Therapeutic potential of low-molecular weight lignin model polymer fractions for treating skin lesions in animals: a pilot study.

Bacterial infections and resistance to antibiotics are increasingly severe problems. In recent years, Staphylococcus species have emerged as important pathogens in animals and humans. Current therapeutic methods against these species have serious disadvantages; therefore new agents with antibacterial potential, such as plant-based substances, are very important in therapy. We report a pilot study with new method of fractioning the dehydrogenate polymer DHP obtained from coniferyl alcohol and application of the low-MW fractions of 200-3000 Da for antibacterial activity in healing animal lesions. In vivo experiments were conducted on the dogs having a skin lesion. Dogs were treated with the suspension containing the low-MW DHP fractions as the active ingredient, in combination with alginate for 7 days. Cytological smears and microbiological analyses of the affected area were performed. Staphylococcus spp. was isolated from lesions in all dogs from our research. The results show that the low-MW DHP suspension in alginate promotes skin healing and reduction of the infection of the lesions in the affected animals. Pharmaceutical composition containing the low-MW DHP fractions exerts a soothing effect on the subject in wound treatment. Reduction in the number of bacteria by 30% and more were noticed in 6 dogs, while in 4 dogs this percentage is above 50%. No side effects were noticed. Synthesized lignin oligomers may have a significant place as antimicrobial and skin healing agents, especially since an increasing number of multidrug-resistant staphylococci are found on the skin lesions in animals.

Two-way reaction of versatile peroxidase with artificial lignin enhances low-molecular weight fractions.

In recent years, versatile peroxidase (VP) has emerged as a promising enzyme for biotechnological applications, as it can oxidize lignin without the external mediators. To gain insights into the breakdown process of artificial lignin by VP, reaction between the two was studied. Degradation products were fractionated using ultrafiltration and analyzed by RP- high performance liquid chromatography with mass detection (HPLC-MS) chromatography. Four fractions were obtained based on their molecular sizes: >10, 3-10, 1-3, and <1 kDa. Interestingly, while VP did not significantly alter the yields of these fractions, the chromatograms revealed the presence of oligomers with different molecular weights (MWs) resulting from the enzymatic activity. The VP exhibits a dual role in its enzymatic activity: both degrading and synthesizing these oligomers. This was confirmed by principal component analysis (PCA). The positive correlations were found between certain oligomers (D1 and D2, D5 and D6, as well as between D7, D10, T2, and T4), suggesting their simultaneous degradation. On the other hand, a negative correlation was found between the monomer and some oligomers (D7, D10, T2, and T4), indicating the decomposition of these oligomers into monomers. These findings shed light on the intricate interplay between VP and artificial lignin, offering valuable insights for potential applications in lignin valorization.

Cell wall response to UV radiation in needles of Picea omorika.

The UV-B represents the minor fraction of the solar spectrum, while UV-C is not contained in natural solar radiation, but both radiation types can cause damaging effects in plants. Cell walls (CWs) are one of the targets for external stressors. Juvenile P. omorika trees were treated either with 21 day-high doses UV-B or with 7 day- UV-C in open-top chambers. Using spectroscopic and biochemical techniques, it was shown that the response to UV radiation includes numerous modifications in needle CW structure: relative content of xylan, xyloglucan, lignin and cellulose decreased; cellulose crystallinity changed; yield of lignin monomers with stronger connection of CC in side chain with the ring increased; re-distribution of inter- and intra-polymer H-bonds occurred. The recovery was mediated by an increase in the activities and changes in isoform profiles of CW bound covalent peroxidases (POD) and polyphenol oxidases (PO) (UV-B), and ionic POD and covalent PO (UV-C). A connection between activities of specific POD/PO isoforms and phenolic species (m- and p-coumaric acid, pinoresinol and cinnamic acid derivatives) was demonstrated, and supported by changes in the sRNA profile. In vivo fluorometry showed phenolics accumulation in needle epidermal CWs. These results imply transversal connections between polymers and changed mechanical properties of needle CW as a response to UV. The CW alterations enabled maintenance of physiological functions, as indicated by the preserved chlorophyll content and/or organization. The current study provides evidence that in conifers, needle CW response to both UV-B and UV-C includes biochemical modifications and structural remodeling.

