Spectroscopic Analyses of the Biofuels-Critical Phytochemical Coniferyl Alcohol and Its Enzyme-Catalyzed Oxidation Products

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, pseudo-kinetic and endpoint analyses, in optical turn on or turn off modes, under acidic or basic conditions. Reactions in microwell plates and 100 μL 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 μM respectively, with linear trend lines over 3 to 4 orders of magnitude. Coniferyl alcohol oxidation was evident within 10 minutes or with 0.01 μg/mL laccase and 2 minutes or 0.001 μg/mL peroxidase. Detection limit improved to 1.0 μM coniferyl alcohol with Km of 978.7 ± 150.7 μM when examined at 260 nm following 30 minutes oxidation with 1.0 μg/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.