Effect of Salts on Laccase-Catalyzed Polymerization of Lignosulfonate.

Enzymatic polymerization of lignosulfonate (LS) has a high potential for various applications ranging from coatings to adhesives. Here, the effect of different ions in low concentrations on enzymatic polymerization of LS was investigated, including salt solutions consisting of mono- and dicarboxylic acids, sulfate, phosphate and chloride with sodium as counter ion. LS polymerization was followed by viscometry and size exclusion (SEC) chromatography. Interestingly, there was only a small effect of ions on the activity of the laccase on standard substrate ABTS, while the effect on polymerization of LS was substantially different. The presence of acetate led to a 39 % higher degree of polymerization (DP) for LS. Small angle X-ray scattering (SAXS) revealed that the structure of the enzyme was largely unaffected by the ions, while the determination of the zeta potential showed that those ions conveying higher negative surface charges onto LS particles showed lower DPs, than those not affecting the surface charge. Further, electron paramagnetic resonance (EPR) spectroscopy showed 5-times higher intensity in phenoxyl radicals for the monovalent ions compared to the divalent ones. It was concluded that the DPs of LS could be tuned in the presence of certain ions, by facilitating the interaction between the laccase substrate-binding site and the LS molecules.

Controlled Enzymatic Synthesis of Polyesters Based on a Cellulose-Derived Triol Monomer: A Design of Experiment Approach.

Regioselective enzymatic polycondensation of the bio-based cellulose derived polyol, Triol-citro, and dimethyl adipate using Candida antarctica Lipase B (CaLB) was investigated. A Design of Experiment approach with MODDE® Pro 13 was used to determine important factors in the branching behavior of this polymer, and reactant ratio, temperature, reaction time and enzyme wt % were the studied factors. Multifunctional polyesters with pendant hydroxy groups were synthesized and fully characterized using 2D NMR techniques to determine degree of branching. Branching was minimal, with a maximum of 16 % observed, and monomer ratio, temperature and reaction time were all determined to be significant factors. In this work, Mn of up to 13 kDa were achieved, while maintaining degree of branching below 15 %, resulting in a linear polyester with the potential to be further functionalized.

Spent brewer's yeast as a selective biosorbent for metal recovery from polymetallic waste streams.

While the amount of electronic waste is increasing worldwide, the heterogeneity of electronic scrap makes the recycling very complicated. Hydrometallurgical methods are currently applied in e-waste recycling which tend to generate complex polymetallic solutions due to dissolution of all metal components. Although biosorption has previously been described as a viable option for metal recovery and removal from low-concentration or single-metal solutions, information about the application of selective metal biosorption from polymetallic solutions is missing. In this study, an environmentally friendly and selective biosorption approach, based on the pH-dependency of metal sorption processes is presented using spent brewer's yeast to efficiently recover metals like aluminum, copper, zinc and nickel out of polymetallic solutions. Therefore, a design of experiment (DoE) approach was used to identify the effects of pH, metal, and biomass concentration, and optimize the biosorption efficiency for each individual metal. After process optimization with single-metal solutions, biosorption experiments with lyophilized waste yeast biomass were performed with synthetic polymetallic solutions where over 50% of aluminum at pH 3.5, over 40% of copper at pH 5.0 and over 70% of zinc at pH 7.5 could be removed. Moreover, more than 50% of copper at pH 3.5 and over 90% of zinc at pH 7.5 were recovered from a real polymetallic waste stream after leaching of printed-circuit boards. The reusability of yeast biomass was confirmed in five consecutive biosorption steps with little loss in metal recovery abilities. This proves that spent brewer's yeast can be sustainably used to selectively recover metals from polymetallic waste streams different to previously reported studies.

Metal recovery from spent lithium-ion batteries via two-step bioleaching using adapted chemolithotrophs from an acidic mine pit lake.