Bacterial cellulose-lignin composite hydrogel as a promising agent in chronic wound healing.

Lignins and lignin-derived compounds are known to have antibacterial properties. The wound healing agents in the form of dressings produce faster skin repair and decrease pain in patients. In order to create an efficient antimicrobial agent in the form of dressing in the treatment of chronic wounds, a composite hydrogel of bacterial cellulose (BC) and dehydrogenative polymer of coniferyl alcohol (DHP), BC-DHP, was designed. Novel composite showed inhibitory or bactericidal effects against selected pathogenic bacteria, including clinically isolated ones. The highest release rate of DHP was in the first hour, while after 24 h there was still slow release of small amounts of DHP from BC-DHP during 72 h monitoring. High-performance liquid chromatography coupled with mass-spectrometry showed that BC-DHP releases DHP oligomers, which are proposed to be antimicrobially active DHP fractions. Scanning electron microscopy and atomic force microscopy micrographs proved a dose-dependent interaction of DHP with BC, which resulted in a decrease of the pore number and size in the cellulose membrane. The Fourier-transform infrared absorption spectra of the BC-DHP showed that DHP was partly bound to the BC matrix. The swelling and crystallinity degree were dose-dependent. All obtained results confirmed BC-DHP composite as a promising hydrogel for wounds healing.

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization.

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20 mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538 µmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0 mmol/g of dry polymer compared to 1.5 mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K m value of 2.61 mM compared to the soluble enzyme. After 7 cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

Lignin model compound in alginate hydrogel: a strong antimicrobial agent with high potential in wound treatment.

Nowadays bacterial resistance to known antibiotics is a serious health problem. In order to achieve more efficient treatment, lately there is an effort to find new substances, such as certain biomaterials, that are non-toxic to humans with antibiotic potential. Lignins and lignin-derived compounds have been proposed to be good candidates for use in medicine and health maintenance. In this study, the antibacterial activity of the lignin model polymer dehydrogenate polymer (DHP) in alginate hydrogel (Alg) was studied. The obtained results show that DHP-Alg has strong antimicrobial activity against several bacterial strains and biofilms and does not have a toxic effect on human epithelial cells. These results strongly suggest its application as a wound healing agent or as an adjunct substance for wound treatments.

Soybean hull peroxidase immobilization on macroporous glycidyl methacrylates with different surface characteristics.

Soybean hull peroxidase (SHP, E.C. 1.11.1.7) was immobilized by a glutaraldehyde and periodate method onto series of macroporous copolymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA), poly(GMA-co-EGDMA) with various surface characteristics and pore size diameters ranging from 44 to 200 nm. Glutaraldehyde immobilization method and poly(GMA-co-EGDMA) named SGE 20/12 with pore sizes of 120 nm gave immobilized enzyme with highest specific activity of 25 U/g. Deactivation studies showed that immobilization increased stability of SHP and that surface characteristics of the used copolymer had a major influence on a stability of immobilized enzyme at high temperatures and in an organic solvent. The highest thermostability was obtained using the copolymer SGE 20/12 with pore size of 120 nm, while the highest stability in dioxane had SHP immobilized onto copolymer SGE 10/4 with pore size of 44 nm. Immobilized SHP showed a wider pH optimum as compared to the native enzyme especially at alkaline pH values and 3.2 times increased K m value for pyrogallol. After 6 cycles of repeated use in batch reactor, immobilized SHP retained 25 % of its original activity. Macroporous copolymers with different surface characteristics can be used for fine tuning of activity and stability of immobilized SHP to obtain a biocatalyst suitable for phenol oxidation or polymer synthesis in organic solvents.

Improved covalent immobilization of horseradish peroxidase on macroporous glycidyl methacrylate-based copolymers.

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g(-1) dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K (m)) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.