Proof of concept for the functionalization of lignins by applying laccase-dehydrogenase enzymatic treatments in biobased materials applications – FuncLIPRO

FuncLIPRO is a trans-disciplinary project, bringing about scientific, technological and financial stakes based on research in the fields of biochemistry (protein production, functional and structural enzyme characterization, protein engineering, biopolymer science), chemistry (analytical, organic and structural analysis, polymer synthesis, material science) and process engineering (process design, product formulations, economical and environmental assessments).
For the production of enzymes, they could be either produced in wild type fungal strian (Pyncoprous cinnabarinus) or by use of heterologous hosts, for isntance in Aspergillus niger.
In order to study the enzyme determinants related to its activity onto lignins, crystals of enzymes will be produced. Crystallization conditions have been already found for PcGDH and for one PcADD. P1 is member of a “BAG” (Block Allocation Group) that will grant access to X-ray beamlines, in order to conduct diffraction experiments at national synchrotron sources (Soleil - Paris and ESRF - Grenoble).
Enzymes will be applied to loignin in pseciifc pH to allow a correct activity of lignin modifying enzymes and lignin will be analyzed by diffrent technics. Among the analytical tools used, screening of the treatment efficiency will be carried out, by comparing the samples obtained from native and enzymatically treated lignins, in terms of (i) molar mass distribution (size-exclusion chromatography, P2) and (ii) functional group content (free hydroxyl groups analysed by 31P NMR, P3). In addition, for fractions with selected profiles (i.e. homogenous lignin fragments containing free phenolic moieties), identification and characterization of lignin degradation products will be performed by LC-MS, GC-MS and NMR (P3).
The best enzymes conditions will be used to provide three different applications, replacing non-renewable chemical compounds and developing eco-friendly approaches (adhesives, coating film and agro-based matérials). Furthermore, data will be collected at the laboratory-scale in order to allow environmental and cost impact assessment

WP2 Production and characterization of enzymes and lignins
(1) Three enzymes from P. cinnabarinus were produced in sufficient quantities to conduct the project: laccase (LAC), oligosaccharide dehydrogenase (ODH) and 2 aryl alcohol dehydrogenases (P1: BBF). The structure-function study of ODH, previously considered as a glucose dehydrogenase, suggests that the physiological substrates could come from the degradation of hemicelluloses (2). Organosolve lignins (lignins closest to natural lignins) were prepared by TNO Institute under MTA and kraft lignins (residues from the paper industry) by FCBA (P4).

WP3 Lignin analyses from enzyme experiments on lignins
(1) Preliminary tests were carried out on laccase treated wood fibers for small scale wet manufacturing of hardboard. The results show that pretreatment of spruce fibers with laccase-mediator systems prior to hot pressing has an impact on the quality of the wood fibers and improves the mechanical strength of the resulting boards while using the lowest enzyme loadings.
(2) First, we implemented laccase from P. cinnabarinus by varying, pH, enzyme concentration and incubation time of the enzyme on organosolve lignin (P1 BBF). The effect of laccase on these lignins was then evaluated by phosphorus NMR (free OH content), HP-SEC (resulting lignin size), FT-IR (lignin compound functions) and Tg (glass transition temperature) techniques and no significant changes were recorded.
(3) In a second step, we turned to the kraft lignins prepared by the FCBA (P1 BBF, P2 FCBA). For example, a first principal component analysis (PCA) based on FT-IR tests (P2 FARE) allowed us to highlight the pH effect, which is very visible in FT-IR, as well as a possible effect of the redox mediator, 1-hydroxybenzotriazole (HBT). Three statistical ANOVA tests were then performed on the FT-IR data and allowed to say that the enzymatic treatment which brings the greatest significant difference is the one corresponding to the treatment with laccase and HBT.
Tests carried out by HP-SEC technique (P3: IJPB) confirmed these results.
Finally, Tg measurements (P5: IP) on these laccase-modified lignins indicate that the most significant Tg changes correspond to the laccase-treated samples. The HBT mediator has an effect that is additive to that of laccase when both are used simultaneously on the lignin.

WP4 Applications of enzymatically modified lignins

Initial application tests have been carried out by P2 (FARE) and P4 (FCBA).
for the preparation of coating films and adhesives (P4), and agro-based materials.

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A next publication is being written to valorize the results concerning the characterization of enzymatically modified lignins and extractives and the development of adhesives. The next step will be to implement laccase with dehydrogenases with sequential treatment or in one step (mixture of enzymes).

1. Sona Garajová, Ilabahen Patel, Anne Lomascolo, Frédéric Legée, Laurent Cézard, Betty Cottyn, Michaël Lecourt, Emmanuel Bertrand, Giuliano Sciara, Sandra Tapin-Lingua, Stéphanie Baumberger, Craig B. Faulds & Eric Record Treatment of wood fibres with laccases: improved hardboard properties through phenolic oligomerization. Eur. J. Wood Prod. 79, 1369–1382 (2021). doi.org/10.1007/s00107-021-01720-3

2. 1. Cerutti G, Gugole E, Montemiglio LC, Turbé-Doan A, Chena D, Navarro D, Lomascolo A, Piumi F, Exertier C, Freda I, Vallone B, Record E, Savino C, Sciara G. Crystal structure and functional characterization of an oligosaccharide dehydrogenase from Pycnoporus cinnabarinus provides insights into fungal breakdown of lignocellulose. Biotechnol Biofuels. 2021 Jul 22;14(1):161. doi: 10.1186/s13068-021-02003-y. PMID: 34294139; PMCID: PMC8296622. biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-021-02003-y

Lignins, the second most abundant polymers on earth, are poorly valorized as combustible energy. This project aims at enzymatically modifying different lignins to produce green biobased products for wood applications in an environmentally friendly and energy-saving process. We propose to exploit in-house enzymes in comparison with industrial one to activate different lignins (organosolv, lignosulfate, Kraft) from two wood essences (pine and birch) and applied them to produce adhesives, biosourced composites and protective coating for the furniture sector. The mode of action of oxidizing enzymes will be studied using a multidisciplinary approach including analytical chemistry, organic synthesis and spectroscopy. In addition, the environmental impact and the economic cost of the biosourced compounds will also be studied in the proposed project.