The demand for lithium-ion batteries (LIBs) has dramatically increased in recent years due to their application in various electronic devices and electric vehicles (EVs). Great amount of LIB waste is generated, most of which ends up in landfills. LIB wastes contain substantial amounts of critical metals (such as Li, Co, Ni, Mn, and Cu) and can therefore serve as valuable secondary sources of these metals. Metal recovery from the black mass (shredded spent LIBs) can be achieved via bioleaching, a microbiology-based technology that is considered to be environmentally friendly, due to its lower costs and energy consumption compared to conventional pyrometallurgy or hydrometallurgy. However, the growth and metabolism of bioleaching microorganisms can be inhibited by dissolved metals. In this study, the indigenous acidophilic chemolithotrophs in a sediment from a highly acidic and metal-contaminated mine pit lake were enriched in a selective medium containing iron, sulfur, or both electron donors. The enriched culture with the highest growth and oxidation rate and the lowest microbial diversity (dominated by Acidithiobacillus and Alicyclobacillus spp. utilizing both electron donors) was then gradually adapted to increasing concentrations of Li+, Co2+, Ni2+, Mn2+, and Cu2+. Finally, up to 100% recovery rates of Li, Co, Ni, Mn, and Al were achieved via two-step bioleaching using the adapted culture, resulting in more effective metal extraction compared to bioleaching with a non-adapted culture and abiotic control.

Multifunctional Casein-Based Wound Dressing Capable of Monitoring and Moderating the Proteolytic Activity of Chronic Wounds.

Every 1.2 s, a diabetic foot ulcer is developed, and every 20 s, one amputation is carried out in diabetic patients. Monitoring and controlling protease activity have been considered as a strategy for more efficient management of diabetic and other chronic wounds. This study aimed to develop a casein-based dressing that, by its disappearance, provides information about the activity of proteases and simultaneously harnesses proteolytic activity. Casein films were fabricated by using an aqueous solution, and heat treatment was successfully deployed as a green and clean approach to confer hydrolytic stability. Our results showed that casein-based films' mechanical characteristics, water absorption, and proteolytic stability could be controlled by the length of the heat treatment, which proved to be a useful tool. An increase in the treatment duration from 30 min to 3 h led to toleration of 2.4 times higher stress, 2 times lower water uptake, and 3.4 times higher proteolytic stability at examined conditions. Selected casein-based structures responded to Bacillus sp. bacteria's protease (BSP) and human neutrophil elastase (HNE) as representatives of bacterial and nonbacterial proteases found in the wounds at 10 and 200 ng mL-1 levels, respectively. The hydrolysis was accompanied by a 36% reduction in proteolytic activity measured by using a casein-based universal protease activity assay. The released casein fragments could scavenge 90% of the examined radicals. In-vitro cell culture studies showed that the hydrolysates were not cytotoxic, and the casein-based film had a favorable interaction with fibroblast cells, indicating its potential as a scaffold in the case that proteolytic activity would not be to the extent that causes its rapid disintegration. In general, these findings hold promise for applying the developed casein-based structure for detecting proteolytic activity without the need for any equipment, kits, or expertise and, more importantly, in a highly economical manner. In the case that the proteolytic activity would not be severe, it could also serve as a substrate for cell adhesion and growth; this would aid in the healing process.

On the Selective Enzymatic Recycling of Poly(pentamethylene 2,5-furanoate)/Poly(lactic acid) Blends and Multiblock Copolymers.

Among novel renewable furanoate-based polyesters, poly(pentamethylene 2,5-furandicarboxylate) (PPeF) shows outstanding gas barrier properties and high flexibility. PPeF blending/copolymerization with another well-known bio-based polymer, poly(lactic acid) (PLA), leads to considerably better mechanical and gas barrier properties of the latter, making it suitable for flexible food packaging applications. In this work, enzymatic depolymerization of PLA/PPeF blends with different compositions (1, 3, 5, 20, 30, and 50 wt % PPeF) and a PLA-PPeF block copolymer (50 wt % PPeF) by cutinase 1 from Thermobifida cellulositilytica (Thc_Cut1) was investigated as a possible recycling strategy. Based on quantification of weight loss and high-performance liquid chromatography (HPLC) analysis of released molecules, faster hydrolysis was seen for PLA/PPeF blends with increasing PPeF content when compared to neat PLA, while the block copolymer (P(LA50PeF50)) was significantly less susceptible to hydrolysis. Surface morphology analysis (via scanning electron microscopy), Fourier transform infrared spectroscopy, and NMR analysis confirmed preferential hydrolysis of the PPeF component. Through crystallization, 2,5-furandicarboxylic acid was selectively recovered from the depolymerized films and used for the resynthesis of the PPeF homopolymer, demonstrating the potential of enzymes for novel recycling concepts. The possibility of selective recovery of 2,5-furandicarboxylic acid from the completely depolymerized films with a 75% yield could bring further evidence of the high value of these materials, both in the form of blends and copolymers, for a sustainable whole packaging life cycle, where PPeF is potentially enzymatically recycled and PLA is mechanically recycled.

