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.

Oxidation of Various Kraft Lignins with a Bacterial Laccase Enzyme.

Modification of kraft lignin (KL), traditionally uses harsh and energy-demanding physical and chemical processes. In this study, the potential of the bacterial laccase CotA (spore coating protein A) for oxidation of KL under mild conditions was assessed. Thereby, the efficiency of CotA to oxidize both softwood and hardwood KL of varying purity at alkaline conditions was examined. For the respective type of wood, the highest oxidation activity by CotA was determined for the medium ash content softwood KL (MA_S) and the medium ash content hardwood KL (MA_H), respectively. By an up to 95% decrease in fluorescence and up to 65% in phenol content coupling of the structural lignin units was indicated. These results correlated with an increase in viscosity and molecular weight, which increased nearly 2 and 20-fold for MA_H and about 1.3 and 6.0-fold for MA_S, respectively. Thus, this study confirms that the CotA laccase can oxidize a variety of KL at alkaline conditions, while the origin and purity of KL were found to have a major impact on the efficiency of oxidation. Under the herein tested conditions, it was observed that the MA_H KL showed the highest susceptibility to CotA oxidation when compared to the other hardwood KLs and the softwood KLs. Therefore, this could be a viable method to produce sustainable resins and adhesives.

A Fungal Ascorbate Oxidase with Unexpected Laccase Activity.

Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.

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.

Developing SyrinOX total antioxidant capacity assay for measuring antioxidants in humans.

Accurate monitoring of the antioxidant status or of oxidative stress in patients is still a big challenge in clinical laboratories. This study investigates the possibility of applying a newly developed total antioxidant capacity assay method based on laccase or peroxidase oxidized syringaldazine [Tetramethoxy azobismethylene quinone (TMAMQ)] which is referred to here as SyrinOX, as a diagnostic tool for monitoring both oxidative stress and antioxidant status in patients. Attempts to adapt the Randox total antioxidant procedure [simultaneous incubation of the radical generating system (metmyoglobin and H(2) O(2) ) and antioxidant sample] for SyrinOX were abandoned after it was discovered that the H(2) O(2) reacted with enzymatically generated TMAMQ and ABTS radicals at a rate of 6.4 × 10(-2) /µM/s and 5.7 × 10(-3) /µM/s respectively. Thus this study for the first time demonstrates the negative effects of H(2) O(2) in the Randox system. This leads to erroneous results because the total antioxidant values obtained are the sum of radicals reduced by antioxidants plus those reacting with the radical generating system. Therefore they should be avoided not only for this particular method but also when using other similar methods. Consequently, SyrinOX is best applied using a three-step approach involving, production of TMAMQ, recovery and purification (free from enzyme and other impurities) and then using TMAMQ for measuring the total antioxidant capacity of samples. Using this approach, the reaction conditions for application of SyrinOX when measuring the total antioxidant capacity of plasma sample were determined to be 50% (v/v) ethanol/50 mM sodium succinate buffer pH 5.5, between 20 and 25 °C for at least 1 h.

Semi-rational engineering of cellobiose dehydrogenase for improved hydrogen peroxide production.

BACKGROUND: The ability of fungal cellobiose dehydrogenase (CDH) to generate H2O2 in-situ is highly interesting for biotechnological applications like cotton bleaching, laundry detergents or antimicrobial functionalization of medical devices. CDH's ability to directly use polysaccharide derived mono- and oligosaccharides as substrates is a considerable advantage compared to other oxidases such as glucose oxidase which are limited to monosaccharides. However CDH's low activity with oxygen as electron acceptor hampers its industrial use for H2O2 production. A CDH variant with increased oxygen reactivity is therefore of high importance for biotechnological application. Uniform expression levels and an easy to use screening assay is a necessity to facilitate screening for CDH variants with increased oxygen turnover. RESULTS: A uniform production and secretion of active Myriococcum thermophilum CDH was obtained by using Saccharomyces cerevisiae as expression host. It was found that the native secretory leader sequence of the cdh gene gives a 3 times higher expression than the prepro leader of the yeast α-mating factor. The homogeneity of the expression in 96-well deep-well plates was good (variation coefficient <15%). A high-throughput screening assay was developed to explore saturation mutagenesis libraries of cdh for improved H2O2 production. A 4.5-fold increase for variant N700S over the parent enzyme was found. For production, N700S was expressed in P. pastoris and purified to homogeneity. Characterization revealed that not only the kcat for oxygen turnover was increased in N700S (4.5-fold), but also substrate turnover. A 3-fold increase of the kcat for cellobiose with alternative electron acceptors indicates that mutation N700S influences the oxidative- and reductive FAD half-reaction. CONCLUSIONS: Site-directed mutagenesis and directed evolution of CDH is simplified by the use of S. cerevisiae instead of the high-yield-host P. pastoris due to easier handling and higher transformation efficiencies with autonomous plasmids. Twelve clones which exhibited an increased H2O2 production in the subsequent screening were all found to carry the same amino acid exchange in the cdh gene (N700S). The sensitive location of the five targeted amino acid positions in the active site of CDH explains the high rate of variants with decreased or entirely abolished activity. The discovery of only one beneficial exchange indicates that a dehydrogenase's oxygen turnover is a complex phenomenon and the increase therefore not an easy target for protein engineering.

