Magnetic Combined Cross-Linked Enzyme Aggregates of Glutamate Oxidase and Catalase by Macromolecular Cross-Linker for Synthesis of α-Ketoglutaric Acid

Cascade catalysis of glutamate oxidase (GLOX) and catalase (CAT) is an important one-pot synthetic route for α-ketoglutarate (α-KG) production. However, as the soluble natural proteins, the instability of free GLOX and CAT and the difficulty of recycling severely limits its application. Here, magnetic dual-enzyme cross-linked enzyme aggregates (combi-CLEAs) of GLOX and CAT were successfully prepared with amino-functionalized Fe 3 O 4 magnetic nanoparticles as the carrier by a macromolecular cross-linking agent. Preparation conditions of the combi-CLEAs were systematically optimized. The highest activity recovery of combi-CLEAs reached 81% under the optimized conditions. Meanwhile, the combi-CLEAs exhibited high stability than free enzymes, including temperature tolerance, pH tolerance, and storage capacity. After incubation at 80 °C for 30 min, the magnetic combi-CLEAs retained 25% of its initial activity whereas the free enzymes maintained only 10%. Moreover, after 6 cycles of use, the combi-CLEAs maintained 74% of its initial activity, indicating excellent reusability. In particular, the combi-CLEAs exhibited high substrate tolerance and conversion efficiency of L-glutamate to α-KG. The total α-KG yield by combi-CLEAs after 6 cycles of use was 5.8 times higher than that of the free enzyme system.