Cao Xudong, Han Ruizhi, Fang Honghui, Ni Ye
School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Sep 25;36(9):1828-1837. doi: 10.13345/j.cjb.200032.
(R)-(+)-1-(1-naphthyl)ethylamine is a key chiral intermediate for the synthesis of calcimimetic drug cinacalcet hydrochloride. ω-Transaminase has been considered to be potential for producing (R)-(+)-1-(1-naphthyl)ethylamine by asymmetric reduction of 1-acetonaphthone. Here, ω-transaminase from Arthrobacter sp. was engineered by combinatorial strategies of random mutagenesis and semi-rational design. Variants F225M, C281I, F225M/C281I with improved catalytic efficiency and thermostability were obtained. Compared with WT, variant F225M/C281I showed 85% increased kcat, 56% decreased Km and 3.42-fold kcat/Km. Furthermore, 22% higher conversion rate was achieved by F225M/C281I at 10 mmol/L 1-acetonaphthone after 24 h. Based on molecular docking and molecular dynamics simulation, improved catalytic efficiency of F225M/C281I could be attributed to its increased Pi-Pi T-shaped interaction with substrate 1-acetonaphthone. Additionally, a slightly higher half-life of F225M/C281I was validated by its lower root-mean-square fluctuation (RMSF) value of loop 134-139 compared with WT.
（R）-（+）-1-（1-萘基）乙胺是合成拟钙剂盐酸西那卡塞的关键手性中间体。ω-转氨酶被认为具有通过对1-乙酰萘进行不对称还原生产（R）-（+）-1-（1-萘基）乙胺的潜力。在此，通过随机诱变和半理性设计的组合策略对节杆菌属的ω-转氨酶进行了工程改造。获得了催化效率和热稳定性提高的变体F225M、C281I、F225M/C281I。与野生型相比，变体F225M/C281I的催化常数（kcat）提高了85%，米氏常数（Km）降低了56%，kcat/Km提高了3.42倍。此外，在24小时后，F225M/C281I在10 mmol/L 1-乙酰萘的条件下转化率提高了22%。基于分子对接和分子动力学模拟，F225M/C281I催化效率的提高可归因于其与底物1-乙酰萘增加的π-π T形相互作用。此外，与野生型相比，F225M/C281I的134-139环的均方根波动（RMSF）值较低，验证了其半衰期略长。
