Chemo-enzymatically prepared lignin nanoparticles for value-added applications.

The global need to develop sustainable materials and products from non-fossil raw material is pushing industry to utilize side-streams more efficiently using green processes. Aromatic lignin, the world's second most abundant biopolymer, has multiple attractive properties which can be exploited in various ways instead of being burnt or used as animal feed. Lignin's poor water solubility and its highly branched and random structure make it a challenging biopolymer to exploit when developing novel technologies for the preparation of tailored nanobiomaterials for value-added applications. The notable number of scientific publications focusing on the formation and modification of technical lignin in nanoparticulate morphology show that these bottlenecks could be solved using lignin in the form of colloidal particles (CLPs). These particles are very stable at wide pH range (4-11) and easily dispersible in organic solvents after stabilized via cross-linking. Negative hydroxyl groups on the CLP surface enable multiple enzymatic and chemical modifications e.g. via polymerization reactions and surface-coating with positive polymers. This contribution highlights how tailored CLPs could be innovatively exploited in different the state-of-the-art applications such as medicine, foods, and cosmetics.

Colloidal Lignin Particles as Adhesives for Soft Materials.

Lignin has interesting functionalities to be exploited in adhesives for medicine, foods and textiles. Nanoparticles (NPs) < 100 nm coated with poly (L-lysine), PL and poly(L-glutamic acid) PGA were prepared from the laccase treated lignin to coat nanocellulose fibrils (CNF) with heat. NPs ca. 300 nm were prepared, ß-casein coated and cross-linked with transglutaminase (Tgase) to agglutinate chamois. Size exclusion chromatography (SEC) and Fourier-transform infrared (FTIR) spectroscopy were used to characterize polymerized lignin, while zeta potential and dynamic light scattering (DLS) to ensure coating of colloidal lignin particles (CLPs). Protein adsorption on lignin was studied by quartz crystal microbalance (QCM). Atomic force microscopy (AFM) was exploited to examine interactions between different polymers and to image NPs with transmission electron microscopy (TEM). Tensile testing showed, when using CLPs for the adhesion, the stress improved ca. 10 and strain ca. 6 times compared to unmodified Kraft. For the ß-casein NPs, the values were 20 and 8, respectively, and for the ß-casein coated CLPs between these two cases. When NPs were dispersed in adhesive formulation, the increased Young's moduli confirmed significant improvement in the stiffness of the joints over the adhesive alone. Exploitation of lignin in nanoparticulate morphology is a potential method to prepare bionanomaterials for advanced applications.

Enzymatically and chemically oxidized lignin nanoparticles for biomaterial applications.

Cross-linked and decolorized lignin nanoparticles (LNPs) were prepared enzymatically and chemically from softwood Kraft lignin. Colloidal lignin particles (CLPs, ca. 200 nm) in a non-malodorous aqueous dispersion could be dried and redispersed in tetrahydrofuran (THF) or in water retaining their stability i.e. spherical shape and size. Two fungal laccases, Trametes hirsuta (ThL) and Melanocarpus albomyces (MaL) were used in the cross-linking reactions. Reactivity of ThL and MaL on Lignoboost™ lignin and LNPs was confirmed by high performance size exclusion chromatography (HPSEC) and oxygen consumption measurements with simultaneous detection of red-brown color due to the formation of quinones. Zeta potential measurements verified oxidation of LNPs via formation of surface-oriented carboxylic acid groups. Dynamic light scattering (DLS) revealed minor changes in the particle size distributions of LNPs after laccase catalyzed radicalization, indicating preferably covalent intraparticular cross-linking over polymerization. Changes in the surface morphology of laccase treated LNPs were imaged by atomic force (AFM) and transmission emission (TEM) microscopy. Furthermore, decolorization of LNPs without degradation was obtained using ultrasonication with H2O2 in alkaline reaction conditions. The research results have high impact for the utilization of Kraft lignin as nanosized colloidal particles in advanced bionanomaterial applications in medicine, foods and cosmetics including different sectors from chemical industry.

Characterization of sulfhydryl oxidase from Aspergillus tubingensis.

