framework Hybrids as Cascade Nanozymes for Ultrasensitive Glutamate Detection.

The hybrid coupling of biocatalysts and chemical catalysts plays a vital role in the fields of catalysis, sensing, and medical treatment due to the integrated advantages in the high activity of natural enzymes and the excellent stability of nanozymes. Herein, a new nanozyme/natural enzyme hybrid biosensor was established for ultrasensitive glutamate detection. The MIL-88B(Fe)-NH2 material with remarkable peroxidase mimic activity and stability was used as a nanozyme and carrier for immobilizing glutamate oxidase (GLOX) through Schiff base reaction to construct a chem-enzyme cascade detector (MIL-88B(Fe)-NH2@GLOX). The resultant MIL-88B(Fe)-NH2@GLOX exhibited a wide linear range (1-100 µM), with a low detection limit of 2.5 µM for glutamate detection. Furthermore, the MIL-88B(Fe)-NH2@GLOX displayed excellent reusability and storage stability. After repeated seven cycles, MIL-88B(Fe)-NH2-GLOX (GLOX was adsorbed on MIL-88B(Fe)-NH2) lost most of its activity, whereas MIL-88B(Fe)-NH2@GLOX still retained 69% of its initial activity. Meanwhile, MIL-88B(Fe)-NH2@GLOX maintained 60% of its initial activity after storage for 90 days, while free GLOX only retained 30% of its initial activity. This strategy of integrating MOF mimics and natural enzymes for cascade catalysis makes it possible to design an efficient and stable chemo-enzyme composite catalysts, which are promising for applications in biosensing and biomimetic catalysis.

Hierarchical micro- and mesoporous ZIF-8 with core-shell superstructures using colloidal metal sulfates as soft templates for enzyme immobilization.

Metal-organic frameworks (MOFs), with large specific surface area and tunable porosity, have gained lots of attention for immobilizing enzymes. However, the intrinsic open channels of most reported MOFs are generally smaller than 2 nm, which significantly prevents the passage of enzymes, and the diffusion efficiency of substrates and products. Here we report a new hierarchical micro-mesoporous zeolitic imidazolate framework-8 (ZIF-8) with core-shell superstructure (HZIF-8) using colloidal hydrated zinc sulfate (ZnSO4·7H2O) as a soft template for enzyme immobilization. The ZnSO4·7H2O forms an aggregation of colloids due to the self-conglobation effect in methanol, which affords a soft template for the formation of HZIF-8. Cytochrome C (Cyt C) was immobilized in interior of HZIF-8 through entrapment during the formation of HZIF-8. The resultant immobilized Cyt C (Cyt C@HZIF-8) exhibited 4-fold and 3-fold higher activity than free Cyt C and Cyt C encapsulated in conventional microporous ZIF-8 (Cyt C@ZIF-8), respectively. Meanwhile, the Km value of Cyt C@HZIF-8 significantly decreased due to the presence of mesopores compared with Cyt C@ZIF-8, indicating enhanced substrate affinity. After 7 cycles, Cyt C@HZIF-8 still maintained 70% of its initial activity whereas Cyt C@ZIF-8 only retained 10% of its initial activity. Moreover, the obtained HZIF-8 showed outstanding performance in co-immobilization of multi-enzyme for the detection of glucose.

Enhanced enzymatic performance of immobilized lipase on metal organic frameworks with superhydrophobic coating for biodiesel production.

Inspired by the interfacial catalysis of lipase, Herein, the hydrophobic ZIF-L coated with polydimethylsiloxane (PDMS) were prepared by chemical vapor deposition (CVD) and used to immobilize lipase from Aspergillus oryzae (AOL) for biodiesel production. The results showed that the PDMS coating enhanced the stability of ZIF-8 and ZIF-L in PBS. Immobilization efficiency of AOL on PDMS-modified ZIF-L was 96% under optimized conditions. The resultant immobilized lipase (AOL@PDMS-ZIF-L) exhibited higher activity recovery (430%) than AOL@ZIF-L. Meanwhile, compared with free lipase, the AOL@PDMS-ZIF-L exhibited better storage stability and thermal stability. After 150 days of storage, the free lipase retained only 20% of its original activity of hydrolyzing p-NPP, while the AOL@PDMS-ZIF-L still retained 90% of its original activity. The biodiesel yield catalyzed from soybean oil by free lipase was only 69%, However, the biodiesel yield by AOL@PDMS-ZIF-L reached 94%, and could still be maintained at 85% even after 5 consecutive cycles. It is believed that this convenient and versatile strategy has great promise in the important fields of immobilized lipase on MOF for biodiesel production.

Three-dimensional ordered magnetic macroporous metal-organic frameworks for enzyme immobilization.

Metal-organic frameworks (MOFs) have been emerged as a promising support for immobilizing enzymes owing to the tunable porosity, high surface area, and structural diversity. However, most of these possess nanometer size and small pores, which are difficult to recover them from the reaction medium and present low immobilization efficiency and protein loading capacity, and high substrate diffusion limitations. Herein, a novel magnetic amino-functionalized zeolitic imidazolate framework-8 (ZIF-8) with 3D highly ordered macroporous structure was synthesized using the assembled polystyrene (PS) nanosphere monoliths as a template. Subsequently, catalase (CAT) molecules were immobilized on the surface of macroporous magnetic ZIF-8 and inside the macropores by precipitation, covalent binding and cross-linking. The resultant immobilized CAT showed high immobilization efficiency (58%) and protein loading capacity (29%), leading to 500% higher activity than the immobilized CAT on ZIF-8 (CAT/ZIF-8). Meanwhile, the immobilized CAT could be easily recovered with a magnet without obvious activity loss. The traditional CAT/ZIF-8 lost its activity after 6 cycles, whereas, the immobilized CAT retained 90% activity of its initial activity after reusing for 8 cycles, indicating excellent reusability. In conclusion, this study provides a facile and efficient approach to immobilize enzymes on/in MOFs with enhanced activity and excellent recyclability.