Applied Microbiology and Biotechnology

, Volume 101, Issue 5, pp 1953–1964 | Cite as

Identification and characterization of a thermostable and cobalt-dependent amidase from Burkholderia phytofirmans ZJB-15079 for efficient synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid

  • Zhe-Ming Wu
  • Ren-Chao Zheng
  • Xiao-Ling Tang
  • Yu-Guo ZhengEmail author
Biotechnologically relevant enzymes and proteins


Enantiomerically pure 3,3,3-trifluoro-2-hydroxy-2-methylpropionic acids are important chiral building blocks for a series of pharmaceuticals. Here, a bacteria strain with 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide-degrading ability was screened and identified as Burkholderia phytofirmans ZJB-15079, from which a novel amidase (Bp-Ami) was cloned and demonstrated to be capable of kinetic resolution of rac-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide to optically pure (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid. Phylogenetic analysis revealed that Bp-Ami was closely located to the acetamidase/formamidase (FmdA_AmdA) family, and it shared high homology with acetamidases. Bp-Ami was found to be the first cobalt-dependent FmdA_AmdA family amidase. The enzyme activity was significantly increased by 37.7-fold in the presence of 1 mM Co2+, with a specific activity of 753.5 U/mg, K m value of 24.73 mM, and k cat /K m value of 22.47 mM−1 s−1. As an enzyme from mesophile, Bp-Ami exhibited extreme thermostability with a half-life of 47.93 h at 80 °C, which was even superior to other reported amidases from thermophiles. The whole cell catalysis of 200 g/L 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide by Escherichia coli harboring Bp-Ami (5 g/L) resulted in 44 % yield and an enantiomeric excess (ee p) of 95 % within 10 min (E = 86). The high substrate tolerance, high specific activity, and extreme thermostability demonstrated the great potential of Bp-Ami for efficient biocatalytic synthesis of (R)-3,3,3-trifluoro-2-hydroxy-2-methylpropionic acid.


Amidase Biochemical characterization Enantioselective resolution (R)-3,3,3-Trifluoro-2-hydroxy-2-methylpropionic acid 



This work was financially supported by the National Natural Science Foundation of China (No. 21202150, No. 21602199), Natural Science Foundation of Zhejiang Province (Y4080334, LY13B060004), and National High Technology Research and Development Program of China (No. 2012AA022201B).

Compliance with ethical standards

Conflict of interest

The author declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7921_MOESM1_ESM.pdf (559 kb)
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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Zhe-Ming Wu
    • 1
    • 2
  • Ren-Chao Zheng
    • 1
    • 2
  • Xiao-Ling Tang
    • 1
    • 2
  • Yu-Guo Zheng
    • 1
    • 2
    Email author
  1. 1.Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and BioengineeringZhejiang University of TechnologyHangzhouPeople’s Republic of China
  2. 2.Engineering Research Center of Bioconversion and Biopurification of Ministry of EducationZhejiang University of TechnologyHangzhouPeople’s Republic of China

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