ω-Transaminase (ω-TA) is an attractive alternative to metal catalysts for the stereoselective amination of prochiral ketones. The narrow substrate scope of an R-ω-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 (μM magnitude), and a wide dynamic range (ɀ-factor > 0.9). A KnowVolution campaign was performed on this enzyme, and screening ~ 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: ~ 0.07 U/mg), and it showed the highest activity on acetonaphthone, p-ethylacetophenone, and phenylacetone.

中文翻译：

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基于荧光的高通量筛选系统，用于R-ω-转氨酶工程及其底物范围的扩展。

ω-转氨酶（ω-TA）是金属催化剂对前手性酮进行立体选择性胺化的一种有吸引力的替代方法。Vanbaalenii分枝杆菌（MvTA）的R-ω-转氨酶的狭窄底物范围限制了其在R-胺合成中的应用。开发了基于荧光的TA活性筛选系统以扩展其底物范围。反应在微量滴定板（MTP）中进行，显示出低背景干扰，高灵敏度（μM大小）和宽动态范围（ɀ因子> 0.9）。对该酶进行了一次KnowVolution活动，并使用该基于荧光的筛选系统筛选了约8000个克隆，从而产生了两个有益的取代（G68Y和F129A）和三个改进的变体（M3，M4和M5）。最好的变体MvTA M5（WT + G68Y + F129A）获得了最高的催化效率（朝向荧光底物NMA），比WT酶高3.2倍。MvTA M5对ee> 99％（R）的六种不同的手性酮表现出明显增强的活性。MvTA M5对乙酰萘甲酮的比活性比WT酶高100倍以上（M5：8.1 U / mg，WT：〜0.07 U / mg），并且对乙酰基萘，对-乙基苯乙酮，和苯丙酮。