Modified polymeric biomaterials with antimicrobial and immunomodulating properties.

The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside) yielded very stable preparations of modified biomaterials. The main emphasis was placed on analyses of their antimicrobial activity and potential immunomodulatory and non-cytotoxic properties towards the CCD841 CoTr model cell line. Cecropin A did not significantly affect the viability or proliferation of the CCD 841 CoTr cells, regardless of its soluble or immobilized form. In contrast, puromycin did not induce a significant decrease in the cell viability or proliferation in the immobilized form but significantly decreased cell viability and proliferation when administered in the soluble form. The covalent immobilization of these two molecules on the surface of biomaterials resulted in stable preparations that were able to inhibit the multiplication of Staphylococcus aureus and S. epidermidis strains. It was also found that the preparations induced the production of cytokines involved in antibacterial protection mechanisms and stimulated the immune response. The key regulator of this activity may be related to TLR4, a receptor recognizing bacterial LPS. In the present study, these factors were produced not only in the conditions of LPS stimulation but also in the absence of LPS, which indicates that cecropin A- and puromycin-modified biomaterials may upregulate pathways leading to humoral antibacterial immune response.

Synthesis and Bioactive Properties of the Novel Coloured Compound Obtained via the Laccase-Mediated Transformation of 5-Aminosalicylic Acid.

Biocatalysis processes based on oxidoreductases, such as fungal laccase, are important for discovering new organic compounds with broad structures and potential applications. They include bioactive compounds, which can be obtained through laccase-mediated oxidation of organic substrates having hydroxyl and/or amino groups especially, e.g., 5-aminosalicylic acid (5-ASA) is characterised for its potential for oxidation by a fungal laccase obtained from a Cerrena unicolor strain. The biotransformation process was optimised in terms of the buffer and co-solvent concentration, buffer pH value, and laccase activity. Selected crude dyes were analysed for their bioactive properties, toxicity, and suitability for the dyeing of wool fibres. The data obtained clearly indicated that a low concentration of the reaction buffer in the pH range from 5 to 6 and in the presence of 10% acetonitrile increased the rate of substrate oxidation and the amount of the product formed. The red-brown compound obtained via laccase-mediated oxidation of 5-aminosalicylic acid showed antioxidant properties and unique antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis strains with the MIC value of 0.125 mg/mL detected for the purest dye. In addition, it was reported to have good wool fibre dyeing properties and no irritant effect after patch tests on a selected group with increased skin sensitivity.

Bioactive Properties of a Novel Antibacterial Dye Obtained from Laccase-Mediated Oxidation of 8-Anilino-1-naphthalenesulfonic Acid.

Fungal laccase obtained from a Cerrena unicolor strain was used as an effective biocatalyst for the transformation of 8-anilino-1-naphthalenesulfonic acid into a green-coloured antibacterial compound, which can be considered as both an antimicrobial agent and a textile dye, simultaneously. The process of biosynthesis was performed in buffered solutions containing methanol as a co-solvent, allowing better solubilisation of substrate. The transformation process was optimised in terms of the buffer pH value, laccase activity, and concentrations of the substrate and co-solvent. The crude product obtained exhibited low cytotoxicity, antibacterial properties against Staphylococcus aureus and Staphylococcus epidermidis, and antioxidant properties. Moreover, the synthesised green-coloured compound proved non-allergenic and demonstrated a high efficiency of dyeing wool fibres.

Structure and Bioactive Properties of Novel Textile Dyes Synthesised by Fungal Laccase.

Novel sustainable processes involving oxidative enzymatic catalysts are considered as an alternative for classical organic chemistry. The unique physicochemical and bioactive properties of novel bio-products can be obtained using fungal laccase as catalyst. Among them are textile biodyes synthesised during oxidation of substrates belonging to the amine and methoxy organic derivatives. The process of synthesis occurs in mild conditions of pH, temperature, and pressure, and without using harmful oxidants. The effect of fungal laccase activity on the substrates mixture transformation efficiency was analysed in terms of antimicrobial dye synthesis on a large scale. Three new phenazine dyes, obtained in the presence of laccase from Cerrena unicolor, were studied for their structure and properties. The phenazine core structure of the products was a result of tri-molecular transformation of aminomethoxybenzoic acid and aminonaphthalene sulfonic acid isomers. One of the compounds from the synthesised dye, namely 10-((2-carboxy-6-methoxyphenyl)amino)-11-methoxybenzo[a]phenazine-8-carboxylic acid, was able to inhibit the growth of Staphylococcus aureus. The high concentration of substrates (5 g/L) was efficiently transformed during 72 h in the mild conditions of pH 4 with the use of laccase with an activity of 200 U per g of the substrates mixture. The new bioactive dye exhibited excellent dyeing properties with concomitant antibacterial and antioxidative activity. The proposed enzyme-mediated synthesis represents an alternative eco-friendly route for the synthesis of novel antimicrobial compounds with high importance for the medical textile industry.