Enzyme-based online monitoring and measurement of antioxidant activity using an optical oxygen sensor coupled to an HPLC system.

It is estimated that up to 50% of the adult population take antioxidant products on a daily basis to promote their health status. Strangely, despite the well-recognized importance of antioxidants, currently there is no international standard index for labeling owing to the lack of standardized methods for antioxidant measurement in complex products. Here, an online high-performance liquid chromatography (HPLC)-based method to detect and measure the total antioxidant capacity of antioxidant samples is presented. In this approach, complex samples containing antioxidants are separated by the HPLC system, which is further coupled to an antioxidant measuring system consisting of an optical oxygen sensor, laccase, and tetramethoxy azobismethylene quinone (TMAMQ). The antioxidants, separated via HPLC, reduce TMAMQ to syringaldazine, which is then reoxidized by laccase while simultaneously consuming O(2). The amount of consumed oxygen is directly proportional to the concentration of antioxidants and is measured by the optical oxygen sensor. The sensor is fabricated by coating a glass capillary with an oxygen-sensitive thin layer made of platinum(II) meso-tetra(4-fluorophenyl)tetrabenzoporphyrin and polystyrene, which makes real-time analysis possible (t(90) = 1.1 s in solution). Four selected antioxidants (3 mM), namely, catechin, ferulic acid, naringenin (used as a control), and Trolox, representing flavonol, hydrocinnamic acid, flavanone, and vitamin E, respectively, were injected into the online antioxidant monitoring system, separated, and then mixed with the TMAMQ/laccase solution, which resulted in oxygen consumption. This study shows that, with the use of such a system, the antioxidant activity of individual antioxidant molecules in a sample and their contribution to the total antioxidant activity of the sample can be correctly assigned.

Editorial on the Special Issue "Chitosan Functional Hydrogels: Synthesis and Applications".

Chitin, a polysaccharide composed of ß-(1-4)-linked 2-deoxy-2-acetamido-d-glucose units, is found in cell walls of different organisms, including crustaceans, fungi, insects, some algae, microorganisms, and some invertebrate animals, and its deacetylation into chitosan confers it with incredible chemical versatility allowing it to be processed into numerous products [...].

A unique two-way approach for the validation of total antioxidant capacity of serum samples.

BACKGROUND: The human body is constantly exposed to a large variety of reactive oxygen species that are implicated in many pathophysiological conditions (atherosclerosis, cancer, neurodegenerative diseases etc.). Monitoring the antioxidant status of biological fluids could be used as an early warning sign 'biomarker' of possible disease onset. However, although several methods have been developed, questionable sensitivity, unreliability and non-reproducibility hamper all, making it difficult to have an internationally accepted standardized method. This study presents and demonstrates the remarkable ability of a newly developed antioxidant capacity assay method based on tetramethoxy azobismethylene quinone (TMAMQ) to measure the total antioxidant capacity of serum samples using three complimentary approaches. DESIGN: Using an UV-Vis spectroscopy and oxygen sensor, the reduction of TMAMQ by serum antioxidants was compared to either the formation of syringaldazine or consumption of oxygen. RESULTS: After adding a fraction of human serum, 4·01 µM TMAMQ was reduced to syringaldazine from a stock of 11·74 µM TMAMQ. Subsequent addition of laccase resulted in the oxidation of the formed syringaldazine back to TMAMQ resulting in an increase in TMAMQ concentration to 11·71 µM (re-establishing almost the same initial concentration of TMAMQ) while consuming 1·04 µM molecular oxygen. CONCLUSIONS: The reduction of TMAMQ by serum samples is directly proportional to the consumption of oxygen and the formation of syringaldazine. This means that either the formation of syringaldazine or oxygen consumption can be used to validate or confirm data obtained through monitoring TMAMQ reduction.

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.

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.

Biotechnological production and high potential of furan-based renewable monomers and polymers.

