Multiple Reaction Monitoring for quantitative laccase kinetics by LC-MS

Abstract

Laccases (EC 1.10.3.2) are enzymes known for their ability to catalyse the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Lignin is a natural phenylpropanoids biopolymer whose degradation in nature is thought to be aided by enzymatic oxidation by laccases. Laccase activity is often measured spectrophotometrically on compounds such as syringaldazine and ABTS which poorly relate to lignin. We employed natural phenolic hydroxycinnamates having different degree of methoxylations, p-coumaric, ferulic and sinapic acid, and a lignin model OH-dilignol compound as substrates to assess enzyme kinetics by HPLC-MS on two fungal laccases Trametes versicolor laccase, Tv and Ganoderma lucidum laccase, Gl. The method allowed accurate kinetic measurements and detailed insight into the product profiles of both laccases. Both Tv and Gl laccase are active on the hydroxycinnammates and show a preference for substrate with methoxylations. Product profiles were dominated by the presence of dimeric and trimeric species already after 10 minutes of reaction and similar profiles were obtained with the two laccases. This new HPLC-MS method is highly suitable and accurate as a new method for assaying laccase activity on genuine phenolic substrates, as well as a tool for examining laccase oxidation product profiles.

Figure 1

Hydroxycinnamic acid and OH-dilignol structures (a). Possible concept for product formation after laccase oxidation: dimer formation from the hydroxycinnamic acids (b) and from the OH-dilignol (c).

Figure 2

Michaelis-Menten curves for Tv and Gl laccase on ferulic acid (a,b) and OH-dilignol (c,d). Three different enzyme dosages (in syringaldazine assay units) are shown: for ferulic acid (a,b): 0.625 μU (green open square), 1.25 μU (red cross) and 2.5 μU (blue open circle). For OH-dilignol (c,d): 1.25 μU (red cross), 2.5 μU (blue open circle) and 5 μU (orange open triangle). Dose response at 10 μM substrate concentration is shown in the inset. For the highest enzyme dose standard deviations are shown.

Figure 3

Laccase oxidation evolution profiles for hydroxycinnamic acids and OH-dilignol: oxidation of sinapic acid with Tv and Gl laccase (a,b); oxidation of ferulic acid with Tv and Gl laccase (c,d); oxidation of p-coumaric acid with Tv and Gl laccase (e,f) and oxidation of OH-dilignol with Tv and Gl laccase (g,h). Chromatograms at different reaction times are shown: 0 minutes (light blue), 10 minutes (yellow), 25 minutes (violet) and 50 minutes (red). Please note that the intensity scale may differ between chromatograms and is adjusted to give optimal display of figures. Panel a includes an example of the MS spectra corresponding to each peak, all ions are observed as [M − H]⁻. All other MS and MS/MS spectra are found in Supplementary Figs S2–S11. Oxidation reaction products are numbered according to substrate and are therefore comparable between enzymes.