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.
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, Kmvalue of 24.73 mM, and kcat/Kmvalue of 22.47 mM-1s-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 (eep) 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.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.|Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Zhe-Ming
ZM
Wu
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.|Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Ren-Chao
RC
Zheng
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.|Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Xiao-Ling
XL
Tang
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. zhengyg@zjut.edu.cn.|Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. zhengyg@zjut.edu.cn.
Yu-Guo
YG
Zheng
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D019117:Burkholderia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003035:Cobalt / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism*; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D006885:Hydroxybutyrates; D011994:Recombinant Proteins; D003035:Cobalt; D000581:Amidohydrolases; C021445:acetamidase; C101846:formamidase
(R)-3,3,3-Trifluoro-2-hydroxy-2-methylpropionic acid; Amidase; Biochemical characterization; Enantioselective resolution