BACKGROUND: Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear. RESULTS: The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initial activity was retained after incubation at 50 °C for 20 h. AtSOX contains a non-covalently bound flavin cofactor. The enzyme oxidised a sulfhydryl group of glutathione to form a disulfide bond, as verified by nuclear magnetic resonance spectroscopy. AtSOX preferred glutathione as a substrate over cysteine and dithiothreitol. The activity of the enzyme was totally inhibited by 10 mM zinc sulphate. Peptide- and protein-bound sulfhydryl groups in bikunin, gliotoxin, holomycin, insulin B chain, and ribonuclease A, were not oxidised by the enzyme. Based on the analysis of 33 fungal genomes, SOX enzyme encoding genes were found close to nonribosomal peptide synthetases (NRPS) but not with polyketide synthases (PKS). In the phylogenetic tree, constructed from 25 SOX and thioredoxin reductase sequences from IPR000103 InterPro family, AtSOX was evolutionary closely related to other Aspergillus SOXs. Oxidoreductases involved in the maturation of nonribosomal peptides of fungal and bacterial origin, namely GliT, HlmI and DepH, were also evolutionary closely related to AtSOX whereas fungal thioreductases were more distant. CONCLUSIONS: AtSOX (55 kDa) is a fungal secreted flavin-dependent enzyme with good stability to both pH and temperature. A Michaelis-Menten behaviour was observed with reduced glutathione as a substrate. Based on the location of SOX enzyme encoding genes close to NRPSs, SOXs could be involved in the secondary metabolism and act as an accessory enzyme in the production of nonribosomal peptides.

Adsorption of Proteins on Colloidal Lignin Particles for Advanced Biomaterials.

Coating of colloidal lignin particles (CLPs), or lignin nanoparticles (LNPs), with proteins was evaluated 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 decisive for the protein adsorption on the lignin surface. The main driving forces for protein adsorption 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 of 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 biomedicine and biodegradable materials, as the protein corona enables tailoring of the CLP surface chemistry for intended applications.

Brachybacterium sp. CH-KOV3 isolated from an oil-polluted environment-a new producer of levan.

Various microorganisms isolated from polluted environments, such as Pseudomonas sp. and Micrococcus sp. can synthesize exopolysaccharides (EPSs) which are natural, non-toxic and biodegradable polymers. EPSs play a key role in protection of microbial cells under various external influences. For humans, these substances have potential use in many industries. EPSs can be applied as a flavor or a fragrance carrier, an emulsifier, a stabilizer, a prebiotic, an antioxidant or an antitumor agent. In this study, we characterized an environmental microorganism that produces EPS, optimized EPS production by this strain and characterized the EPS produced. Isolate CH-KOV3 was identified as Brachybacterium paraconglomeratum. The sucrose level in the growth medium greatly influenced EPS production, and the highest yield was when the microorganism was incubated in media with 500g/L of sucrose. The optimal temperature and pH were 28°C and 7.0, respectively. The nuclear magnetic resonance (NMR) results and GC-MS analysis confirmed that the residues were d-fructofuranosyl residues with ß-configuration, where fructose units are linked by ß-2,6-glycosidic bonds, with ß-2,1-linked branches. All these data indicate that the investigated EPS is a levan-type polysaccharide. Thus, it was concluded that Brachybacterium sp. CH-KOV3 could constitute a new source for production of the bioactive polysaccharide, levan.

Versatile peroxidase as a valuable tool for generating new biomolecules by homogeneous and heterogeneous cross-linking.

The modification and generation of new biomolecules intended to give higher molecular-mass species for biotechnological purposes, can be achieved by enzymatic cross-linking. The versatile peroxidase (VP) from Pleurotus eryngii is a high redox-potential enzyme with oxidative activity on a wide variety of substrates. In this study, VP was successfully used to catalyze the polymerization of low molecular mass compounds, such as lignans and peptides, as well as larger macromolecules, such as protein and complex polysaccharides. Different analytical, spectroscopic, and rheological techniques were used to determine structural changes and/or variations of the physicochemical properties of the reaction products. The lignans secoisolariciresinol and hydroxymatairesinol were condensed by VP forming up to 8 unit polymers in the presence of organic co-solvents and Mn(2+). Moreover, 11 unit of the peptides YIGSR and VYV were homogeneously cross-linked. The heterogeneous cross-linking of one unit of the peptide YIGSR and several lignan units was also achieved. VP could also induce gelation of feruloylated arabinoxylan and the polymerization of ß-casein. These results demonstrate the efficacy of VP to catalyze homo- and hetero-condensation reactions, and reveal its potential exploitation for polymerizing different types of compounds.

Discovery of novel secreted fungal sulfhydryl oxidases with a plate test screen.

Sulfhydryl oxidases (SOX) are FAD-dependent enzymes capable of oxidising free thiol groups and forming disulphide bonds. Although the quantity of scientific papers and suggested applications for SOX is constantly increasing, only a limited number of microbial SOX have been reported and are commercially available. Hence, the aim of this study was to develop a fast and reliable qualitative plate test for screening novel secreted fungal SOX. The screening was based on the Ellman's reagent, i.e. 5,5'-dithiobis[2-nitrobenzoic acid]. Altogether, 32 fungal strains from an in-house culture collection were screened. A total of 13 SOX-producing strains were found positive in the plate test screen. The novel SOX producers were Aspergillus tubingensis, Chaetomium globusum, Melanocarpus albomyces, Penicillium aurantiogriseum, Penicillium funiculosum, Coniophora puteana and Trametes hirsuta. Six of the discovered SOX were partially characterised by determination of isoelectric point, pH optimum and substrate specificity. A. tubingensis was identified as the most efficient novel SOX producer.

