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.
Highlights
Lignin is a natural plant biopolymer composed of aromatic units in the form of phenylpropanoids, i.e. p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S)[1]
The objective of the present work was to develop a real time, highly sensitive and accurate methodology based on HPLC-MS to assess laccase activity on monomeric phenolics and a dimeric OH-lignol-compound and assess whether the kinetic rates and catalytic efficiencies of laccases depend on the substitutions on the phenolic ring structure and/or on the molecular structure as a whole beyond the monomeric phenol moiety
The Trametes versicolor (Tv) laccase is a widely studied high redox potential laccase, whereas the Ganoderma lucidum (Gl) laccase represents a newer laccase, we have found to work well in relation to enhancing cellulose catalysed lignocellulosic degradation[17]
Summary
Lignin is a natural plant biopolymer composed of aromatic units in the form of phenylpropanoids, i.e. p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S)[1]. Since syringaldazine and ABTS are poorly related to lignin, the use of them provide insufficient information about the actual oxidative kinetics of laccase on true natural phenols and “lignin-like” phenolic structures Another drawback of using these substrates is that the oxidation products formed after laccase oxidation tend to precipitate quickly even at modest concentrations[11] creating an unstable assay with the risk of measuring wrong enzyme activity values. Multiple Reaction Monitoring is based on identification and quantification of specific fragment ions from a predetermined precursor list of parent masses and offers several advantages including high level of specificity, high sensitivity and low susceptibility to interfering compounds[15,16] The latter is important in biological samples with highly complex matrices. From the type of the reactions monitored in this study, it is expected that oxidation products will primarily be dimeric and possibly similar to the structures in Fig. 1 panel b and c
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