Abstract
Laccases are multi-copper oxidases that use molecular oxygen as the electron acceptor to oxidize phenolic and indirectly also non-phenolic substrates by mechanisms involving radicals. Due to their eco-friendliness and broad substrate specificity, laccases span a wide range of biotechnological applications. We have heterologously expressed a laccase from the coprophilic basidiomycete Coprinopsis cinerea (CcLcc9) in the methylotrophic yeast Pichia pastoris. The recombinant CcLcc9 (rCcLcc9) oxidized 2,6-dimethoxyphenol in the neutral pH range, and showed thermostability up to 70°C. The rCcLcc9 efficiently oxidized veratryl alcohol to veratraldehyde in the presence of low molecular weight mediators syringyl nitrile, methyl syringate and violuric acid, which are syringyl-type plant phenolics that have shown potential as natural co-oxidants for lignocellulosic materials. In addition, rCcLcc9 is able to depolymerize biorefinery hardwood lignin in the presence of methyl syringate and syringyl nitrile as indicated by gel permeation chromatography, and infrared spectral and nucleic magnetic resonance analyses. Furthermore, we showed that several added-value aromatic compounds, such as vanillin, vanillic acid, syringaldehyde, syringic acid and p-hydroxybenzoic acid, were formed during sequential biocatalytic chemical degradation of biorefinery lignin, indicating that rCcLcc9 harbors a great potential for sustainable processes of circular economy and modern biorefineries.
Highlights
New sustainable technologies and raw materials are key elements in transition towards bio-based economy
The highest laccase activity was predicted in 7 days cultivation at pH 6.5–7.0 when laccase was induced by 0.5% methanol when rCclcc9 was expressed by AOX1 promoter (Figure 3B)
Further design of the optimum condition for methanol induced recombinant CcLcc9 (rCcLcc9) predicted 30% increase of activity on the day 12, it was not experimentally verified (Supplementary Table S1)
Summary
New sustainable technologies and raw materials are key elements in transition towards bio-based economy. The aromatic structure of lignin makes it a promising bio-based alternative for substitution of nonrenewable fossil-based chemical resources (Agustin et al, 2021; Haldar and Purkait, 2021). Despite being the largest resource of renewable aromatic biopolymer, the structural heterogeneity and recalcitrance of lignin has limited its valorization into chemicals and aromatic building blocks in Neutral Laccase in Lignin LMS-Oxidation larger scale. Oxidative enzyme-based depolymerization and bioconversion of lignin is environmentally friendly and sustainable alternative for chemical processing to overcome the recalcitrance of lignin. The substrate range of laccases includes a variety of phenolic compounds such as mono-, di- and polyphenols, methoxy-substituted phenols and aromatic amines. As the majority of lignin comprise of non-phenolic aromatic structures, laccase-mediator system (LMS) has potential in initiating lignin degradation in vitro, and possibly in vivo (Marinovíc et al, 2018)
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