Tailor-made alkaliphilic and thermostable fungal laccases for industrial wood processing

Background: During the kraft process to obtain cellulosic pulp from wood, most of the lignin is removed by high‑ temperature alkaline cooking, released in the black liquors and usually incinerated for energy. However, kraft lignins are a valuable source of phenolic compounds that can be valorized in new bio‑based products. The aim of this work is to develop laccases capable of working under the extreme conditions of high temperature and pH, typical of the industrial conversion of wood into kraft pulp and fibreboard, in order to provide extremophilic biocatalysts for depoly‑ merising kraft lignin, and enzyme‑assisted technologies for kraft pulp and fibreboard production. Results: Through systematic enzyme engineering, combining enzyme‑directed evolution and rational design, we changed the optimal pH of the laccase for oxidation of lignin phenols from acidic to basic, enhanced the catalytic activity at alkaline pH and increased the thermal tolerance of the enzyme by accumulating up to eight mutations in the protein sequence. The extremophilic laccase variants show maximum activity at 70 °C and oxidize kraft lignin at pH 10. Their integration into industrial‑type processes saves energy and chemicals. As a pre‑bleaching stage, the enzymes promote kraft pulp bleachability and significantly reduce the need for chlorine dioxide compared to the industrial sequence. Their application in wood chips during fibreboard production, facilitates the defibering stage, with less energy required. Conclusions: A set of new alkaliphilic and thermophilic fungal laccases has been developed to operate under the extreme conditions of high temperature and pH typical of industrial wood conversion processes. For the first time basidiomycete laccases of high‑redox potential show activity on lignin‑derived phenols and polymeric lignin at pH 10. Considering the extreme conditions of current industrial processes for kraft pulp and fibreboard production, the new tailor‑made laccases constitute a step forward towards turning kraft pulp mills into biorefineries. Their use as biocata‑ lysts in the wood conversion sector is expected to support the development of more environmentally sound and efficient processes, and more sustainable products. Keywords: Laccase, Enzyme directed evolution, Extremophilic properties, Lignin, Kraft pulping, Fibreboard, Biorefinery