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

Abstract

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 depolymerising 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 biocatalysts in the wood conversion sector is expected to support the development of more environmentally sound and efficient processes, and more sustainable products.

Fungal Laccases: Fundamentals, Engineering and Classification Update

“…In fact, fungal laccases have traditionally been much more widely studied than their counterparts, particularly those secreted by white-rot basidiomycetes [9]. Some of these laccases have the highest redox potentials described so far [10,11], which gives them superior oxidation capacities on a wider range of substrates and, thus, a higher applicability as biocatalysts. With this in mind, this review focuses on fungal laccases, with special emphasis on basidiomycete laccases.…”

“…Today, the use of chlorine dioxide (ClO 2 ) as a bleaching agent in modern paper mills can be reduced by the application of enzymes such as laccases and xylanases [78][79][80][81]. Laccases, in the presence of redox mediators, can be used in a pre-bleaching step for removing the color caused by lignin, allowing us to reduce the use of bleaching chemicals with the additional benefit that cellulose is not degraded during the process [11,42,80]. In addition, laccases can be applied for deinking recycled paper, thus reducing the environmental impact [82].…”

“…In silico screening of genomes and databases allows for the discovery of wild-type enzymes from extremophiles with potential properties of interest under harsh industrial conditions [116]. Alternatively, it is possible to endow native enzymes with new functionalities under nonnatural (extreme) conditions by using protein engineering to adapt the enzymes to the target industrial process [11,117].…”

“…More recently, we have developed alkaliphilic and thermophilic high-redox-potential laccases for wood conversion processes. These evolved enzymes are able to boost kraft pulp bleaching, achieving 11% ClO 2 savings; improve the de-fibering of wood chips in fiberboard production with less energy required; and depolymerize kraft lignins at pH 10 [11,85].…”

Recombinant Oxidase from Armillaria tabescens as a Potential Tool for Aflatoxin B1 Degradation in Contaminated Cereal Grain

“…For example, P. sordida was reported to be able to infect humans [ 37 ]. Laccases are well-known enzymes catabolizing many compounds including lignin, and are considered for use in wood processing and the textile industry [ 38 ] because of the broader specificity of this group of enzymes compared to other aflatoxin-degrading enzymes and their activity at extreme pH values and temperatures [ 39 , 40 ], which are not typical for feed processing. Moreover, in spite of the successful results demonstrated in some studies [ 27 , 36 , 40 ], the heterologous expression of laccases is rather difficult due to their complex copper-dependent active site [ 38 , 40 ] that results in a rapid proteolytic cleavage of these enzymes during their post-translational modification.…”