Wang Chun-Ning, Qiu Shuai, Fan Fang-Fang, Lyu Chang-Jiang, Hu Sheng, Zhao Wei-Rui, Mei Jia-Qi, Mei Le-He, Huang Jun
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.
School of Biological and Chemical Engineering, Ningbo Tech University, Ningbo, China.
Biotechnol J. 2023 Oct;18(10):e2300120. doi: 10.1002/biot.202300120. Epub 2023 Jul 9.
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.
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 /K ) toward 1-acetylnaphthalene and the stability (half-life t ) 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.
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.
高浓度有机溶剂中的生物催化已被应用于生产各种工业产品，具有诸多优势。然而，在有机溶剂中使用酶常常会出现失活或催化活性及稳定性降低的情况。来自土曲霉的一种R-选择性ω-氨基转氨酶（AtATA）对1-乙酰萘表现出活性。然而，AtATA的有机溶剂耐受性并不理想，而这对于提高疏水底物1-乙酰萘的溶解度是必需的。因此，提高酶在有机溶剂中的耐受性至关重要。
采用区域随机突变与组合突变相结合的方法来提高AtATA在有机溶剂中的耐受性。酶表面区域是经历可逆构象转变的结构元件，从而影响酶在有机溶剂中的稳定性。在此，选择包含三个环的三个表面区域作为潜在的突变区域。并获得了“最佳”突变体T23I/T200K/P260S（M3）。在不同浓度的二甲基亚砜（DMSO）中，M3对1-乙酰萘的催化效率（k /K ）和稳定性（半衰期t ）均高于野生型AtATA。通过在15%、25%、35%和45% DMSO条件下进行20纳秒的分子动力学（MD）模拟，均方根波动（RMSF）值降低的结果表明，突变体M3的柔韧性更低，获得了更稳定的蛋白质结构，并且与野生型相比，其在有机溶剂中的稳定性更高。此外，M3被用于转化1-乙酰萘以合成（R）-（+）-1-（1-萘基）乙胺（（R）-NEA），（R）-NEA是用于治疗继发性甲状腺功能亢进和高钙血症的盐酸西那卡塞的中间体。此外，在20毫升的放大实验中，在25% DMSO条件下，10毫摩尔的1-乙酰萘在10小时内可转化为（R）-NEA，产率为85.2%，且具有严格的R-立体选择性（对映体过量（e.e.）值>99.5%）。
通过区域随机突变确定了有益的突变位点，以调整AtATA在有机溶剂中的稳定性。“最佳”突变体T23I/T200K/P260S（M3）在（R）-NEA的合成中具有巨大的潜在应用价值。
