An enzymatic process to a-ketoglutarate from L -glutamate: the coupled system L -glutamate dehydrogenase/NADH oxidase Peter O ¨ dman, a,b William B. Wellborn b and Andreas S. Bommarius b, * a Department of Chemical Engineering, Lund University, S-22100 Lund, Sweden b School of Chemical and Biomolecular Engineering, Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332-0363, USA Received 10 June 2004; accepted 7 July 2004 Available online 11 September 2004 Abstract—a-Ketoglutarate, employed to treat mild chronic renal insufficiency, was obtained through enzymatic oxidation of mono- sodium glutamate (MSG) catalyzed by L-glutamate dehydrogenase (L-gluDH) coupled with NADH oxidase for the regeneration of NADH back to NAD + . The irreversible reduction of molecular oxygen to water by NADH oxidase is demonstrated to drive oxi- dation of MSG to a-ketoglutarate to completion. L-gluDH was found to be inhibited by all three oxidative deamination products, a- ketoglutarate, NADH, and ammonia. As the pH in the current system was balanced by sodium, not ammonia, and NADH was recycled to NAD + , inhibition of L-gluDH by a-ketoglutarate is believed to present the biggest challenge to an efficient process. In a batch experiment, we achieved a volumetric productivity of 1 g/(LÆd). Ó 2004 Elsevier Ltd. All rights reserved. 1. Introduction Keto acids are the nitrogen-free analogs of amino acids, and are transaminated to form the respective amino acid in the body. This improves nitrogen balance at a lower nitrogen intake and corresponds with relief of the symp- toms of uremia while maintaining good nutrition. 1 Thus, a-ketoglutarate is beneficial as a component of endoperitoneal solutions for the conservative treatment of mild chronic renal insufficiency in hemodialysis patients and, in combination with calcium carbonate, of hyperphosphatemia. 2–4 Long-term co-administration of keto acids, erythropoietin, and low-protein diet was also associated with a delay in progression of renal insufficiency and a reduction in proteinuria. a-Ketoglutarate is most advantageously produced by oxidation of inexpensive L-glutamate (MSG), a flavor enhancer available at huge scale. The current process involves oxidation of MSG in air with a copper com- plex. The oxidation of MSG, however, can be afforded enzymatically, by catalysis with L-glutamate dehydroge- nase (L-gluDH), with simultaneous reduction of NAD + to NADH. Herein, we demonstrate the feasibility to couple L-gluDH with NADH oxidase to regenerate the co-factor NADH back to NAD + and to drive the ther- modynamically unfavorable equilibrium from L-gluta- mate to a-ketoglutarate. This is accompanied by the irreversible four-electron reduction of molecular oxygen to water. Recently, we published the characterization of a novel water-forming NADH oxidase from Lactobacil- lus sanfranciscensis, which accomplishes this task. 5,6 Herein, we use this protein in conjunction with L-gluDH from Clostridium symbiosum 7,8 expressed in Escherichia coli, to transform MSG to the sodium salt of a-ketoglut- arate (Fig. 1). 0957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetasy.2004.07.055 * Corresponding author. Tel.: +1 404 385 1334; fax: +1 404 894 2291; e-mail: andreas.bommarius@chbe.gatech.edu NH 3 H + NaO COOH O O O 2 NADH NAD + H 2 O NaO NH 2 COOH O H 2 O L-gluDH NADH Oxidase + ½ + + MSG α-ketoglutarate Figure 1. Process to a-ketoglutarate from monosodium glutamate (MSG) catalyzed by L-gluDH/NADH oxidase. Tetrahedron: Asymmetry 15 (2004) 2933–2937 Tetrahedron: Asymmetry