Engineering of a bi-enzymatic reaction for efficient production of the ascorbic acid precursor 2-keto-L-gulonic acid

The enzymatic production of the vitamin C precursor 2-keto-L-gulonate (2-KLG) from 2,5-diketo-D-gluconate (2,5-DKG) was catalyzed by coupling 2,5-diketo-D-gluconic acid reductase (2,5-0KG reductase) via its coenzyme to glucose dehydrogenase. This bi-enzymatic process shows complicated inhibition patterns caused by reaction products, NADP(+) and NADPH. The bioconversion was optimized by modeling. Rate equations were derived for both enzymes and solved simultaneously by a numerical method to predict 2-KLG production. A series of batch conversions were used to verify the model and construct isocharts to determine optimal process parameters. The following key parameters for a fast and efficient conversion were found: (1) the NADP(H) concentration, (2) the volumetric activity of 2,5-DKG reductase, (3) the ratio of synthetic enzyme activity to regenerate enzyme activity and (4) the glucono-1,5-lactone concentration. By modeling the space-time yield of the process was nearly doubled and the coenzyme concentration reduced threefold. (C) 2013 Elsevier B.V. All rights reserved.