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Characterization of Four New Distinct ω-Transaminases from Pseudomonas putida NBRC 14164 for Kinetic Resolution of Racemic Amines and Amino Alcohols

Applied Biochemistry and Biotechnology volume 181, pages 972–985 (2017)Cite this article

Abstract

Four uncharacterized ω-transaminases (ωTAs) from Pseudomonas putida NBRC 14164 have been identified and cloned from the pool of fully sequenced genomes. The genes were functionally expressed in Escherichia coli BL21, and the enzymes were purified and characterized. Four TAs showed highly (S)-selective ωTA activity and converted (S)-α-methylbenzylamine and pyruvate to acetophenone and l-Ala. The maximum activity of cloned enzymes was in the pH range of 8.0–8.5 (Pp36420), 8.5–9.5 (Pp21050), 9.0–9.5 (PpspuC), and 9.5–10.5 (PpbauA), and the optimal temperatures were at 35 °C (Pp36420, Pp21050, and PpspuC) and 50 °C (PpbauA), respectively, with K M of 161.3 mM (Pp21050), 136.7 mM (PpbauA), 398.5 mM (Pp36420), and 130.9 mM (PpspuC) and yielding a catalytic efficiency k cat/K M of 0.015, 0.003, 0.012, and 0.023 mM−1 s−1. Several racemic amines and amino alcohols were resolved by the cloned ωTAs; perfect conversions (48–50 %) were obtained by at least one enzyme, and the residual substrates were left with 97–99 % ee. Kinetic resolution of racemic phenylglycinol was done with PpspuC in a 100-mL scale. Enaniomeric excess of (S)-phenylglycinol reached 99 % with 45 % isolated yield. The high enantioselectivity and large substrate spectra of the cloned PpTAs showed an attractive potency for biotechnology application in production of chiral amines and amino alcohols.

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Acknowledgments

This work was financially supported by the Qualified Personnel Foundation of Taiyuan University of Technology (grant no. tyut-rc201484a) and the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (STIP) (grant no. 2015132).

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  1. Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, No.79 West Yingze Street, Taiyuan, Shanxi, 030024, China

    Hua-Lei Wu, Jian-Dong Zhang, Chao-Feng Zhang, Xiao-Jun Fan, Hong-Hong Chang & Wen-Long Wei

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  1. Hua-Lei Wu
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Wu, HL., Zhang, JD., Zhang, CF. et al. Characterization of Four New Distinct ω-Transaminases from Pseudomonas putida NBRC 14164 for Kinetic Resolution of Racemic Amines and Amino Alcohols. Appl Biochem Biotechnol 181, 972–985 (2017). https://doi.org/10.1007/s12010-016-2263-9

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  • DOI: https://doi.org/10.1007/s12010-016-2263-9

Keywords

  • Transaminase
  • Kinetic resolution
  • Enantioselectivity
  • Chiral amino alcohol
  • Pseudomonas putida