title: Removal of L-alanine from the production of L-2-aminobutyric acid by introduction of alanine racemase and D-amino acid oxidase.
abstract: 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.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Rd, Shanghai 200032, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.forename: Rongsheng
authors.initials: R
authors.lastname: Tao
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Yu
authors.initials: Y
authors.lastname: Jiang
authors.forename: Xin
authors.initials: X
authors.lastname: Lin
authors.forename: Yunliu
authors.initials: Y
authors.lastname: Yang
authors.forename: Huabao
authors.initials: H
authors.lastname: Zheng
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000411:Alanine Racemase / Q000235:genetics / Q000378:metabolism*; D000613:Aminobutyrates / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D001711:Biotransformation; D003605:D-Amino-Acid Oxidase / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D012248:Rhodotorula / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D000613:Aminobutyrates; D001426:Bacterial Proteins; D005656:Fungal Proteins; C012223:alpha-aminobutyric acid; D003605:D-Amino-Acid Oxidase; D000411:Alanine Racemase; D000409:Alanine