Further Step in the Transition from Conventional Plasticizers to Versatile Bioplasticizers Obtained by the Valorization of Levulinic Acid and Glycerol.

In the last two decades, the use of phthalates has been restricted worldwide due to their well-known toxicity. Nonetheless, phthalates are still widely used for their versatility, high plasticization effect, low cost, and lack of valuable alternatives. This study presents the fully bio-based and versatile glycerol trilevulinate plasticizer (GT) that was obtained by the valorization of glycerol and levulinic acid. The mild-conditions and solvent-free esterification used to synthesize GT was optimized by investigating the product by Fourier transform infrared and NMR spectroscopy. An increasing content of GT, from 10 to 40 parts by weight per hundred parts of resin (phr), was tested with poly(vinyl chloride), poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(lactic acid), and poly(caprolactone), which typically present complicated processability and/or mechanical properties. GT produced a significant plasticization effect on both amorphous and semicrystalline polymers, reducing their glass-transition temperature and stiffness, as observed by differential scanning calorimetry measurements and tensile tests. Remarkably, GT also decreased both the melting temperature and crystallinity degree of semicrystalline polymers. Furthermore, GT underwent enzyme-mediated hydrolysis to its initial constituents, envisioning a promising prospective for environmental safety and upcycling. Furthermore, 50% inhibitory concentration (IC50) tests, using mouse embryo fibroblasts, proved that GT is an unharmful alternative plasticizer, which makes it potentially applicable in the biomedical field.

Dihydropyrimidinase from Saccharomyces kluyveri can hydrolyse polyamides.

In Saccharomyces kluyveri, dihydropyrimidinase (DHPaseSK) is involved in the pyrimidine degradation pathway, which includes the reversible ring cleavage between nitrogen 3 and carbon 4 of 5,6-dihydrouracil. In this study, DPHaseSK was successfully cloned and expressed in E. coli BL-21 Gold (DE3) with and without affinity tags. Thereby, the Strep-tag enabled fastest purification and highest specific activity (9.5 ± 0.5 U/mg). The biochemically characterized DHPaseSK_Strep had similar kinetic parameters (Kcat/Km) on 5,6-dihydrouracil (DHU) and para-nitroacetanilide respectively, with 7,229 and 4060 M-1 s-1. The hydrolytic ability of DHPaseSK_Strep to polyamides (PA) was tested on PA consisting of monomers with different chain length (PA-6, PA-6,6, PA-4,6, PA-4,10 and PA-12). According to LC-MS/TOF analysis, DHPaseSK_Strep showed a preference for films containing the shorter chain monomers (e.g., PA-4,6). In contrast, an amidase from Nocardia farcinica (NFpolyA) showed some preference for PA consisting of longer chain monomers. In conclusion, in this work DHPaseSK_Strep was demonstrated to be able to cleave amide bonds in synthetic polymers, which can be an important basis for development of functionalization and recycling processes for polyamide containing materials.

The Biomodified Lignin Platform: A Review.

A reliance on fossil fuel has led to the increased emission of greenhouse gases (GHGs). The excessive consumption of raw materials today makes the search for sustainable resources more pressing than ever. Technical lignins are mainly used in low-value applications such as heat and electricity generation. Green enzyme-based modifications of technical lignin have generated a number of functional lignin-based polymers, fillers, coatings, and many other applications and materials. These bio-modified technical lignins often display similar properties in terms of their durability and elasticity as fossil-based materials while also being biodegradable. Therefore, it is possible to replace a wide range of environmentally damaging materials with lignin-based ones. By researching publications from the last 20 years focusing on the latest findings utilizing databases, a comprehensive collection on this topic was crafted. This review summarizes the recent progress made in enzymatically modifying technical lignins utilizing laccases, peroxidases, and lipases. The underlying enzymatic reaction mechanisms and processes are being elucidated and the application possibilities discussed. In addition, the environmental assessment of novel technical lignin-based products as well as the developments, opportunities, and challenges are highlighted.