Loosening of globular structure under alkaline pH affects accessibility of ß-lactoglobulin to tyrosinase-induced oxidation and subsequent cross-linking.

Globular proteins such as ß-lactoglobulin (BLG) are poorly accessible to enzymes. We have studied susceptibility of BLG to oxidation by Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) tyrosinases and subsequent intermolecular cross-linking with respect to pH-induced structural changes. We evaluated pH-induced structural changes in BLG using circular dichroism, tryptophan fluorescence and small angle X-ray scattering (SAXS) measurements, where after these results were correlated with the analysis of cross-linking by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Oxygen consumption measurement and changes in radii of gyration determined by SAXS during the enzyme-induced oxidation at the respective reaction conditions were also followed. Intermolecular cross-linking of BLG by TrTyr was found at pH 9 but not at pH 7.5. AbTyr was unable to catalyze cross-linking at pH 7.5 or pH 9. Increased accessibility and cross-linking by TrTyr was addressed to loosening of the three dimensional structure of the protein, increased flexibility of the backbone as well as partial hydrolysis. In addition to basic research of the effect of protein folding on enzymatic cross-linking the research results have significance on the exploitation of TrTyr at alkaline conditions.

Reactivity of bacterial and fungal laccases with lignin under alkaline conditions.

The ability of Streptomyces ipomoea laccase to polymerize secoisolariciresinol lignan and technical lignins was assessed. The reactivity of S. ipomoea laccase was also compared to that of low redox fungal laccase from Melanocarpus albomyces using low molecular mass p-coumaric, ferulic and sinapic acid as well as natural (acetosyringone) and synthetic 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) mediators as substrates. Oxygen consumption measurement, MALDI-TOF MS and SEC were used to follow the enzymatic reactions at pH 7, 8, 9 and 10 at 30°C and 50°C. Polymerization of lignins and lignan by S. ipomoea laccase under alkaline reaction conditions was observed, and was enhanced in the presence of acetosyringone almost to the level obtained with M. albomyces laccase without mediator. Reactivities of the enzymes towards acetosyringone and TEMPO were similar, suggesting exploitation of the compounds and low redox laccase in lignin valorization under alkaline conditions. The results have scientific impact on basic research of laccases.

Protein analysis by 31p NMR spectroscopy in ionic liquid: quantitative determination of enzymatically created cross-links.

Cross-linking of ß-casein by Trichoderma reesei tyrosinase (TrTyr) and Streptoverticillium mobaraense transglutaminase (Tgase) was analyzed by (31)P nuclear magnetic resonance (NMR) spectroscopy in ionic liquid (IL). According to (31)P NMR, 91% of the tyrosine side chains were cross-linked by TrTyr at high dosages. When Tgase was used, no changes were observed because a different cross-linking mechanism was operational. However, this verified the success of the phosphitylation of phenolics within the protein matrix in the IL. Atomic force microscopy (AFM) in solid state showed that disk-shaped nanoparticles were formed in the reactions with average diameters of 80 and 20 nm for TrTyr and Tgase, respectively. These data further advance the current understanding of the action of tyrosinases on proteins on molecular and chemical bond levels. Quantitative (31)P NMR in IL was shown to be a simple and efficient method for the study of protein modification.

Effect of protein structural integrity on cross-linking by tyrosinase evidenced by multidimensional heteronuclear magnetic resonance spectroscopy.

Enzymatic cross-linking of proteins can be catalyzed either by transferase-type enzymes, e.g., transglutaminases, or by oxidoreductases, e.g., tyrosinases or laccases. Three-dimensional structure of protein substrate plays a key role in these reactions, that is, the reactivity and end product are strongly modulated by the accessibility of target amino acid residues to the cross-linking enzyme. Typically structural integrity of protein can be distorted by heat, pH, or mechanical action, as well as by varying ionic concentration of the solution. In this study we used partially unfolded protein (wild-type DrkN SH3) and its structurally stabilized mutant (T22G) to investigate the impact of folded/unfolded conformations on cross-linking by Trichoderma reesei tyrosinase. Our results clearly showed formation of intermolecular cross-links solely between unfolded conformations, making them superior substrates to folded proteins when using tyrosinase as a cross-linking enzyme. Multidimensional heteronuclear magnetic resonance experiments in solution state were employed to investigate cross-linked end-products. The results presented in this study form basis for application development in food, medical, cosmetic, textile, packing and other sectors. In addition, the outcome of this study has a high value for the basic understanding of reaction mechanism of tyrosinases on proteins.

Structure of the polyphenolic component of suberin isolated from potato (Solanum tuberosum var. Nikola).

