Joint BioEnergy Institute (JBEI), Emeryville, CA 94550, USA.
Molecules. 2009 Nov 23;14(11):4758-78. doi: 10.3390/molecules14114758.
Lignin composition (monolignol types of coniferyl, sinapyl or p-coumaryl alcohol) is causally related to biomass recalcitrance. We describe multiwavelength (220, 228, 240, 250, 260, 290, 295, 300, 310 or 320 nm) absorption spectroscopy of coniferyl alcohol and its laccase- or peroxidase-catalyzed products during real time kinetic, pseudokinetic and endpoint analyses, in optical turn on or turn off modes, under acidic or basic conditions. Reactions in microwell plates and 100 microL volumes demonstrated assay miniaturization and high throughput screening capabilities. Bathochromic and hypsochromic shifts along with hyperchromicity or hypochromicity accompanied enzymatic oxidations by laccase or peroxidase. The limits of detection and quantitation of coniferyl alcohol averaged 2.4 and 7.1 muM respectively, with linear trend lines over 3 to 4 orders of magnitude. Coniferyl alcohol oxidation was evident within 10 minutes or with 0.01 microg/mL laccase and 2 minutes or 0.001 microg/mL peroxidase. Detection limit improved to 1.0 microM coniferyl alcohol with Km of 978.7 +/- 150.7 microM when examined at 260 nm following 30 minutes oxidation with 1.0 microg/mL laccase. Our assays utilized the intrinsic spectroscopic properties of coniferyl alcohol or its oxidation products for enabling detection, without requiring chemical synthesis or modification of the substrate or product(s). These studies facilitate lignin compositional analyses and augment pretreatment strategies for reducing biomass recalcitrance.
木质素组成（松柏醇、丁香醇或对香豆醇醇）与生物质的抗降解性有关。我们描述了在实时动力学、拟动力学和终点分析中，在光学开启或关闭模式下，在酸性或碱性条件下，多波长（220、228、240、250、260、290、295、300、310 或 320nm）吸收光谱对松柏醇及其漆酶或过氧化物酶催化产物的研究，微井板和 100μL 体积的反应显示了分析的小型化和高通量筛选能力。漆酶或过氧化物酶的酶促氧化伴随着红移和蓝移以及增色或减色。松柏醇的检测限和定量限平均值分别为 2.4 和 7.1μM，线性趋势线跨越 3 到 4 个数量级。松柏醇氧化在 10 分钟内或 0.01μg/mL 漆酶和 2 分钟或 0.001μg/mL 过氧化物酶下即可检测到。当在 260nm 下，在 1.0μg/mL 漆酶作用下氧化 30 分钟后，检测限提高到 1.0μM 松柏醇，Km 为 978.7±150.7μM。我们的测定利用了松柏醇或其氧化产物的固有光谱特性来进行检测，而不需要对底物或产物进行化学合成或修饰。这些研究促进了木质素组成分析，并增强了降低生物质抗降解性的预处理策略。
