Production of D-glutamate from L-glutamate with glutamate racemase and L-glutamate oxidase

We studied production of D-glutamate from L-glutamate using a bioreactor co nsisting of two columns of sequentially connected immobilized glutamate rac emase (EC 5.1.1.3, from Bacillus subtilis no 3336) and L-glutamate oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6): L-glutamate was racemized by t he glutamate racemase column, and then L-glutamate was oxidized by the L-gl utamate oxidase column. Consequently only D-glutamate remained, and was eas ily separated from the alpha-ketoglutarate formed by anion-exchange chromat ography. Both enzymes were highly stabilized by immobilization. The pH and temperature optima of immobilized glutamate racemase (pH 8, 40 degrees C) w ere similar to those of immobilized L-glutamate oxidase (pH 7, 50 degrees C ). Accordingly, we connected the two columns tandemly to do both enzyme rea ctions under the same conditions. Actually 4.5 mu mol of D-glutamate was pr oduced and isolated from 10 mu mol of L-glutamate, about 90% of the theoret ical yield.