ω-转氨酶定点突变及生物催化制备(S)-1-(2-氟苯基)乙胺

Site-specific mutation of ω-transaminase and the biocatalytic preparation of (S)-1-(2-fluorophenyl) ethylamine

摘要:

(S)-1-(2-氟苯基)乙胺是药物合成中重要的手性砌块，ω-转氨酶被认为是一种催化制备手性胺的绿色、高效催化剂。本研究通过基因挖掘手段从NCBI数据库获得来源于氧化亚铁假古布尔班克氏菌(Pseudogulbenkiania ferrooxidans)的新型ω-转氨酶(PfTA)，对其进行半理性设计，得到一株酶活性提高的突变株Y168R/R416Q，其可高选择性催化2-氟苯乙酮合成(S)-1-(2-氟苯基)乙胺，反应10 h后产率达83.58%，对映体过量值(enantiomeric excess, ee)大于99%。与野生型(wild type, WT)相比，突变株Y168R/R416Q的比酶活提高了11.65倍，达到47.04 U/mg，催化效率k_cat/K_m提高20.9倍。分子对接和结构模拟分析表明，突变后酶的活性口袋拓宽，底物2-氟苯乙酮进入酶活性中心的空间位阻减小，从而有效提高了突变体的催化效率。

Abstract:

(S)-1-(2-fluorophenyl) ethylamine plays a crucial role as a chiral building block in pharmaceutical synthesis. ω-transaminases are widely recognized as environmentally friendly and efficient catalysts for the preparation of chiral amines. In this study, we isolated a novel ω-transaminase, PfTA, from Pseudogulbenkiania ferrooxidans through gene mining in the NCBI database. By employing semi-rational design, we obtained a Y168R/R416Q variant with enhanced enzyme activity. This variant exhibited the ability to catalyze the synthesis of (S)-1-(2-fluorophenyl) ethylamine from 2-fluorophenone, achieving a yield of 83.58% and an enantioselectivity exceeding 99% after a 10 h reaction. Compared to the wild type, the specific enzyme activity of the Y168R/R416Q variant reached 47.04 U/mg, which represents an increase of 11.65 times. Additionally, the catalytic efficiency, as measured by k_cat/K_m, was increased by 20.9 times. Molecular docking and structural simulation analysis revealed that the primary factor contributing to the improved catalytic efficiency is the expansion of the enzyme's active pocket and the alleviation of steric hindrance.