Production of 2-Keto- l -Gulonic Acid from d -Glucose by Two-Stage Fermentation

Abstract

A practical method for the production of calcium 2-keto- l -gulonate (an intermediate in the Reichstein synthesis of l -ascorbic acid) from d -glucose has been established by using a two-stage fermentation system. d -Glucose was first converted to calcium 2,5-diketo- d -gluconate by a mutant strain of Erwinia sp. in a medium containing d -glucose, corn steep liquor, (NH 4 ) 2 HPO 4 , and CaCO 3 . After a 26-h cultivation, 328.6 mg of calcium 2,5-diketo- d -gluconate per ml was obtained, with a 94.5% yield from d -glucose. This broth was used directly for the next conversion without removal of cells by treatment with sodium dodecyl sulfate. The stereospecific reduction of calcium 2,5-diketo- d -gluconate to calcium 2-keto- l -gulonate was performed with a mutant strain of Corynebacterium sp. When the cell growth reached a maximum (about 16 h) in a medium containing d -glucose, corn steep liquor, NaNO 3 , KH 2 PO 4 , and trace elements, NaNO 3 was added to the culture, and then the calcium 2,5-diketo- d -gluconate broth was fed over a period of about 50 h. Since the mutant strain requires a hydrogen donor for reduction, the calcium 2,5-diketo- d -gluconate broth was mixed with d -glucose before being fed. The results of four two-stage fermentations in 10-m 3 conventional fermentors showed that an average of 106.3 mg of calcium 2-keto- l -gulonate per ml was obtained, with a 84.6% yield from d -glucose, the starting material of calcium 2,5-diketo- d -gluconate production. Calcium 2-keto- l -gulonate was stable in the broth. Neither 2-keto- d -gluconic acid nor 5-keto- d -gluconic acid was detected in the final broth.