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.
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Acknowledgments
This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KSCX2-EW-G-7, KSCX2-YW-G-075-14, KSCX2-EW-G-8), the Huzhou Municipal Science and Technology Project (2010ZD1006), the “365” Outstanding Scientific and Technological Innovation Team of Huzhou (2010KC01), and the Hi-Tech industrialized seed fund projects by Pudong New Area and Chinese Academy of Sciences (No. PKC2010-03). This work was also supported in part by National Basic Research Program of China (973: 2007CB707803, 2011CBA00806).
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Li Zhu and Rongsheng Tao contributed equally to this work.
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Zhu, L., Tao, R., Wang, Y. et al. Removal of l-alanine from the production of l-2-aminobutyric acid by introduction of alanine racemase and d-amino acid oxidase. Appl Microbiol Biotechnol 90, 903–910 (2011). https://doi.org/10.1007/s00253-011-3127-4
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DOI: https://doi.org/10.1007/s00253-011-3127-4