Affiliation:
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
Abstract
AbstractBackgroundBiocatalysis 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 ResultsThe 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 (kcat/Km) toward 1‐acetylnaphthalene and the stability (half‐life t1/2) 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.ConclusionThe 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.
Funder
National Natural Science Foundation of China
Subject
Molecular Medicine,Applied Microbiology and Biotechnology,General Medicine
Cited by
3 articles.
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