Arca-Ramos A, Ammann E M, Gasser C A, Nastold P, Eibes G, Feijoo G, Lema J M, Moreira M T, Corvini P F-X
Department of Chemical Engineering, Institute of Technology, University of Santiago de Compostela, 15782, Santiago de Compostela, Spain.
Institute for Ecopreneurship, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, Muttenz, 4132, Switzerland.
Environ Sci Pollut Res Int. 2016 Feb;23(4):3217-28. doi: 10.1007/s11356-015-5564-6. Epub 2015 Oct 21.
Enzymes immobilization is a useful way to allow enzyme reuse and increase their stability. A high redox potential laccase from Trametes versicolor (TvL) and a low redox potential, but commercially available low-cost laccase from Myceliophthora thermophila (MtL), were successfully immobilized and co-immobilized onto fumed silica nanoparticles (fsNP). Enzyme loads of 1.78 ± 0.07, 0.69 ± 0.03, and 1.10 ± 0.01 U/mg fsNP were attained for the optimal doses of TvL, MtL, and co-immobilized laccases, respectively. In general, the laccase-fsNP conjugates showed a higher resistance against an acidic pH value (i.e., pH 3), and a higher storage stability than free enzymes. In addition, immobilized enzymes exhibited a superior long-term stability than free laccases when incubated in a secondary effluent from a municipal wastewater treatment plant (WWTP). For instance, the residual activity after 2 weeks for the co-immobilized laccases and the mixture of free laccases were 40.2 ± 2.5% and 16.8 ± 1.0%, respectively. The ability of the laccase-fsNP to remove a mixture of (14)C-bisphenol A (BPA) and (14)C-sodium diclofenac (DCF) from spiked secondary effluents was assessed in batch experiments. The catalytic efficiency was highly dependent on both the microbial source and state of the biocatalyst. The high redox potential TvL in free form attained a four-fold higher percentage of BPA transformation than the free MtL. Compared to free laccases, immobilized enzymes led to much slower rates of BPA transformation. For instance, after 24 h, the percentages of BPA transformation by 1000 U/L of a mixture of free laccases or co-immobilized enzymes were 67.8 ± 5.2 and 27.0 ± 3.9%, respectively. Nevertheless, the use of 8000 U/L of co-immobilized laccase led to a nearly complete removal of BPA, despite the unfavorable conditions for laccase catalysis (pH ~ 8.4). DCF transformation was not observed for any of the enzymatic systems, showing that this compound is highly recalcitrant toward laccase oxidation under realistic conditions.
酶固定化是实现酶重复利用并提高其稳定性的一种有效方法。分别成功地将来自云芝的高氧化还原电位漆酶（TvL）和来自嗜热毁丝霉的低氧化还原电位但具有商业可用性的低成本漆酶（MtL）固定化以及共固定化到气相二氧化硅纳米颗粒（fsNP）上。对于TvL、MtL和共固定化漆酶的最佳剂量，酶负载量分别达到1.78±0.07、0.69±0.03和1.10±0.01 U/mg fsNP。总体而言，漆酶 - fsNP缀合物对酸性pH值（即pH 3）表现出更高的耐受性，并且比游离酶具有更高的储存稳定性。此外，当在城市污水处理厂（WWTP）的二级出水中孵育时，固定化酶比游离漆酶表现出更好的长期稳定性。例如，共固定化漆酶和游离漆酶混合物在2周后的残余活性分别为40.2±2.5%和16.8±1.0%。在分批实验中评估了漆酶 - fsNP从加标的二级出水中去除（14）C - 双酚A（BPA）和（14）C - 双氯芬酸钠（DCF）混合物的能力。催化效率高度依赖于微生物来源和生物催化剂的状态。游离形式的高氧化还原电位TvL实现的BPA转化百分比比游离MtL高四倍。与游离漆酶相比，固定化酶导致BPA转化速率慢得多。例如，24小时后，1000 U/L的游离漆酶混合物或共固定化酶对BPA的转化百分比分别为67.8±5.2和27.0±3.9%。然而，尽管存在不利于漆酶催化的条件（pH约8.4），使用8000 U/L的共固定化漆酶仍导致BPA几乎完全去除。对于任何酶系统都未观察到DCF转化，这表明该化合物在实际条件下对漆酶氧化具有高度抗性。
