DWI Leibniz Institute for Interactive Materials, Forckenbeckstrasse 50, 52056, Aachen, Germany.
Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
Chembiochem. 2019 Jun 3;20(11):1458-1466. doi: 10.1002/cbic.201800807. Epub 2019 Apr 15.
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 (k =(6.0±0.2) s ) compared with that of wild-type M. albomyces laccase (k =(2.11±0.07) s ).
迄今为止，商业漆酶制剂已应用于食品、纺织和造纸及纸浆工业（pH 值较温和）。漆酶在染料分子的合成或氧化木质素处理中很有吸引力，这些反应发生在高 pH 值（≥8.0）下。到目前为止，已有报道称一种真菌漆酶在碱性 pH 值下具有活性。在此，报道了通过知识获取定向进化（KnowVolution）对来自白腐菌（Melanocarpus albomyces）的漆酶（MaL）进行工程改造，以提高其在 pH（≥9.0）下对 2,6-二甲氧基苯酚的底物活性。通过知识获取定向进化（KnowVolution）活动，确定了关键位置 Leu365 和 Leu513 以提高碱性耐受性。这两个位置都位于 T1Cu 位点附近。分子对接和模拟研究表明，这两个取代基以协同方式作用，在更高 pH 值下稳定并提高漆酶活性。最终变体 MaL-M1（L365E/L513M）的动力学特性研究表明，在 pH 值为 9.8 时，其 k 值（k =（6.0±0.2）s）比野生型白腐菌漆酶（k =（2.11±0.07）s）提高了三倍。