Laccase Properties, Physiological Functions, and Evolution.

Discovered in 1883, laccase is one of the first enzymes ever described. Now, after almost 140 years of research, it seems that this copper-containing protein with a number of unique catalytic properties is widely distributed across all kingdoms of life. Laccase belongs to the superfamily of multicopper oxidases (MCOs)-a group of enzymes comprising many proteins with different substrate specificities and diverse biological functions. The presence of cupredoxin-like domains allows all MCOs to reduce oxygen to water without producing harmful byproducts. This review describes structural characteristics and plausible evolution of laccase in different taxonomic groups. The remarkable catalytic abilities and broad substrate specificity of laccases are described in relation to other copper-containing MCOs. Through an exhaustive analysis of laccase roles in different taxa, we find that this enzyme evolved to serve an important, common, and protective function in living systems.

Novel textile dye obtained through transformation of 2-amino-3-methoxybenzoic acid by free and immobilised laccase from a Pleurotus ostreatus strain.

Transformation of 2-amino-3-methoxybenzoic acid into novel and eco-friendly orange dye (N15) was performed using native and immobilised laccase (LAC) from Pleurotus ostreatus strain. A several parameters affecting laccase-mediated transformation efficiency included the selection of type and pH value of buffer, reaction temperature, substrate and laccase concentration as well as the type of carrier and LAC storage conditions were evaluated. The optimal conditions for N15 dye synthesis were 40 mM sodium-tartrate buffer pH 5.5 containing 3 mM of the substrate, efficiently transformed by 2 U of free laccase per 1 mmol of the substrate. Laccase was immobilised on porous Purolite® carriers, which had never been tested as a support for oxidoreductases. Immobilised laccase, characterised by a high immobilisation yield, was obtained by adsorption of laccase on a porous acrylic carrier with octadecyl groups (C18) incubated in optimum conditions of 40 mM phosphate buffer pH 7.0 containing 1 mg of laccase per 1 g of the carrier (wet mass). The immobilised LAC showed the highest storage stability for 21 days and higher thermostability at 40 ℃ and 60 ℃ in comparison to its native form. The N15 dye showed good dyeing properties towards natural fibres, and the dyed fibre demonstrated resistance to different physicochemical factors during use, which was confirmed by commercial quality tests. The N15 dye is a phenazine, i.e. a heterogenic compound containing amino-, methoxy-, and three carboxyl functional groups with the molecular weight of approximately 449.37 U.

[Biologically active compounds and methods of their removal through biocatalysis]. / Zwiazki biologicznie aktywne i metody ich usuwania na drodze biokatalizy.

Pharmaceuticals and other biologically active substances are produced in increasing numbers. Because of increased usage and improper storage, they pass into surface water, ground water and drinking water directly or through wastewaters. This is a threat to many living organisms, including humans, because of hormonal imbalances primarily related to reproductive processes or the problem of microbial drug resistance. Due to the scale of the emission and limited possibilities of decomposition of these pollutants by physico-chemical methods it is necessary to develop new efficient processes. One of the proposed solutions is the use of tools offered by biocatalysis. Thanks to the biocatalysis process, a wide range of biologically active compounds can be removed, by using of enzymes with low substrate specificity and operating in environmentally friendly conditions. Recent studies indicate the effectiveness of those methods used in the removal of pollutants of different chemical structure, with the formation of non-toxic metabolites.

Laccase-mediated synthesis of a phenoxazine compound with antioxidative and dyeing properties--the optimisation process.

This study demonstrates the optimisation of the main parameters of the laccase-mediated biosynthesis of high-intensity-coloured orange phenoxazine compound, 2-amino-3-oxo-3H-phenoxazine-8-sulfonic acid, and the antioxidative and dyeing properties. Among optimised parameters were the pH value, the activity of laccase, and the high concentration of the precursor as the necessary step in terms of dye synthesis scale-up. The high concentration of the precursor of ca. 10 g/L can be transformed totally by laccase at the activity of 30 U/g during 12 hours, in an optimised and standardised process in nearly 100% yield of synthesis. The obtained dye exhibited good dyeing properties determined according to the ISO standards. Antioxidative activities were detected for phenoxazinone dye using two independent methods, the chemiluminescence assay and the ABTS free radical-scavenging test, with the values of EC50 for the tested phenoxazine dye amounting 189.8 µg/mL and 1428 µg/mL, respectively. Despite the presence of the phenoxazine core in the structure of this dye, no antibacterial capacity was noted.

