Surfaces exhibiting antimicrobial activity were prepared for potential medical application. A polycationic lipopeptide polymyxin B was selected as the bioactive agent for covalent immobilization onto the surface. First, by using sol–gel technology the inert glass substrate was functionalized by a silane coating with epoxide rings to which the peptide was coupled by means of a catalyst. Preparation of the coating and presence of the peptide on the surface were followed by FTIR, XPS and AFM analyses. The obtained material showed antimicrobial effect indicating that in spite of immobilization the peptide has retained its bioactivity. The coated surface was able to reduce bacterial cell counts of the Gram-negative bacterium Escherichia coli by more than five orders of magnitude in 24 h of incubation. It can be concluded that bioactive coatings with covalently bound polycationic peptides have potential for application on medical devices where leakage into the surrounding is not allowed in order to prevent bacterial growth and biofilm formation.

Waste bovine hooves and horns were enzymatically hydrolysed into soluble products intended for foliar fertilizer. With the powdered keratin at 50°C and pH 8 between 34 to nearly 60% of nitrogen was solubilized in 5 h, depending on the enzyme concentration. The reaction could further be improved by steam pretreatment of the keratin, resulting in 98% solubilisation of the nitrogen. The products of hydrolysis consisted of a mixture of soluble proteins, peptides, and free amino acids. Among the latter, 18 common amino acids were detected. Several of them were previously recognized to have a positive effect on plants. Nonpolar neutral, basic, and sulphur amino acids were present in relatively large amounts, while proline and tryptophan were not found. Comparison with other protein hydrolysates aimed for fertilizer suggests that keratin degradation products, obtained by enzymatic hydrolysis, have potential to be used for foliar fertilization, alone or in a combination with another complementary hydrolysate of a different source, such as skin or plant proteins.

Candida antarctica lipase B (CAL-B)-catalysed regioselective deacetylation of 2′,3′,5′-tri-O-acetyl-1-β-d-arabinofuranosyluracil (1) and 2′,3′,5′-tri-O-acetyl-9-β-d-arabinofuranosyladenine (2) was studied. The choice of the reaction medium allowed the regioselective formation of products bearing different degree of acetylation: in isopropanol, CAL-B catalysed the formation of the corresponding 2′-O-acetylated arabinonucleosides, while hydrolyses afforded the 2′,3′-di-O-acetylated products. In particular, the procedure herein described allows a simple and efficient preparation of the reported vidarabine prodrug 2′,3′-di-O-acetyl-9-β-d-arabinofuranosyladenine, avoiding the utilisation of protective groups. Moreover, to achieve full deacetylation of the assayed substrates, a set of commercial hydrolases and fungal keratinases from Doratomyces microsporus (DMK) and Paecilomyces marquandii (PMK) were tested. While only PMK and DMK catalysed the quantitative complete deacetylation of 1, DMK accomplished full deacetylation of 2 in shorter time than the other assayed enzymes.

Versatile peroxidase from white rot fungus Bjerkandera adusta was over-expressed in a soluble form in Escherichia coli. In the constructed enzyme model based on the selected gene from B. adusta, the active sites for oxidation of Mn2+ ions and for oxidation of aromatic substrates were identified, both characteristic for versatile peroxidase. For over-expression of the recombinant enzyme different host strains, media formulations, growth temperatures, and fusion partners were tested. With the bacterial strain BL21(DE3)pLysS cultivated at 25°C in auto-induction medium and presence of heme, a soluble peroxidase with incorporated heme and activity against different substrates was obtained. By exploiting an appropriate expression system and providing suitable culture conditions, the recombinant fungal peroxidases in soluble form can be produced in bacteria.

A protocol for isolation of genomic DNA from a filamentous fungus was optimised and a genomic library was constructed. Various methods for preparing genomic DNA from different fungi are known: simplified methods to allow processing of large sample numbers and methods to increase the quality of DNA, which is especially important for synthesis of a genomic DNA library. An isolation of high-molecular-weight DNA from a fungus is a challenging process since cell wall composition and cellular components differ with the fungal species. A procedure for isolation of high-molecular-weight DNA from the keratinolytic filamentous fungus Doratomyces microsporus, strain MZKI B399, is described and compared to the method used for filamentous bacteria and to the method including commercially available DNA extraction buffer. The isolation protocol that gave the desired quality of DNA was optimised in the following steps: disruption of cells by grinding in liquid nitrogen, followed by removal of polysaccharides and proteins by a phenol extraction buffer and finally precipitation of DNA with isopropanol. The obtained DNA was used to produce the bacteriophage genomic DNA library of the fungus.