Engineering a Carboxyl Methyltransferase for the Formation of a Furan-Based Bioplastic Precursor.

FtpM from Aspergillus fumigatus was the first carboxyl methyltransferase reported to catalyse the dimethylation of dicarboxylic acids. Here the creation of mutant R166M that can catalyse the quantitative conversion of bio-derived 2,5-furandicarboxylic acid (FDCA) to its dimethyl ester (FDME), a bioplastics precursor, was reported. Wild type FtpM gave low conversion due to its reduced catalytic efficiency for the second methylation step. An AlphaFold 2 model revealed a highly electropositive active site, due to the presence of 4 arginine residues, postulated to favour the binding of the dicarboxylic acid over the intermediate monoester. The R166M mutation improved both binding and turnover of the monoester to permit near quantitative conversion to the target dimethyl ester product. The mutant also had improved activity for other diacids and a range of monoacids. R166M was incorporated into 2 multienzyme cascades for the synthesis of the bioplastics precursor FDME from bioderived 5-hydroxymethylfurfural (HMF) as well as from poly(ethylene furanoate) (PEF) plastic, demonstrating the potential to recycle waste plastic.

Bio-upcycling of multilayer materials and blends: closing the plastics loop.

The urge to discover and develop new technologies for closing the plastic carbon cycle is motivating industries, governments, and academia to work closely together to find suitable solutions in a timely manner. In this review article, a combination of uprising breakthrough technologies is presented highlighting their potential and complementarity to be integrated one with the other, therefore providing a potential solution to efficiently solve the plastics problem. First, modern approaches for bio-exploration and engineering of polymer-active enzymes are presented to degrade polymers into valuable building blocks. Special focus is placed on the recovery of components from multilayered materials since these complex materials can only be recycled insufficiently or not at all by existing technologies. Then, the potential of microbes and enzymes for resynthesis of polymers and reuse of building blocks is summarized and discussed. Finally, examples for improvement of the bio-based content and enzymatic degradability and future perspectives are given.

Improving Properties of Starch-Based Adhesives with Carboxylic Acids and Enzymatically Polymerized Lignosulfonates.

A novel strategy for improving wet resistance and bonding properties of starch-based adhesives using enzymatically polymerized lignosulfonates and carboxylic acids as additives was developed. Therefore, lignosulfonates were polymerized by laccase to a molecular weight of 750 kDa. Incorporation of low concentrations (up to 1% of the starch weight) of 1,2,3,4-butanetetracarboxylic acid (BTCA) led to further improvement on the properties of the adhesives, while addition of greater amounts of BTCA led to a decrease in the properties measured due to large viscosity increases. Great improvements in wet-resistance from 22 to 60 min and bonding times (from 30 to 20 s) were observed for an adhesive containing 8% enzymatically polymerized lignin and 1% BTCA. On the other hand, the addition of citric acid (CA) deteriorated the properties of the adhesives, especially when lignosulfonate was present. In conclusion, this study shows that the addition of the appropriate amount of enzymatically polymerized lignosulfonates together with carboxylic acids (namely BTCA) to starch-based adhesives is a robust strategy for improving their wet resistance and bonding times.

Bioelectrochemical methanation by utilization of steel mill off-gas in a two-chamber microbial electrolysis cell.

