Precipitated-crosslinked multi-enzyme hybrid nanoflowers for efficient synthesis of α-ketoglutaric acid

Cascade catalysis of glutamate oxidase (GLOX) and catalase (CAT) to perform one-pot synthetic route for α-ketoglutarate (α-KG) production offers several advantages including simplicity of operation and the generation of few reaction by-products. Nevertheless, the instability of free GLOX and CAT, the high production cost and the difficulty of recycling severely limits its industrial utilisation. Here, catalase-inorganic hybrid nanoflowers were first prepared, and cross-linked with GLOX precipitates by a macromolecular cross-linking agent dextran polyaldehyde to form a novel dual enzyme precipitation-cross-linking hybrid nanoflower (GLOX@CAT-HNFs). The resultant GLOX@CAT-HNFs exhibited higher catalytic activity than conventional combined cross-linked enzyme aggregates (combi-CLEAs) and hybrid nanoflowers (GLOX&CAT-HNFs). The GLOX@CAT-HNFs exhibited 92 % activity recovery whereas combi-CLEAs and GLOX&CAT-HNFs was 87 % and 72 %, respectively. Meanwhile, the GLOX@CAT-HNFs showed better thermal stability, pH and storage stability than free enzymes. After incubation at 60 °C for 100 min, GLOX@CAT-HNFs maintained 70 % of its initial activity while free enzyme was only 18.52 %. Furthermore, after 7 cycles of use, GLOX@CAT-HNFs maintained 68.79 % of its initial activity, indicating excellent reusability. Benefiting from the excellent stability and reusability of GLOX@CAT-HNFs, a nearly 100 % (99.64 %) conversion of L-glutamate to α-KG was achieved, over 1.79 times higher than that of the free GLOX system (55.53 %). This work provides a feasibility for constructing a high-performance cascade catalyst of multiple enzymes.