Of the 25 million tons of plastic waste produced every year in Europe, 40% of these are not reused or recycled, thus contributing to environmental pollution, one of the major challenges of the 21st century. Most of these plastics are made of petrochemical-derived polymers which are very difficult to degrade and as a result, a lot of research efforts have been made on more environmentally friendly alternatives. Bio-based monomers, derived from renewable raw materials, constitute a possible solution for the replacement of oil-derived monomers, with furan derivatives that emerged as platform molecules having a great potential for the synthesis of biobased polyesters, polyamides and their copolymers. This review article summarizes the latest developments in biotechnological production of furan compounds that can be used in polymer chemistry as well as in their conversion into polymers. Moreover, the biodegradability of the resulting materials is discussed.

Enzyme Catalyzed Copolymerization of Lignosulfonates for Hydrophobic Coatings.

Enzymatic polymerization of lignin can generate a variety of value-added products concomitantly replacing fossil-based resources. In line with this approach, a laccase from the thermophilic fungus Myceliophthora thermophila (MtL) was used to couple a hydrophobicity enhancing fluorophenol (FP) molecule, namely 4-[4-(trifluoromethyl)phenoxy]phenol (4,4-F3MPP), as a model substrate onto lignosulfonate (LS). During the coupling reaction changes in fluorescence, phenol content, viscosity and molecular weight (size exclusion chromatography; SEC) were monitored. The effects of enzymatic coupling of FP onto LS on hydrophobicity were investigated by the means of water contact angle (WCA) measurement and determination of swelling capacity. Full polymerization of LS resulting in the production of water-insoluble polymers was achieved at a pH of 7 and 33°C. Incorporation of 2% (w/v) of FP led to an increase in WCA by 59.2% while the swelling capacity showed a decrease by 216.8%. Further, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis indicated successful covalent coupling of the FP molecule onto LS by an emerging peak at 1,320 cm-1 in the FTIR spectrum and the evidence of Fluor in the XPS spectrum. This study shows the ability of laccase to mediate the tailoring of LS properties to produce functional polymers.

Harnessing the Power of Enzymes for Tailoring and Valorizing Lignin.

Lignin, a structural component of lignocellulosic plants, is an alternative raw material with enormous potential to replace diminishing fossil-based resources for the sustainable production of many chemicals and materials. Unfortunately, lignin's heterogeneity, low reactivity, and strong intra- and intermolecular hydrogen interactions and modifications introduced during the pulping process present significant technical challenges. However, the increasing ability to tailor lignin biosynthesis pathways by targeting enzymes and the continued discovery of more robust biocatalysts are enabling the synthesis of novel valuable products. This review summarizes how enzymes involved in lignin biosynthesis pathways and microbial enzymes are being harnessed to produce chemicals and materials and to upgrade lignin properties for the synthesis of a variety of value-added lignin industrial products.

Lignin-Based Pesticide Delivery System.

The potential of lignosulfonates as widely underutilized byproducts of the pulp and paper industry for the synthesis of a biodegradable pesticide carrier system was assessed in this study. Design of experiment software MODDE Pro was for the first time applied to optimize lignosulfonate granule production using Myceliophthora thermophila laccase as a biocatalyst. Enzymatic cross-linking was monitored using size exclusion chromatography coupled online to multiangle laser light scattering, viscosity measurement, and enzyme activity. The determined optimal and experimentally confirmed incubation conditions were: 33 °C, 30 cm3/min O2 supply, and 190 min reaction time. The granules were thereafter loaded with 2 g/kg 3,6-dichloro-2-methoxybenzoic acid (Dicamba), a broad-spectrum herbicide. According to the HPLC analysis, complete release of Dicamba was achieved after 48 h of release. This study showed the green production of a 100% lignosulfonate-based biodegradable solid carrier with potential application in agriculture.

Controlled enzymatic hydrolysis and synthesis of lignin cross-linked chitosan functional hydrogels.

This study presents a novel fully enzymatic process for the controlled depolymerisation of fungal and shrimp chitosan, and their subsequent use in the synthesis of lignin cross-linked chitosan (CTS) hydrogels. Cellobiosehydrolase (CBH) was used to depolymerize CTS resulting in decrease in average molecular weight (Mw) of shrimp CTS from 140 kDa and degree of deacetylation (DD %) from 91.3% to an average MW of 15 kDa and 16% DD. Similarly, fungal chitosan average molecular weight decreased from 92 kDa and the degree of deacetylation (DD) of 48.3% to 12 kDa and a DD of 13%. The depolymerized CTS were completely soluble in water and miscible with lignosulfonates without encountering the usual problem of formation of flocs. Introduction of laccase into a lignosulfonate-chitosan mixture resulted in the oxidation and generation of lignin reactive phenoxyl radicals that cross-linked with CTS-NH2 reactive groups resulting in the increase of Mw from 20 kDa to >500 kDa and viscosity from 20 mPa to >500 mPa. This resulted in the formation of stable lignin-cross-linked hydrogels with elongation at break of 111% and tensile strength of 7 mPa. The produced functional hydrogels have potential application in food and biomedical industries as e.g. as oxygen barriers in packaging or as functional wound dressing or tissue engineering platforms.

