Joint BioEnergy Institute, Emeryville, CA 94550, USA.
Molecules. 2010 Mar 11;15(3):1645-67. doi: 10.3390/molecules15031645.
We instituted a quasi-quality assurance program for demonstrating coniferyl alcohol's fluorescence and fluorescence diminishment following enzymatic oxidation. The magnitude of diminishment was a measure of catalysis. High throughput screening was performed in pseudo-kinetic and endpoint modes by measuring the fluorescence at 416 nm following excitation at 290, 310 or 340 nm. Dose-response tracings were linear between two and three orders of magnitude with average limits of detection and quantitation of 1.8 and 6.9 mM coniferyl alcohol, respectively. Oxidation was evident with 0.025 mg/mL laccase or 0.003 mg/mL peroxidase or inside 5 min using 0.5 mg/mL laccase or 5 mM substrate. Sodium chloride inhibited (IC50, 25 mM) laccase oxidation of coniferyl alcohol. Fluorescence from 10 concentrations (1 to 1000 mM) of coniferyl alcohol was stable for 24 hours over 14 excitation/emission cycles at 3 different combinations of excitation and emission wavelengths. In conclusion, coniferyl alcohol absorption and fluorescence assays should facilitate biomass lignin analyses and improve delignification.
我们制定了一个半质量保证计划，以证明松柏醇的荧光和酶氧化后的荧光减弱。减弱的幅度是催化作用的衡量标准。通过在 290nm、310nm 或 340nm 激发后测量 416nm 处的荧光，以准动力学和终点模式进行高通量筛选。在两个到三个数量级之间，剂量反应轨迹呈线性，平均检测限和定量限分别为 1.8mM 和 6.9mM 松柏醇。用 0.025mg/mL 漆酶或 0.003mg/mL 过氧化物酶或 0.5mg/mL 漆酶或 5mM 底物在 5 分钟内即可观察到氧化。氯化钠抑制（IC50，25mM）漆酶氧化松柏醇。松柏醇的 10 个浓度（1 至 1000mM）的荧光在 3 种不同的激发和发射波长组合下，在 14 个激发/发射循环中稳定 24 小时。总之，松柏醇的吸收和荧光测定法应该有助于生物质木质素分析并提高脱木质素效率。
