Production of Aspartic Acid and Enzymatic Alteration in Pyruvate Kinase Mutants of Brevibacterium flavum

抄録

A pyruvate kinase-lacking mutant of Brevibacteriumflavum produced 22.6 g/liter of L-aspartic acid with glutamic acid as a by-product, when cultured for 48 hr in a medium containing 100 g/litei of glucose. The production clearly depended on the amount of biotin added. This strain, 70, was derived by several steps of mutation from wild strain 2247 producing glutamate, successively via a citrate synthase-defective glutamate auxotroph, strain 214, a prototrophic revertant, strain 15-8 producing 10 g/liter of L-aspartic acid, and an S-(2-aminoethyl)-L-cysteine-resistant mutant, strair 1-231, having low pyruvate kinase and homoserine dehydrogenase and producing lysine. Strain 70. amethionine-insensitive revertant from strain 1-231, had a normal level of homoserine dehydrogenase but no pyruvate kinase. Its citrate synthase activity was about half that of the wild strain at saturated concentrations of the substrates with Michaelis constants for oxalacetate and acetyl- GoA of 1 10 and 6 times as high as those of the wild-type enzyme, respectively. The mutational step for these alterations in citrate synthase was strain 15-8. Phosphoenolpyruvate carboxylase of strain 70 showed 1.5-fold higher activity in the crude extract at saturated concentrations of phosphoenolpyruvate, a lower Michaelis constant (1.5mM). for the substrate, phosphoenolpyruvate, less sensitivity to the feedback inhibition by aspartate, and higher sensitivities to the activators, acetyl- CoA and fructose-1, 6-bisphosphate, than those of the wild strain. The concentrations of aspartate giving 50% inhibition were 6.2- and 4.5-fold higher in the absence and presence of acetyl-CoA. respectively.