Enhancing the organic solvent resistance of ω-amine transaminase for enantioselective synthesis of (R)-(+)-1(1-naphthyl)-ethylamine

Chun-Ning Wang; Shuai Qiu; Fang-Fang Fan; Chang-Jiang Lyu; Sheng Hu; Wei-Rui Zhao; Jia-Qi Mei; Le-He Mei; Jun Huang

doi:10.1002/biot.202300120

Enhancing the organic solvent resistance of ω-amine transaminase for enantioselective synthesis of (R)-(+)-1(1-naphthyl)-ethylamine

Biotechnol J. 2023 Oct;18(10):e2300120. doi: 10.1002/biot.202300120. Epub 2023 Jul 9.

Authors

Chun-Ning Wang¹, Shuai Qiu¹, Fang-Fang Fan¹, Chang-Jiang Lyu¹, Sheng Hu², Wei-Rui Zhao², Jia-Qi Mei³, Le-He Mei^{2

4

5}, Jun Huang¹

Affiliations

¹ Key Laboratory of Chemical and Biological Processing Technology for Farm Products of Zhejiang Province, Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, China.
² School of Biological and Chemical Engineering, Ningbo Tech University, Ningbo, China.
³ Hangzhou Huadong Medicine Group Co. Ltd, Hangzhou, China.
⁴ College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China.
⁵ Jinhua Advanced Research Institute, Jinhua, China.

PMID: 37337619
DOI: 10.1002/biot.202300120

Abstract

Background: Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. An R-selective ω-amine transaminase from Aspergillus terreus (AtATA) exhibited activity toward 1-acetylnaphthalene. However, AtATA displayed unsatisfactory organic solvent resistance, which is required to enhance the solubility of the hydrophobic substrate 1-acetylnaphthalene. So, improving the tolerance of enzymes in organic solvents is essential.

Main methods and results: The method of regional random mutation combined with combinatorial mutation was used to improve the resistance of AtATA in organic solvents. Enzyme surface areas are structural elements that undergo reversible conformational transitions, thus affecting the stability of the enzyme in organic solvents. Herein, three surface areas containing three loops were selected as potential mutation regions. And the "best" mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency (k_cat /K_m ) toward 1-acetylnaphthalene and the stability (half-life t_1/2 ) were higher than the wild-type (WT) of AtATA. The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics (MD) simulations under 15%, 25%, 35%, and 45% DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was an intermediate of Cinacalcet Hydrochloride for the treatment of secondary hyperthyroidism and hypercalcemia. Moreover, in a 20-mL scale-up experiment, 10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 85.2% yield and a strict R-stereoselectivity (enantiomeric excess (e.e.) value >99.5%) within 10 h under 25% DMSO.

Conclusion: The beneficial mutation sites were identified to tailor AtATA's organic solvents stability via regional random mutation. The "best" mutant T23I/T200K/P260S (M3) holds great potential application for the synthesis of (R)-NEA.

Keywords: (R)-(+)-1(1-naphthyl)-ethylamine; MD simulations; organic solvent stability; regional random mutation; ω-amine transaminase.

Abstract

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