期刊
CHEMBIOCHEM
卷 20, 期 11, 页码 1458-1466出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201800807
关键词
directed evolution; laccases; molecular dynamics; pH; protein engineering
资金
- FuPol (Functionalization of Polymers) Alliance [FKZ: 031A227F]
- JARA-HPC from RWTH Aachen University [RWTH0116]
- German Federal Ministry of Education and Research (BMBF)
To date, commercial laccase preparations are used in the food, textile, and paper and pulp industries (mild pH). Laccases are attractive in the synthesis of dye molecules or oxidative lignin treatment, which take place at high pH (>= 8.0). So far, one fungal laccase has been reported to be active at alkaline pH. Herein, engineering of the fungal laccase from Melanocarpus albomyces (MaL) for increased activity toward the substrate 2,6-dimethoxyphenol at pH (>= 9.0) is reported. Through a knowledge-gaining directed evolution (KnowVolution) campaign, the key positions Leu365 and Leu513 were identified to increase alkaline tolerance. Both positions are located in close proximity of the T1Cu site. Molecular docking and simulations studies reveal that both substitutions act in a synergic way to stabilize and improve laccase activity at higher pH. Kinetic characterization of the final variant MaL-M1 (L365E/L513M) revealed at pH 9.8 a threefold improved k(cat) (k(cat)=(6.0 +/- 0.2) s(-1)) compared with that of wild-type M. albomyces laccase (k(cat)=(2.11 +/- 0.07) s(-1)).
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据