Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People's Republic of China.
The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Appl Microbiol Biotechnol. 2020 Apr;104(7):2999-3009. doi: 10.1007/s00253-020-10444-y. Epub 2020 Feb 17.
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.
ω-转氨酶（ω-TA）是一种有吸引力的金属催化剂替代品，可用于对前手性酮进行立体选择性氨化。分枝杆菌（Mycobacterium vanbaalenii）来源的 R-ω-转氨酶（MvTA）的底物范围较窄，限制了其在 R-胺合成中的应用。开发了一种基于荧光的 TA 活性筛选系统来扩展其底物范围。反应在微孔板（MTP）中进行，显示出低背景干扰、高灵敏度（μM 数量级）和宽动态范围（ɀ 因子>0.9）。对该酶进行了 KnowVolution 实验，使用这种基于荧光的筛选系统筛选了约 8000 个克隆，得到了两个有益的取代（G68Y 和 F129A）和三个改进的变体（M3、M4 和 M5）。最佳变体 MvTA M5（WT+G68Y+F129A）的催化效率最高（针对荧光底物 NMA），比 WT 酶高 3.2 倍。MvTA M5 对六种不同的前手性酮的活性显著增强，ee 值>99%（R）。MvTA M5 对苯乙酮的比活度比 WT 酶高 100 多倍（M5：8.1 U/mg，WT：~0.07 U/mg），对苯乙酮、对乙基苯乙酮和苯丙酮的活性最高。
