Search for natural laccase mediator and its application for green chemistry

漆酶介体系统是木质素最有效的降解系统之一。然而，从木质素降解真菌培养中获得的天然漆酶介质尚未见报道。为了解决这个问题，我们考虑了寻找介质的分析系统。首先，我们开发了一种利用漆酶-介质体系处理的未漂白硫酸盐浆的破裂能力（亮度和Kappa数）的测定系统。然而，由于操作复杂，我们拒绝了这个系统。其次，我们设计了一个利用蒽氧化的检测系统。漆酶只有在有介质存在的情况下才能氧化蒽，如1-羟基苯并三唑（HBT）。因此，该系统是一种简便、快速的高效液相色谱分析系统。作为中介物的候选物，我们制备了色曲霉漆酶（Trametes versicolor漆酶，Fr. B）的超滤渗透液及其真菌的水提取物（Fr. F）。它们可以作为漆酶氧化蒽的介质（降解率F: 67.3%, B: 75.5%）。然后，将这些提取物分离成己烷溶性（Hex）和乙酸乙酯溶性（EtOAc）。其中3个组分具有介质活性（降解率：F-Hex: 70.0%, B-Hex: 70.0%, B-EtOAc: 75.6%）。这些萃取物中存在介质的可能性很大。

Laccase-mediator system is one of the most powerful lignin degrading systems. However, the natural laccase mediator from the culture of lignin-degrading fungi has been not reported yet. To address this question, we considered the assay systems for searching the mediator. First, we developed an assay system using the breaching abilities (brightness and Kappa number) of unbleached kraft pulp treated with laccase-mediator system. However, we rejected this system for the complicated operation. Second, we planed an assay system by using of anthracene oxidation. Laccase could only oxidized anthracene in the presence of mediator, for example 1-hydroxybenzotriazole (HBT). Consequently, this system was simple and quick assay system by using HPLC.For the mediator candidates, we prepared the ultrafiltration permeate of Trametes versicolor laccase (Fr. B) and water extracts of its fungus (Fr. F). They could act as the laccase mediator for anthracene oxidation (Degradation rate, F : 67.3%, B : 75.5 %). Then, these extracts were fractionated to hexane-soluble (Hex) and ethyl acetate-soluble (EtOAc). Three fractions of them have mediator activities (Degradation rate, F-Hex : 70.0%, B-Hex : 70.0 %, B-EtOAc : 75.6 %). The existence of mediator in these extractives is seen as a strong possibility.