Carbon capture and utilization has been proposed as one strategy to combat global warming. Microbial electrolysis cells (MECs) combine the biological conversion of carbon dioxide (CO2) with the formation of valuable products such as methane. This study was motivated by the surprising gap in current knowledge about the utilization of real exhaust gas as a CO2 source for methane production in a fully biocatalyzed MEC. Therefore, two steel mill off-gases differing in composition were tested in a two-chamber MEC, consisting of an organic substrate-oxidizing bioanode and a methane-producing biocathode, by applying a constant anode potential. The methane production rate in the MEC decreased immediately when steel mill off-gas was tested, which likely inhibited anaerobic methanogens in the presence of oxygen. However, methanogenesis was still ongoing even though at lower methane production rates than with pure CO2. Subsequently, pure CO2 was studied for methanation, and the cathodic biofilm successfully recovered from inhibition reaching a methane production rate of 10.8 L m-2d-1. Metagenomic analysis revealed Geobacter as the dominant genus forming the anodic organic substrate-oxidizing biofilms, whereas Methanobacterium was most abundant at the cathodic methane-producing biofilms.

Chitosan: Sources, Processing and Modification Techniques.

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is derived from chitin. Chitin is found in cell walls of crustaceans, fungi, insects and in some algae, microorganisms, and some invertebrate animals. Chitosan is emerging as a very important raw material for the synthesis of a wide range of products used for food, medical, pharmaceutical, health care, agriculture, industry, and environmental pollution protection. This review, in line with the focus of this special issue, provides the reader with (1) an overview on different sources of chitin, (2) advances in techniques used to extract chitin and converting it into chitosan, (3) the importance of the inherent characteristics of the chitosan from different sources that makes them suitable for specific applications and, finally, (4) briefly summarizes ways of tailoring chitosan for specific applications. The review also presents the influence of the degree of acetylation (DA) and degree of deacetylation (DDA), molecular weight (Mw) on the physicochemical and biological properties of chitosan, acid-base behavior, biodegradability, solubility, reactivity, among many other properties that determine processability and suitability for specific applications. This is intended to help guide researchers select the right chitosan raw material for their specific applications.

Role of Surface Enhancement in the Enzymatic Cross-Linking of Lignosulfonate Using Alternative Downstream Techniques.

Lignosulfonate (LS), one of the byproducts of the paper and pulp industry, was mainly used as an energy source in the last decade until the valorization of lignin through different functionalization methods grew in importance. Polymerization using multicopper oxidase laccase (from the Myceliophthora thermophila fungus) is one of such methods, which not only enhances properties such as hydrophobicity, flame retardancy, and bonding properties but can also be used for food and possesses pharmaceutical-like antimicrobial properties and aesthetic features of materials. Appropriate downstream processing methods are needed to produce solids that allow the preservation of particle morphology, a vital factor for the valorization process. In this work, an optimization of the enzymatic polymerization via spray-drying of LS was investigated. The response surface methodology was used to optimize the drying process, reduce the polymerization time, and maximize the dried mass yield. Particles formed showed a concave morphology and enhanced solubility while the temperature sensitivity of spray-drying protected the phenol functionalities beneficial for polymerization. Using the optimized parameters, a yield of 65% in a polymerization time of only 13 min was obtained. The experimental values were found to be in agreement with the predicted values of the factors (R 2: 95.2% and p-value: 0.0001), indicating the suitability of the model in predicting polymerization time and yield of the spray-drying process.

Optimized biogenic sulfuric acid production and application in the treatment of waste incineration residues.

The biological leaching of metals from different waste streams by bacteria is intensively investigated to address metal recycling and circular economy goals. However, usually external addition of sulfuric acid is required to maintain the low pH optimum of the bacteria to ensure efficient leaching. Extremely acidophilic Acidithiobacillus spp. producing sulfuric acid and ferric iron have been investigated for several decades in the bioleaching of metal-containing ores. Their application has now been extended to the extraction of metals from artificial ores and other secondary sources. In this study, an optimized process for producing biogenic sulfuric acid from elemental sulfur by two sulfur-oxidizing species, A. thiooxidans and A. caldus and their combinations, was investigated in batch and stirred tank experiments. Using a combined culture of both species, 1.05 M and 1.4 M biogenic sulfuric acid was produced at 30 °C and 6% elemental sulfur in batch and semi continuous modes, respectively. The acid produced was then used to control the pH in a heap bioleaching system in which iron- and sulfur-oxidizing A. ferrooxidans was applied to biologically leach metals from waste incineration residuals. Metals like Cu, Ni, Al, Mn, and Zn were successfully recovered by 99, 93, 84, 77 and 68%, respectively within three weeks of heap bioleaching. Overall, a potential value recovery of incorporated metals >70% could be achieved. This highlights the potential and novelty of applying extremely acidophilic sulfur-oxidizing bacteria for cheap and efficient production of biogenic sulfuric acid and its use in pH control.

