Kashima Keita, Fujisaki Tomoyuki, Serrano-Luginbühl Sandra, Khaydarov Abbos, Kissner Reinhard, Ležaić Aleksandra Janošević, Bajuk-Bogdanović Danica, Ćirić-Marjanović Gordana, Schuler Lukas D, Walde Peter
Department of Materials, ETH Zurich Vladimir-Prelog-Weg 5 8093 Zürich Switzerland
Department of Materials Chemistry and Bioengineering, National Institute of Technology, Oyama College 771 Ohaza-Nakakuki Oyama Tochigi 323-0806 Japan.
RSC Adv. 2018 Sep 26;8(58):33229-33242. doi: 10.1039/c8ra05731a. eCollection 2018 Sep 24.
The laccase (TvL)-catalysed oligomerisation of the aniline dimer -aminodiphenylamine (PADPA) was investigated in an aqueous medium of pH = 3.5, containing 80-100 nm-sized anionic vesicles formed from AOT, the sodium salt of bis(2-ethylhexyl)sulfosuccinic acid. If run under optimal conditions, the reaction yields oligomeric products which resemble the emeraldine salt form of polyaniline (PANI-ES) in its polaron state, known to be the only oxidation state of linear PANI which is electrically conductive. The vesicles serve as "templates" for obtaining products with the desired PANI-ES-like features. For this complex, heterogeneous, vesicle-assisted, and enzyme-mediated reaction, in which dissolved dioxygen also takes part as a re-oxidant for TvL, small changes in the composition of the reaction mixture can have significant effects. Initial conditions may not only affect the kinetics of the reaction, but also the outcome, , the product distribution once the reaction reaches its equilibrium state. While a change in the reaction temperature from ≈ 25 to 5 °C mainly influenced the rate of reaction, increase in enzyme concentration and the presence of millimolar concentrations of chloride ions were found to have significant undesired effects on the outcome of the reaction. Chloride ions, which may originate from the preparation of the pH = 3.5 solution, inhibit TvL, such that higher TvL concentrations are required than without chloride to yield the same product distribution for the same reaction runtime as in the absence of chloride. With TvL concentrations much higher than the elaborated value, the products obtained clearly were different and over-oxidised. Thus, a change in the activity of the enzyme was found to have influence not only on kinetics but also led to a change in the final product distribution, molecular structure and electrical properties, which was a surprising find. The complementary analytical methods which we used in this work were UV/vis/NIR, EPR, and Raman spectroscopy measurements, in combination with a detailed HPLC analysis and molecular dynamics simulations. With the results obtained, we would like to recall the often neglected or ignored fact that it is important to describe and pay attention to the experimental details, since this matters for being able to perform experiments in a reproducible way.
在pH = 3.5的水性介质中，研究了漆酶（TvL）催化的苯胺二聚体 - 氨基二苯胺（PADPA）的低聚反应，该介质含有由双（2-乙基己基）磺基琥珀酸钠（AOT）形成的80 - 100纳米大小的阴离子囊泡。如果在最佳条件下进行反应，反应会生成类似于处于极化子状态的聚苯胺翡翠盐形式（PANI-ES）的低聚产物，已知这是线性聚苯胺唯一具有导电性的氧化态。这些囊泡作为“模板”来获得具有所需PANI-ES样特征的产物。对于这个复杂的、非均相的、囊泡辅助且酶介导的反应，其中溶解的氧气也作为TvL的再氧化剂参与反应，反应混合物组成的微小变化可能会产生显著影响。初始条件不仅可能影响反应动力学，还会影响反应结果，即反应达到平衡状态时的产物分布。虽然反应温度从约25℃变为5℃主要影响反应速率，但发现酶浓度的增加和毫摩尔浓度氯离子的存在对反应结果有显著的不良影响。可能源于pH = 3.5溶液制备过程的氯离子会抑制TvL，因此与不存在氯离子时相比，需要更高的TvL浓度才能在相同的反应运行时间内产生相同的产物分布。当TvL浓度远高于精心确定的值时，得到的产物明显不同且过度氧化。因此，发现酶活性的变化不仅影响动力学，还导致最终产物分布、分子结构和电学性质发生变化，这是一个令人惊讶的发现。我们在这项工作中使用的互补分析方法包括紫外/可见/近红外光谱、电子顺磁共振和拉曼光谱测量，以及详细的高效液相色谱分析和分子动力学模拟。根据获得的结果，我们想再次强调一个经常被忽视或忽略的事实，即描述并关注实验细节很重要，因为这对于能够以可重复的方式进行实验至关重要。
