Wrocław University of Technology, Faculty of Chemistry, Department of Bioorganic Chemistry, Wrocław, Poland.
Bioresour Technol. 2010 Apr;101(7):2076-83. doi: 10.1016/j.biortech.2009.11.077. Epub 2009 Dec 23.
Cerrena unicolor laccase was immobilized by adsorption and covalent bonds formation on silica-gel carriers, functionalized with different organosilanes and surface densities. The effects of protein concentration, pH value of the coupling mixture and the enzyme purity on immobilization efficiency of the best carrier, moderately modified (0.75 mmol/g carrier) with 3-aminopropyltriethoxysilane were investigated. Activity of the best biocatalysts, expressed in ABTS oxidation, was 4028 U/mL of the carrier or 3530 U/mg of bound protein. Properties of immobilized laccase were determined to find excellent thermal stability improvement; t(1/2) for freely suspended enzyme was 2-3 min at 80 degrees C, whereas after immobilization over 100 min. Kinetic experiments in both batch and packed-bed reactors gave only four times lower k(cat)/K(m) value than for the native enzyme. A packed-bed reactor with silica-gel-bound laccase beads appeared to be very efficient in ABTS oxidation and its exceptional potentials were shown in the continuous decolorization of indigo carmine for 18 days without loss in activity. This system offers perfect ability to degrade recalcitrant dyes, but we can also envisage its use, with ABTS acting as a mediator, in regeneration of nicotinamide cofactors.
杂色云芝漆酶通过吸附和共价键形成固定在硅胶载体上，这些载体用不同的有机硅烷和表面密度进行了功能化。研究了蛋白质浓度、偶联混合物的 pH 值以及酶纯度对最佳载体（用 3-氨丙基三乙氧基硅烷适度修饰（0.75mmol/g 载体））固定化效率的影响。以 ABTS 氧化表示的最佳生物催化剂的活性为 4028U/mL 载体或 3530U/mg 结合蛋白。测定了固定化漆酶的性质，以发现其优异的热稳定性提高；自由悬浮酶的 t(1/2)在 80°C 下为 2-3 分钟，而固定化后超过 100 分钟。批式和填充床反应器中的动力学实验得出的 k(cat)/K(m)值比天然酶低四倍。硅胶结合漆酶珠的填充床反应器在 ABTS 氧化中表现出非常高的效率，其在连续 18 天对靛蓝胭脂红的脱色中没有活性损失，显示出了其特殊的潜力。该系统提供了降解难处理染料的完美能力，但我们也可以设想在使用 ABTS 作为介体的烟酰胺辅酶再生中使用它。
