Simultaneous Production of Amino Acid Dehydrogenases and Glucose Dehydrogenase by Bacillus Megaterium and Their Utilization for L-Valine Synthesis with Nadh Regeneration

Abstract

An improved method for the simultaneous production of valine dehydrogenase and glucose dehydrogenase by Bacillus megaterium (ATCC 39118) is described. The highest yields in volumetric activities (8200 U.S^-1' of glucose dehydrogenase and 7200 U.S¹ of valine dehydrogenase) were obtained using a fed batch cultivation technique with glucose, yeast extract and corn steep liquor in the feed medium. The main characteristics (stability, optimal pH, Michaelis constants, substrate and product inhibitions) of valine dehydrogenase and glucose dehydrogenase from crude extracts were determined. B. megaterium crude extract was suitable for synthesis of L-valine from $aL-keto isovalerate with glucose dehydrogenase as the NADH-regenerating enzyme and the conditions of the conversion have been optimized. $aL-Keto acid was supplied in fed batch mode in order to avoid substrate inhibition and was not involved in side reactions. With the optimized system, a concentration of 95 mM L-valine was obtained in 45 hours with a molar conversion yield close to 100%.

Key Words:

• Amino acid dehydrogenase
• glucose dehydrogenase
• L-valine
• NADH regeneration
• Bacillus megaterium.