Fifty-eight fungi have been tested for their ability to degrade a recalcitrant synthetic polymer polyamide-6, generally known as nylon-6. Most of them were isolated from a factory producing nylon-6. After preliminary screening, 12 strains were selected for submerged culture in a medium with nylon fibres as the only N-source. No degradation was observed with the isolates from the factory. Wood degrading fungi from a culture collection, however, degraded nylon after incubation for several weeks. Bjerkandera adusta disintegrated the fibres most efficiently, starting with the small transverse grooves, which deepened into cracks. The superficial layers crumbled to leave a thin inner core of the fibre, which finally broke down into fragments. The remaining insoluble part of the nylon showed a decrease in number average molecular mass from 16900 to 5600 during a 60-day incubation. Its thermal properties, such as shifts in melting points and broadening of the melting endotherms, were altered. The reduction of the amount of nylon and the composition of the liquid phase indicated that part of the polymer was degraded into soluble products. After 50 days, the total nitrogen content of the soluble fraction was 10-fold higher than in the control sample. Manganese peroxidase, presumably responsible for the degradation, was detected in the liquid phase. The study shows that only white rot fungi are able to break down nylon-6. For the first time this polymer was shown to be disrupted by B. adusta. The extent of the biodegradation indicates its potential for application in nylon waste reduction.

We screened wild fungal isolates for melanolytic activity and found that Sporotrichum pruinosum was the most promising of the very limited number of fungi that decolourised synthetic melanin. We used a submerged aerobic process to produce a skin depigmentation enzyme by this strain, and found that in the medium the presence of Mn 2+ ions was necessary, the limitation of carbon source was beneficial, and Zn 2+ ions were inhibitory. Cultivation in a stirred bioreactor required immobilization of mycelium and use of low stirring velocity. A partially purified enzyme was prepared and tested for depigmentation of human skin corneocytes and whole epidermis of phototypes III and V. This is the first study demonstrating the effective enzymatic degradation of the skin melanin rather than inhibition of its synthesis. This opens the possibility of using melanolytic enzymes in cosmetic skin lightening.

A textile dye Reactive Black 5 was used in screening 25 fungal strains for their decolourising ability. The most promising strains were tested in a medium containing specific constituents of a dye bath in order to approach real application conditions. It was shown that the concentrations of the constituents had to be reduced to allow fungal growth. Decolourisation started in cultures of Geotrichum candidum but was not complete. Only Bjerkandera adusta was able to decolourise the black-blue colour through violet and red to pale yellow. After 17days spectral absorption coefficients, α, at three wavelengths, 620, 525 and 436nm almost reached the permitted values. A partly purified manganese peroxidase prepared from B. adusta was tested for decolourisation of several artificial dye baths. The constituents seemed not to be inhibitory to the enzyme and no dilution was necessary. Evaluation of decolourisation gave different results, depending on the method used. The most efficient decolourisation on a percentage basis was observed in the dye bath of the anthraquinone dye Reactive Blue 19, followed by the diazo dye Reactive Black 5. However, based on absorbance units, the largest reduction was achieved with the Reactive Black 5 and Acid Orange 7 dye baths. Comparing the α values after 120h fungal and enzymatic treatments of Reactive Black 5 dye bath the enzyme showed about 1.5 times greater colour reduction than the fungus. Given the tolerance to the constituents and concentration of dye baths, the enzyme proved to be a promising tool for their treatment.

Decoloration of the synthetic textile dye Reactive Black 5 by the cultivated fungi, Bjerkandera adusta, Irpex lacteus, and Hypoxylon ftagiforme was studied. The most effective fungus in shaken flask experiments was B. adusta, which was able to decolorise the dye from black-blue to a yellow color in less than 10 days. This fungus was chosen for cultivation in a 5 L stirred tank bioreactor with immobilised mycelium. Here decoloration of the dye in an initial concentration of 0.2 g/L from black-blue to intense yellow was reached in 20 days. Considering the spectral absorption coefficients at three different wavelengths, the final liquid was decolorised to an almost ecologically acceptable degree. Measurement of activities of the oxidative enzymes laccase, lignin peroxidase, manganese dependent- and manganese independent peroxidases showed that initially lignin peroxidases and subsequently manganese dependent peroxidases are presumably responsible for the decoloration of the diazo dye Reactive Black 5 by B. adusta.