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Substrate profiling of cyclohexylamine oxidase and its mutants reveals new biocatalytic potential in deracemization of racemic amines

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Abstract

A cyclohexylamine oxidase (CHAO) of bacterial origin was previously shown to be a potentially useful catalyst in the deracemization of racemic primary amines. To further explore the properties and application of this enzyme, five single-amino acid substitution mutants (L199A, M226A, Y321A, Y321F, and L353M) were created based on superimposition of the tertiary structure of CHAO and the monoamine oxidase (MAO) B homolog. The substrate specificity of the purified wild-type and five mutant enzymes were examined towards 38 structurally diverse amines. All the enzymes exhibited better activity for primary amines than secondary and tertiary amines and in general exhibited high stereoselectivity. Among the mutant enzymes, M226A displayed an enhanced activity (5–400 %) towards most substrates, and L353M showed 7–445 % higher activity towards primary aliphatic amines with cycloalkane or aromatic moieties. Kinetic parameters revealed that both Y321 mutants showed higher catalytic efficiency towards cyclooctanamine, whereas the wild-type CHAO (wt CHAO) was most efficient towards cyclohexylamine. The wt CHAO or variant L353M in combination with a borane–ammonia complex as reducing agent was applied to the deracemization of 1-aminotetraline to give the (R)-enantiomer, a precursor of an antidepressant drug Norsertraline, in good yield (73–76 %), demonstrating their application potential in chiral amine synthesis.

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Acknowledgment

This work was financially supported by the National Natural Science Foundation of China (grant no. 21072151) and National Key Basic Research and Development Program (973 Program, grant no. 2011CB710801). Support by Chinese Academy of Sciences Visiting Professorship for Senior International Scientists to P.C.K. Lau (grant no. 2011T1S28) is gratefully acknowledged. I. Ahmad Mirza is gratefully acknowledged for the suggestion of choice of mutants. Past and present members of Lau’s laboratory are thanked for their various assistances.

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Authors and Affiliations

  1. National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin, 300308, People’s Republic of China

    Guangyue Li, Jie Ren, Dalong Zhang, Qiaqing Wu, Jinhui Feng & Dunming Zhu

  2. National Research Council Canada, 6100 Royalmount Avenue, Montreal, QC, H4P 2R2, Canada

    Peter C. K. Lau

  3. Departments of Chemistry and Microbiology & Immunology, McGill University, Montreal, QC, H3A 2B4, Canada

    Peter C. K. Lau

  4. FQRNT Centre in Green Chemistry and Catalysis, Montreal, QC, Canada

    Peter C. K. Lau

  5. Department of Life Science & Biotechnology and ORDIST, Kansai University, Suita, Osaka, 564-8680, Japan

    Hiroaki Iwaki & Yoshie Hasegawa

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  1. Guangyue Li
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  2. Jie Ren
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  3. Hiroaki Iwaki
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  4. Dalong Zhang
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  5. Yoshie Hasegawa
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  6. Qiaqing Wu
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  7. Jinhui Feng
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  8. Peter C. K. Lau
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  9. Dunming Zhu
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Correspondence to Peter C. K. Lau or Dunming Zhu.

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Li, G., Ren, J., Iwaki, H. et al. Substrate profiling of cyclohexylamine oxidase and its mutants reveals new biocatalytic potential in deracemization of racemic amines. Appl Microbiol Biotechnol 98, 1681–1689 (2014). https://doi.org/10.1007/s00253-013-5028-1

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  • DOI: https://doi.org/10.1007/s00253-013-5028-1

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