Preparation of α-Ketoglutaric Acid by a Dual Enzyme Cascade Bioenzymatic Method.

α-Ketoglutaric acid (α-KG) is an important intermediate in the tricarboxylic acid cycle and is widely used in organic synthesis, nutritional fortification agents, and other fields. In this study, we co-expressed L-glutamate oxidase (LGOX) from Streptomyces ghanaensis and catalase (CAT) from Escherichia coli and optimized the expression intensity of LGOX and CAT as well as the whole-cell catalytic conditions, which achieved 87.97% conversion of monosodium glutamate and 96.77 g/L yield of α-KG in the whole-cell catalytic process in 12 h. Further, for the LGOX and CAT dual enzyme system, five immobilization methods were designed, and ten batches of ZIF-8-GA immobilized cells with catalytic efficiency of more than 80% were prepared, and the immobilized cells were catalyzed for 12 h, with the conversion rate of monosodium glutamate of 88.46% and the yield of α-KG of 79.61 g/L. The use of a dual-enzyme system with a single microorganism for α-KG production overcomes the limitations of existing methods relying on exogenous catalase (CAT) addition. By employing a novel immobilization technique, this approach enables continuous, batch-based, low-cost production, offering a new solution for the green and efficient biomanufacturing of α-ketoglutaric acid.