Stimulation of the Antioxidative and Antimicrobial Potential of the Blood Red Bracket Mushroom Pycnoporus sanguineus (Higher Basidiomycetes).

The antioxidative and antibacterial properties of low-molecular-weight secondary metabolite subfractions (ex-LMS) from cultures of Pycnoporus sanguineus cultivated under different temperature conditions (25°C [ex-LMSa] and 30°C [ex-LMSb]) were assessed. The antioxidative properties were studied using chemiluminometric measurement, an ABTS assay, and a DPPH reduction rate assay with Trolox and ascorbic acid as the control. The values noted for the ex-LMSb were significantly higher than those for ex-LMSa: 97%, 52%, and 31% for chemiluminometric measurement, the ABTS assay, and the DPPH assay, respectively, at a concentration of 50 µg/mL. Half-maximal effective concentrations reached 4.17 µg/mL for chemiluminometric measurement, 47.25 µg/mL for the ABTS assay, and 51.46 µg/mL for DPPH assay. Toxicity tests against Vibrio fischeri yielded 99.8% for ex-LMSa and 99.85% for ex-LMSb. Antibacterial activity toward Staphylococcus aureus was observed in the ex-LMSb fractions (inhibition zone, 23.5 mm; minimum inhibitory concentration, 0.12 mg/mL). Scanning electron microscopy images exhibited severe disruption of the bacterial cells treated with ex-LMSb compared with the control. The results obtained suggest that the extracellular fluid isolated from P. sanguineus-submerged cultures might be a good source of antioxidative and antibacterial compounds. In addition, the increase in the culture temperature evidently enhanced the bioactive properties of the preparation.

Exopolysaccharide from Ganoderma applanatum as a promising bioactive compound with cytostatic and antibacterial properties.

A new exopolysaccharide preparation isolated from stationary cultures of the white rot fungus Ganoderma applanatum (GpEPS) was tested in terms of its bioactive properties including its cytotoxic and immunostimulatory effect. The results indicate that the tested GpEPS (at concentrations above 22.85 µg/mL and 228.5 µg/mL) may exhibit selective activity against tumor cells (cell lines SiHa) and stimulate production of TNF-α THP-1-derived macrophages at the level of 752.17 pg/mL. The GpEPS showed antibacterial properties against Staphyloccoccus aureus and a toxic effect against Vibrio fischeri cells (82.8% cell damage). High cholesterol-binding capacity and triglycerides-binding capacity (57.9% and 41.6% after 24 h of incubation with the tested substances, resp.) were also detected for the investigated samples of GpEPS.

New bioactive fungal molecules with high antioxidant and antimicrobial capacity isolated from Cerrena unicolor idiophasic cultures.

Three bioactive fractions, extracellular laccase (ex-LAC), crude endopolysaccharides (c-EPL), and a low molecular subfraction of secondary metabolites (ex-LMS), were isolated from the idiophasic cultures of the white rot fungus Cerrena unicolor. For the first time, we determined the antioxidant properties of these samples by chemiluminometric measurement (a) and assessment of the scavenging effect on ABTS (b) and the DPPH reduction rate (c). The highest reducing capability was found for the ex-LMS fraction: 39-90% for (a), 20-90% for (b), and 10-59% for (c) at the concentration of 6.25-800 µg/mL. The scavenging abilities of the C. unicolor c-EPL were between 36 and 70% for (a), 2 and 60% for (b), and 28 and 32% for (c) at the concentration of 6.25-800 µg/mL. A very high prooxidative potential was observed for the ex-LAC probes. The preliminary toxicity tests were done using the Microtox system and revealed the following percentage of the toxic effect against Vibrio fischeri: 85.37% for c-EPL, 50.67% for ex-LAC, and 99.8% for ex-LMS, respectively. The ex-LAC sample showed the antibacterial activity against Escherichia coli, c-EPL against Staphylococcus aureus, and ex-LMS against both bacterial strains, respectively, but the stronger inhibitory effect was exerted on S. aureus.

Structure/redox potential relationship of simple organic compounds as potential precursors of dyes for laccase-mediated transformation.

