Fluorescence-based high-throughput screening system for R-ω-transaminase engineering and its substrate scope extension
Fluorescence-based high-throughput screening system for R-ω-transaminase engineering and its substrate scope extension
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基于荧光的R-β-转氨酶工程高通量筛选系统及其底物范围扩展
DOI：
10.1007/s00253-020-10444-y
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发表时间：
2020-02-17
影响因子：
5
通讯作者：
Zheng, Yu-Guo
中科院分区：
文献类型：
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作者：
Cheng, Feng;Chen, Xiu-Ling;Zheng, Yu-Guo
欧米伽-转氨酶(omega-TA)是一种很有吸引力的金属催化剂，用于前手性酮的立体选择性胺化反应。范巴伦分枝杆菌的R-omega-转氨酶(MvTA)底物范围较窄，限制了其在R-胺合成中的应用。开发了一种基于荧光的TA活性筛选系统，以扩大其底物范围。这些反应是在微滴定板(MTP)中进行的，表现出低背景干扰、高灵敏度(Mu M量级)和宽动态范围(因子>；0.9)。用这种基于荧光的筛选系统筛选了8000个克隆，得到了两个有益的替换(G68Y和F129A)和三个改进的变异体(M3、M4和M5)。MvTA M5(WT+G68Y+F129A)对荧光底物NMA的催化效率最高，是WT酶的3.2倍。MvTA M5对具有e.e的6种不同的前手性酮表现出显著的活性增强。>；99%(R)。MvTA M5的比活力是WT酶的100多倍(M5：8.1U/mg，WT：接近0.07U/mg)，对对乙基苯乙酮、对乙基苯乙酮和苯丙酮表现出最高的活性。
omega-Transaminase (omega-TA) is an attractive alternative to metal catalysts for the stereoselective amination of prochiral ketones. The narrow substrate scope of an R-omega-transaminase from Mycobacterium vanbaalenii (MvTA) limits its application in R-amine synthesis. A fluorescence-based TA activity screening system was developed to extend its substrate scope. The reactions were conducted in microtiter plates (MTPs) and displayed low background interference, high sensitivity (mu M magnitude), and a wide dynamic range (-factor > 0.9). A KnowVolution campaign was performed on this enzyme, and screening similar to 8000 clones with this fluorescence-based screening system resulted in two beneficial substitutions (G68Y and F129A) and three improved variants (M3, M4, and M5). The best variant, MvTA M5 (WT+G68Y+F129A), achieved the highest catalytic efficiency (toward fluorogenic substrate NMA) which was 3.2-fold higher than that of the WT enzyme. MvTA M5 exhibited significantly enhanced activity toward six different prochiral ketones with e.e. > 99% (R). The specific activity of MvTA M5 was more than 100 times higher than that of the WT enzyme toward acetonaphthone (M5: 8.1 U/mg, WT: similar to 0.07 U/mg), and it showed the highest activity on acetonaphthone, p-ethylacetophenone, and phenylacetone.