Suberin is present in the underground parts of vegetables and in the bark of trees. Characterization of suberin and the structure of its polyphenolic component have been hampered by insolubility of the polymers. Thus, enzymatically isolated and extractive free suberin enriched fraction from potato, Solanum tuberosum var. Nikola, and the chemically further fractionated phenolics were characterized in solid state by FTIR, DSC, and elemental analysis to identify the groups and to verify success of isolation. For MW and quantitative determination of the groups, polymers were solubilized in ionic liquid derivatized and analyzed by GPC and (31)P NMR. Suberin enriched fraction, MW = ca. 44 x 10(3) g/mol, is a mixture of carbohydrates and polyesters of aliphatic long chain hydroxy fatty acids and diacids linked via ester bonds to the phenolics, MW = ca. 27 x 10(3) g/mol, formed by guaiacyl- and p-hydroxyphenyl structures. Phenolics in peels may be important sources of antioxidants for various applications.

Formation of protein-oligosaccharide conjugates by laccase and tyrosinase.

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.

Oxidation of peptides and proteins by Trichoderma reesei and Agaricus bisporus tyrosinases.

The capability of a novel tyrosinase from Trichoderma reesei (TrTyr) to catalyse the oxidation and oxidative cross-linking of l-tyrosine (l-Y) and tyrosine side-chains in GYG and EGVYVHPV peptides, in bovine serum albumin (BSA) and beta-casein proteins as well as in proteinaceous wool fibres was studied by oxygen consumption measurement, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase high-performance liquid chromatography (RP-HPLC), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence microscopy. TrTyr was compared to the well-characterised tyrosinase from Agaricus bisporus (AbTyr) in terms of oxidation and cross-linking. According to the results obtained TrTyr was capable of cross-linking peptides and proteins more efficiently than AbTyr. However, the size and three-dimensional structure of the proteinaceous substrates proved to be crucial for the success of the enzymatic catalysis. Random coil beta-casein could be cross-linked by TrTyr already in three hours, but large and compact BSA was not cross-linked even in 24h. TrTyr could also be used to incorporate a diphenolic compound, l-dihydroxyphenyl alanine (l-dopa), into protein fibres whereas incorporation of a monophenol, l-Y was less efficient. Thus TrTyr is a potential tool for protein cross-linking and/or modification.

Effect of protein structure on laccase-catalyzed protein oligomerization.

Laccase-catalyzed oligomerization of proteins was studied using Trametes hirsuta laccase (ThL) and coactosin as a model system. The reaction mechanism was elucidated using free amino acids and the tripeptide Gly-Leu-Tyr as substrates. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography (HPLC) as well as oxygen consumption measurements and SDS-PAGE were used to study the reactions. Of the 15 selected amino acids, ThL was found to oxidize tryptophan (Trp), tyrosine (Tyr), and cysteine (Cys), of which the reactions with Tyr and Cys have been described earlier. ThL was able to link four full-length coactosins, whereas coactosin that was truncated from its C-terminus remained unpolymerized. Of the four tyrosine residues present in coactosin, only the tyrosine in the C-terminus was found to be reactive. Polymerization between tyrosine side-chains was unambiguously shown using different oligomers of Gly-Leu-Tyr as parent ions in MALDI-TOF/TOF MS fragment ion analyses.

Laccase-catalyzed polymerization of tyrosine-containing peptides.

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.

Enhanced ligand affinity for receptors in which components of the binding site are independently mobile.

Using calmodulin antagonism as a model, it is demonstrated that, under circumstances in which binding sites are motionally independent, it is possible to create bifunctional ligands that bind with significant affinity enhancement over their monofunctional counterparts. Suitable head groups were identified by using a semiquantitative screen of monofunctional tryptophan analogs. Two bifunctional ligands, which contained two copies of the highest-affinity head group tethered by rigid linkers, were synthesized. The bifunctional ligands bound to calmodulin with a stoichiometry of 1:1 and with an affinity enhancement over their monofunctional counterparts; the latter bound with a stoichiometry of 2:1 ligand:protein. A lower limit to the effective concentrations of the domains of calmodulin relative to each other (0.2-2 mM) was determined. A comparable effective concentration was achieved for bifunctional ligands based on higher-affinity naphthalene sulphonamide derivatives.

Quaternary structure built from subunits combining NMR and small-angle x-ray scattering data.

A new principle in constructing molecular complexes from the known high-resolution domain structures joining data from NMR and small-angle x-ray scattering (SAXS) measurements is described. Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The residual dipolar coupling data serves to reduce angular degrees of freedom, and the small-angle scattering data serves to confine the translational degrees of freedom. The complex built by this method was found to be consistent with the known crystal structure. The study demonstrates how approximate tertiary structures of modular proteins or quaternary structures composed of subunits can be assembled from high-resolution structures of domains or subunits using mutually complementary NMR and SAXS data.