Bio-based adhesives offer a sustainable alternative to formaldehyde-based adhesives, which is the most common product applied in wood-based panel manufacturing. Despite the undeniable interest in lignin as a bulk chemical for the synthesis of bio-based adhesives, the main problem is its low reactivity. Therefore, it is crucial to study techniques to increase their reactivity, such as demethylation. One of the parameters to monitor the success of the demethylation is the methanol concentration measured in the reaction medium and complementing these results with the use of non-quantitative analyses such as FT-IR, MALDI-ToF and SEC. In order to maximize the action of the enzyme, a preliminary screening was performed with laccases with different redox potentials, which were added to organosolv lignin in the form of purified enzymes and cocktails. Trametes versicolor laccase was selected as the best candidate for the demethylation of organosolv lignin, with a methanol released concentration of 107 mg/L. Based on these results, parameters affecting both enzyme action and enzyme-substrate interaction were evaluated. Optimal working conditions were strongly defined by the use of surfactants that enhanced lignin solubility (Tween-80) and mediators that favored enzyme catalysis to participate directly in the enzyme catalytic cycle (HBT). However, it was also observed that factors such as pH, temperature and aeration have a direct impact on the enzymatic demethylation of lignin. Considering the operational variables that allowed better demethylation values, the amount of methanol released increased to 363 mg/L, which means an improvement of 239 %. These results were also supported by the increase in molecular weight observed in the SEC analysis and the reduction of the intensity of the FT-IR bands related to the methyl/methoxyl groups.
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