Removal of l -alanine from the production of l -2-aminobutyric acid by introduction of alanine racemase and d -amino acid oxidase

l -2-Aminobutyric acid can be synthesized in a transamination reaction from l -threonine and l -aspartic acid as substrates by the action of threonine deaminase and aromatic aminotransferase, but the by-product l -alanine was produced simultaneously. A small amount of l -alanine increased the complexity of the l -2-aminobutyric acid recovery process because of their extreme similarity in physical and chemical properties. Acetolactate synthase has been introduced to remove the pyruvate intermediate for reducing the l -alanine concentration partially. To eliminate the remnant l -alanine, alanine racemase of Bacillus subtilis in combination with d -amino acid oxidase of Rhodotorula gracilis or Trigonopsis variabilis respectively was introduced into the reaction system for the l -2-aminobutyric acid synthesis. l -Alanine could be completely removed by the action of alanine racemase of B. subtilis and d -amino acid oxidase of R. gracilis ; thereby, high-purity l -2-aminobutyric acid was achieved. The results revealed that alanine racemase could discriminate effectively between l -alanine and l -2-aminobutyric acid, and selectively catalyzed l -alanine to d -alanine reversibly. d -Amino acid oxidase then catalyzed d -alanine to pyruvate stereoselectively. Furthermore, this method was also successfully used to remove the by-product l -alanine in the production of other neutral amino acids such as l -tertiary leucine and l -valine, suggesting that multienzymatic whole-cell catalysis can be employed to provide high purity products.