Enzymatic synthesis of wet-resistant lignosulfonate-starch adhesives.

This work describes a new method for improving the properties, mainly the wet-resistance, of starch-based adhesives using enzymatically polymerized lignosulfonates. A correlation of viscosity with molecular weight was found, allowing simple control of enzymatic polymerization of lignosulfonates. Incorporation of lignosulfonates polymerized from 29 kDa to > 4500 kDa using laccase led to a considerable increase in wet-resistance (from 15 to 20 min for the laminating glue and from 150 to 1200 min for the bag glue) while not affecting (for the laminating glue) or even improving the bonding time (from 80 to 60 s for the bag glue). Finally, the effect of active laccase in the final adhesive was investigated by enzymatic inactivation using NaN3 before formulation of the glue, as well as by extra laccase addition. In conclusion, this study shows that enzymatically polymerized lignosulfonate is a robust strategy for improving wet resistance of starch-based adhesives.

Residue-Specific Incorporation of the Non-Canonical Amino Acid Norleucine Improves Lipase Activity on Synthetic Polyesters.

Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.

Cutinase-Catalyzed Polyester-Polyurethane Degradation: Elucidation of the Hydrolysis Mechanism.

Polyurethanes (PU) are one of the most-used classes of synthetic polymers in Europe, having a considerable impact on the plastic waste management in the European Union. Therefore, they represent a major challenge for the recycling industry, which requires environmentally friendly strategies to be able to re-utilize their monomers without applying hazardous and polluting substances in the process. In this work, enzymatic hydrolysis of a polyurethane-polyester (PU-PE) copolymer using Humicola insolens cutinase (HiC) has been investigated in order to achieve decomposition at milder conditions and avoiding harsh chemicals. PU-PE films have been incubated with the enzyme at 50 °C for 168 h, and hydrolysis has been followed throughout the incubation. HiC effectively hydrolysed the polymer, reducing the number average molecular weight (Mn) and the weight average molecular weight (Mw) by 84% and 42%, respectively, as shown by gel permeation chromatography (GPC), while scanning electron microscopy showed cracks at the surface of the PU-PE films as a result of enzymatic surface erosion. Furthermore, Fourier Transform Infrared (FTIR) analysis showed a reduction in the peaks at 1725 cm-1, 1164 cm-1 and 1139 cm-1, indicating that the enzyme preferentially hydrolysed ester bonds, as also supported by the nuclear magnetic resonance spectroscopy (NMR) results. Liquid chromatography time-of-flight/mass spectrometry (LC-MS-Tof) analysis revealed the presence in the incubation supernatant of all of the monomeric constituents of the polymer, thus suggesting that the enzyme was able to hydrolyse both the ester and the urethane bonds of the polymer.

Enzymatic Conversion of Lignosulfonate into Wood Adhesives: A Next Step towards Fully Biobased Composite Materials.

This study investigates the effect of the enzymatic polymerization of lignosulfonate for the formulation of a lignosulfonate-based adhesive. For this, beech lamellas were glued together and tested according to the EN 302-1 standard. The results showed that the laccase-polymerized lignosulfonate-based wood adhesives (LS-p) had similar mechanical properties as a standard carpenter's glue (PVAc-based D3 class white glue), as no significant difference in tensile shear strength between these two adhesive types was found. However, carpenter's glue showed almost 100% wood failure, while with the lignosulfonate-based wood glue, the samples failed, mainly in the glueline. Pre-polymerization of LS-p is the most critical factor to achieve the required viscosity, which is also connected to the wetting properties and the resulting tensile shear strength. The longer the pre-polymerization, the higher the viscosity of the LS-p adhesive, with the tensile shear strength reaching a plateau. The presented data show the potential of using enzymatically pre-polymerized lignosulfonate as a well-performing wood adhesive. Further development and optimization of the pre-polymerization process is required, which is also important to push towards upscaling and practical applications.