The lack of renewable resources and their inefficient use is a major challenge facing the society. Lignin is a natural biopolymer obtained mainly as a by-product from pulp-and paper-making industry, and is primarily burned to produce energy. However, the interest for using lignin in more advanced applications has increased rapidly. In particular, lignin based nanoparticles could find potential use in functional surface coatings, nanoglues, drug delivery, and microfluidic devices. In this work, a straightforward method to produce lignin nanoparticles from waste lignin obtained from kraft pulping is introduced.Spherical lignin nanoparticles were obtained by dissolving soft wood kraft lignin in tetrahydrofuran (THF) and subsequently introducing water into the system through dialysis. No chemical modification of the lignin was needed. Water acts as a nonsolvent reducing lignin's degrees of freedom causing the segregation of hydrophobic regions to compartments within the forming nanoparticles. The final size of the nanoparticles depended on the pre-dialysis concentration of dissolved lignin. The stability of the nanoparticle dispersion as a function of time, salt concentration and pH was studied. In pure water and room temperature the lignin nanoparticle dispersion was stable for over two months, but very low pH or high salt concentration induced aggregation. It was further demonstrated that the surface charge of the particles could be reversed and stable cationic lignin nanoparticles were produced by adsorption of poly(diallyldimethylammonium chloride) (PDADMAC).

Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOES of the backbone protons between the wild-type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly.

Different possibilities for protein crosslinking are examined in this review, with special emphasis on enzymatic crosslinking and its impact on food structure. Among potential enzymes for protein crosslinking are transglutaminase (TG) and various oxidative enzymes. Crosslinking enzymes can be applied in cereal, dairy, meat, and fish processing to improve the texture of the product. Most of the current commercial applications are based on TG. The reaction mechanisms of the crosslinking enzymes differ, which in turn results in different technological properties.

Fundamentals of nanoprecipitation process to form colloidal lignin particles (CLPs) from tetrahydrofuran (THF)-water solvent system were studied, and applied in establishment of a robust reactor design for scaled-up CLP production. Spherical lignin particles with an average diameter of 220 nm could be produced by the new reactor design. Evaporation was applied for removal of THF, concentration of the CLP dispersions, and finally for drying of the CLPs into flake like dry form. The dried CLPs could be re-dispersed in water to restore their colloidal form by applying short physical agitation. Salt triggered sedimentation of the particles was also investigated as a way for reducing the energy consumption related to water evaporation from the CLP dispersions. Aqueous thermal post-treatments were demonstrated to yield structural reinforcement of the CLP structure against solvation in various lignin solvents. In summary, the presented work pushes forward the conceptual design of large-scale CLP production, and addresses some of the foreseen technical challenges.

Proteins and certain carbohydrates contain phenolic moieties, which are potential sites for modification of the function of the biopolymers. In this study, the capability of two different fungal oxidative enzymes, laccase from Trametes hirsuta (ThL) and tyrosinase from Trichoderma reesei (TrT), to catalyze formation of hetero-cross-linking between tyrosine side chains of alpha-casein and phenolic acids of hydrolyzed oat spelt xylan (hOSX) was studied. Formation of reaction products was followed by size exclusion chromatography (SEC), fluorescence spectroscopy, and SDS-PAGE, using specific staining methods for proteins and protein-carbohydrate conjugates. ThL and TrT were observed to differ significantly in their ability to catalyze the formation of protein-carbohydrate conjugates or the linking of the small molecular weight phenolic compounds to alpha-casein. The efficiency of these enzymes to directly cross-link protein also differed notably. TrT was able to cross-link alpha-casein more efficiently than ThL. ThL-catalyzed casein cross-linking was significantly enhanced by ferulic acid, p-coumaric acid, and also hOSX. The main reaction products by ThL appeared to be phenolic acid-bridged alpha-caseins. Indications of hetero-cross-link formation between alpha-casein and hOSX by both oxidative enzymes could be visualized by glycoprotein-specific staining in the SDS-PAGE analysis, although ThL was observed to be more effective in the heteroconjugate formation than TrT.