The aim of this study was to examine the ability of an extracellular fungal laccase (LAC) to form colored products from simple non-colored organic precursors. Thirty different phenolic and non-phenolic precursors (o-, m-, and p-methoxy-, hydroxy-, sulfonic-, and amino-substituted) were tested as single and coupled substrates in a LAC-catalyzed oxidation. The findings show that LAC catalyzes the formation of colored products (from yellow/brown to red and blue) by oxidation of single substrates that are benzene derivatives containing at least two substituents comprised of amino, hydroxy, and methoxy groups. All precursors were tested by cyclic voltammetry and the correlation between their structure and redox potential, and the possibility of their transformation into colored products by fungal LAC was found. Colored products were yielded from single substrates possessing a value of the oxidation peak (E(o)) lower than 1,150 mV vs. normal hydrogen electrode (NHE). Substrates with an oxidation peak higher than 1,150 mV vs. NHE were transformed by LAC into colored compounds only in the presence of an additional precursor characterized by a low value of E(o) and the presence of reactive substituents such as methoxy, hydroxy, and amino groups. Therefore, additional hydroxylation, methoxylation, and amination of phenolic and non-phenolic substrates may represent a strategy to increase the range of these compounds as potential dyes precursors.

Genetic characterization of the CcpA-dependent, cellobiose-specific PTS system comprising CelB, PtcB and PtcA that transports lactose in Lactococcus lactis IL1403.

Lactose metabolism is one of the most important areas of research on Lactic Acid Bacteria (LAB). In rapidly acidifying industrial Lactococcus lactis strains, lactose is transported by a lactose-specific phosphotransferase system (PTS) encoded by a plasmid. However, an alternative lactose catabolic pathway was evidenced in the plasmid-cured, and thus initially lactose-negative L. lactis IL1403. We showed that in this strain the chromosomally-encoded cellobiose-specific PTS system comprising the celB, ptcB and ptcA genes is also able to transport lactose. By expression studies in the wild type IL1403 strain and IBB550, its ccpA-deficient derivative, we demonstrated that celB, ptcB and ptcA are tightly regulated by the general catabolite repression system, whereas celB additionally requires the presence of cellobiose to be fully induced. The comparison of expression levels of sugar catabolic genes indicated that the efficiency of CcpA-mediated catabolic repression depends on conservation of the cre sequence, and that in the case of perfect matching with the cre consensus, CcpA still drives a strong repression even under non-repressing conditions.

Whole-cell fungal transformation of precursors into dyes.

BACKGROUND: Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. RESULTS: An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. CONCLUSIONS: This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important products. The use of immobilized fungal biomass limits free migration of cells and facilitates their reuse in a continuous system for precursor transformation.

Risk of tick-borne bacterial diseases among workers of Roztocze National Park (south-eastern Poland).

The objective of the present study was to assess the risk of borreliosis and anaplasmosis (ehrlichiosis) among the forestry workers of the Roztocze National Park (south-eastern Poland) by examination of Ixodes ricinus ticks living in park area with PCR method, and by the serological and clinical examination of the workers. In 406 examined ticks, the prevalence of infection with Borrelia burgdorferi sensu lato was 11.5 %. The nested PCR reaction for determining the genospecies showed that the most common was Borrelia burgdorferi sensu stricto (55.3 % of total positive) followed by Borrelia afzelii (38.3 %). As many as 6.1 % out of 115 examined ticks showed the presence of Anaplasma phagocytophilum DNA. The infection rate was high in males and females (14.3 % and 11.1 % respectively) and low in nymphs (1.5 %). In 46 out 113 examined forestry workers (40.7 %) the presence of specific IgG and/or IgM antibodies against Borrelia burgdorferi sensu lato was found in ELISA test, while only 4 out of 56 urban blood donors showed a positive response (p<0.0001). The prevalence of IgG antibodies against Anaplasma phagocytophilum determined with the use of indirect immunofluorescence test (IFA) was 17.7 % in forestry workers compared to 5.4 % in reference group of blood donors (p<0.05). No correlation was found between the presence of antibodies to A. phagocytophilum and to B. burgdorferi s. l. Clinical investigations of 113 forestry workers showed 3 cases of borreliosis (2.7 %) and no cases of anaplasmosis (ehrlichiosis). In conclusion, forestry workers of the Roztocze National Park in south-eastern Poland are often exposed to Ixodes ricinus ticks infected with Borrelia burgdorferi and Anaplasma phagocytophilum, and show a high proportion of asymptomatic borreliosis and anaplasmosis (ehrlichiosis) manifested by a positive serologic response, while the number of clinical cases is relatively low.