Effects of enzymes on the refining of different pulps.

Comparative studies of the effects of two commercial enzyme formulations on fiber refining were conducted. Extensive basic characterisation of the enzymes involved, assessment of their hydrolytic activities on different model substrates as well as on different pulps (softwood sulfate, softwood sulfite, hardwood sulfate) were evaluated. Both enzyme formulations showed endoglucanase as well as some xylanase and ß-glucosidase activity. In addition, Enzyme A reached a CMC end viscosity of 19.5 mPa compared to 11.1 mPa for Enzyme B. Reducing sugar release almost doubled from 695 µmol mL-1 for hardwood sulfate pulp to 1300 µmol mL-1 for softwood sulfite pulp with Enzyme B under the same conditions. Enzyme A increased the degree of refining even under non-ideal conditions from 23 °SR to up to 50 °SR. Further characterization of hand sheets, made from enzyme pre-treated and refined cellulose fibers with Enzyme A and B, showed that Enzyme A had the best effects leading to hand sheets with increased tensile strength and low air permeability. In summary, the increase in the degree of refining seen for Enzyme A correlated to higher xylanase and ß-glucosidase activity and lower endoglucanase activity.

Antioxidant activity assay based on laccase-generated radicals.

A novel antioxidant activity assay was developed using laccase-oxidized phenolics. In a three-step approach, phenolic compounds were first oxidized by laccase. Laccase was then inhibited using 80% (v/v) methanol which also stabilized the oxidized phenolics which were then used to measure antioxidant activities of ascorbic acid and Trolox. From a number of laccase-oxidized phenolics screened for potential use in the measurement of antioxidant activities, syringaldazine emerged the best, giving results comparable to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, which is currently used in conventional methods. Like DPPH radicals, two moles of stoichiometric oxidized syringaldazine were reduced by one mole of either ascorbic acid or Trolox. For the first time we show that antioxidant activity can be correlated to oxygen consumption by laccase. Reduction of one molecule of oxygen corresponded to oxidation of four molecules of syringaldazine which in turn is reduced by two molecules of Trolox or ascorbic acid. This study therefore demonstrates the great potential of using laccase-oxidized syringaldazine for the measurement of antioxidant activity.

Enzymes as Green Catalysts and Interactive Biomolecules in Wound Dressing Hydrogels.

Hydrogels are 3D hydrophilic polymer networks that absorb and hold huge amounts of water. Although hydrogels have traditionally been synthesized using chemical and physical methods, rapid developments in enzyme technology that, like chemical-based methods, enable the formation of stable covalent bonds are fast emerging as alternative 'green catalyst' tools. Enzymes show great potential for the synthesis of complex multifunctional wound dressing hydrogels (WDHs) ex situ and in situ as well as in acting as interactive molecules to promote the wound healing process. This review presents advances in the use of enzymes to synthesize WDHs and their fascinating role as bioactive molecules promoting the wound healing process, preventing microbial infection, and providing in situ, in-built infection-detection and diagnostic systems.

Anti-inflammatory and anti-oxidant properties of laccase-synthesized phenolic-O-carboxymethyl chitosan hydrogels.

A bioactive O-carboxymethyl chitosan (CMCS) hydrogel crosslinked with natural phenolics with potential application in wound dressings was synthesized using a laccase from Myceliophthora thermophila (MTL). The highest degree of cross-linking (49.7%) was achieved with catechol. All the phenolic-CMCS hydrogels synthesized showed excellent anti-oxidant properties with a free radical scavenging activity up to 4-fold higher than in the absence of the phenolics, as quantified by the di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) assay. In addition, the hydrogels produced showed an anti-inflammatory effect as evidenced by the inhibition of enzymes [myeloperoxidase (MPO), matrix-metalloproteinase-1 (MMP-1) and human neutrophil elastase (HNE)] over-expressed in chronic wounds. Sinapyl-CMCS hydrogels showed an MMP-1 inhibition of 37%. Further, the phenolic-CMCS hydrogels did not affect the viability of the NIH 3T3 mouse fibroblast cell line and were also able to slowly release human fibroblast growth factor 2, reaching 48.3% over a period of 28days. This study thus shows the possibility of synthesizing multifunctional bioactive chitosan based hydrogels with anti-oxidant and anti-inflammatory properties using natural occurring phenolics as crosslinkers.