Laccase (EC 1.10.3.2) is a multicopper enzyme belonging to the blue multicopper oxidase family. The most studied laccases are of fungal and plant origin [1][2][3][4][5], however, some bacterial laccases [6,7] have also been isolated. In addition, these enzymes have been found in some insects [8][9][10]. Laccases catalyze the oxidation of a wide range of organic and inorganic substances [11]. Typical organic substrates are aromatic compounds, such as different phenols, anilines and benzenethiols [11][12][13][14][15][16][17]. Laccases catalyze single-electron oxidation of the substrate, with concomitant reduction of molecular oxygen to water as shown in Scheme 1 [18]:Attempts to utilize the reactivity of laccase on phenolic substrates have been made, e.g., in pulp and paper, textile and food applications. Denim bleaching with a laccase-mediator system has been launched in the textile industry. The other applications, i.e., kraft pulp bleaching and detoxification have been only tested in laboratory or pilot scale [19]. Interest in the use of laccases in food processing is also increasing [20][21][22][23]. Laccases have been tested in bread making, where they can improve bread volume [24]. They can cross-link pentosans and arabinoxylans via ferulic acid (FA) side-chains [25,26]. It has been suggested that this kind of cross-linking of feroylated carbohydrates by laccase is similar to the peroxidase-catalyzed reaction, the aromatic ring of FA being the initiating site for enzymatic oxidation [25]. It has also been shown that laccase can cross-link whey proteins in the presence of phenolic acid [27]. However, in order to be able to develop new applications for laccases in foods, it is crucial also to understand enzymatic reactions on proteins at the molecular level. At present, laccase-catalyzed reactions resulting in oxidation of proteins are poorly understood. Very Laccase-catalyzed polymerization of tyrosine and tyrosine-containing peptides was studied in the presence and absence of ferulic acid (FA). Advanced spectroscopic methods such as MALDI-TOF MS, EPR, FTIR microscopy and HPLC-fluorescence, as well as more conventional analytical tools: oxygen consumption measurements and SDS ⁄ PAGE were used in the reaction mechanism studies. Laccase was found to oxidize tyrosine and tyrosine-containing peptides, with consequent polymerization of the compounds. The covalent linkage connecting the compounds was found to be an ether bond. Only small amounts of dityrosine bonds were detected in the polymers. When FA was added to the reaction mixtures, it was found to be incorporated into the polymer structure. Thus, in addition to homopolymers, different heteropolymers containing two or four FA residues were formed in the reactions.

Coating of colloidal lignin particles (CLPs), or lignin nanoparticles (LNPs), with proteins was investigated in order to establish a safe, self-assembly-mediated modification technique to tune their surface chemistry. Gelatin and poly-L-lysine formed the most pronounced protein corona on the CLP surface, as determined by dynamic light scattering (DLS) and zeta potential measurements. Spherical morphology of individual protein coated CLPs was confirmed by transmission electron (TEM) and atomic force (AFM) microscopy. A mechanistic adsorption study with several random coiled and globular model proteins was carried out using quartz crystal microbalance with dissipation monitoring (QCM-D). The three-dimensional (3D) protein fold structure and certain amino acid interactions were highly dependent on the protein adsorption on the lignin surface. The main driving forces of protein-lignin affinity were electrostatic, hydrophobic, and Van der Waals interactions, and hydrogen bonding. The relative contributions of these interactions were highly dependent on the ionic strength of the surrounding medium. Capillary electrophoresis (CE) and Fourier transform infrared spectroscopy (FTIR) provided further evidence about the adsorption-enhancing role of specific amino acid residues such as serine and proline. These results have high impact on the utilization of lignin as colloidal particles in 2 biomedicine and biodegradable materials, as the protein corona enables tailoring of the CLP surface chemistry for intended applications.

Three‐dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel β‐sheet structure of the CBD fold was preserved in all engineered peptides. The three‐dimensional NMR‐based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N‐terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.