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Changes in electrophoresis pattern after interaction of supercoiled plasmid pBR322 DNA with avarol was studied at a micromolar concentration of reactants under mild reaction conditions. Interactions of avarol with linear high-molecular CT-DNA at millimolar concentrations were analyzed by electrophoresis and UV spectrophotometry. It was observed that avarol is capable of quenching ethidium bromide fluorescence in DNA bands. An increase in the absorbance of DNA was detected. The results indicate the binding of avarol to DNA and/or modification of nucleotide bases.

The quinone avarone, isolated from the marine sponge Dysidea avara, possesses the ability to chemically modify proteins. In this work, modification of lectin isolated from the coral Gerardia savaglia by avarone was examined. The techniques used for studying the modification were: SDS PAGE, isoelectric focusing and hemagglutination testing. The results of the SDS PAGE indicate dimerization of the protein. A shift of the pI toward lower value occurs upon modification. The change of the hemagglutination activity of the protein confirms that chemical modification of G. savaglia lectin by avarone changes its ability to interact with the membrane of erythrocytes.

Amyloglucosidase from A. niger was covalently immobilized onto poly (GMA-co-EGDMA) by the glutaraldehyde and periodate method. The immobilization of amyloglucosidase after periodate oxidation gave a preparate with the highest specific activity reported so far on similar polymers. The obtained immobilized preparates show the same pH optimum, but a higher temperature optimum compared with the soluble enzyme. The kinetic parameters for the hydrolysis of soluble starch by free and both immobilized enzymes were determined.

The avarone/avarol quinone/hydroquinone couple shows considerable antitumor activity. In this work, covalent modification of b-lactoglobulin by avarone and its derivatives as well as by the synthetic steroidal quinone 2,5(10)-estradiene-1,4,17-trione and its derivatives were studied. The techniques for studying chemical modification of b-lactoglobulin by quinones were: UV/Vis spectrophotometry, SDS PAGE and isoelectrofocusing. SDS PAGE results suggest that polymerization of the protein occurs. It could be seen that the protein of 18 kD gives the bands of 20 kD, 36 kD, 40 kD, 45 kD, 64 kD and 128 kD depending on modification agent. The shift of the pI of the protein (5.4) upon modification toward lower values (from pI 5.0 to 5.3) indicated that lysine amino groups are the principal site of the reaction of b-lactoglobulin with the quinones.

The avarone/avarol quinone/hydroquinone couple, as well as their derivatives show considerable antitumor activity. In this work, covalent modifications of b-lactoglobulin, isolated from cow milk, by avarone, its model compound 2-tert-butyl-1,4-benzoquinone, and several of their alkylthio derivatives were studied. The techniques applied for assaying the modifications were: UV/VIS spectrophotometry, SDS PAGE and isoelectrofocusing. The results of the SDS PAGE suggest that polymerisation of the protein occurs. The shift of the pI of the protein upon modification toward lower values indicates that lysine amino groups are the principal site of the reaction of b-lactoglobulin with the quinones.

Rat brain Na,K-ATPase partially purified by SDS from synaptic plasma membranes (SPM) was immobilized by adsorption on nitrocellulose (NC), polyvinylidene fluoride (PVDF) and glass fiber (GF) membranes. Partial SDS solubilization increased the enzyme activity by 40 %. With regard to the preservation of the enzyme activity, nitrocellulose was shown to be the optimal support for the immobilization. The enzyme showed the highest percentage activity (14 %) after 30 min of SPM adsorption, at 20°C under the vaccum, with 25 mg of proteins per NC disc filter. In addition, adsorption on NC stabilizes the Na,K-ATPase, since the activity was substantial 72 h after adsorption at 20°C. After adsorption, the sensitivity of the enzyme to HgCl2and CdCll2 inhibition was higher. The results show that immobilized Na,K-ATPase SPM can be used as a practical model for the detection of metal ions in different samples.

Chalkbrood is a disease of honey bee brood caused by the fungal parasite Ascosphaera apis. Many factors such as genetics, temperature, humidity and nutrition influence the appearance of clinical symptoms. Poor nutrition impairs the immune system, which favors the manifestation of symptoms of many honey bee diseases. However, a direct link between dietary ingredients and the symptoms of chalkbrood disease has not yet been established. We show here that the elemental composition of chalkbrood mummies and healthy larvae from the same infected hives differ, as well as that mummies differ from larvae from healthy hives. Chalkbrood mummies had the highest concentration of macroelements such as Na, Mg, P, S, K and Ca and some microelements such as Rb and Sn, and at the same time the lowest concentration of B, As, Sr, Ag, Cd, Sb, Ba and Pb. Larvae from infected hives contained less Pb, Ba, Cs, Sb, Cd, Sr, As, Zn, Cu, Ni, Co, Mn, Cr, V and Al in contrast to healthy larvae from a disease-free apiary. This is the first study to demonstrate such differences, suggesting that an infection alters the larval nutrition or that nutrition is a predisposition for the outbreak of a chalkbrood infection. Though, based on results obtained from a case study, rather than from a controlled experiment, our findings stress the differences in elements of healthy versus diseased honey bee larvae.

Research background. Inulinases are used for fructooligosaccharide production and they are of interest for both scientific community and industry. Black aspergilli represent a diverse group of species that has use for enzyme production, in particular some species are known as potent inulinase producers. Finding new potential producers from the environment is as important as improving the production with known strains. Safe use of enzymes produced by aspergilli in food industry is placed ahead of their benefit for inulinase production. Experimental approach. Here we show a specific approach to finding/screening of newly isolated fungal inulinase producers that combines a newly developed screening method and an equally important assessment of the toxigenic potential of the fungus. In this study 39 black aspergilli collected from different substrates in Serbia were identified and assessed for inulinase production. Results and conclusions. The most common species were Aspergillus tubingensis (51.2%), followed by A. niger (23.1%), A. welwitschiae (23.1%) and A. uvarum (2.6%). The isolates for inulinase production were selected using a cheap and easy, fast and non-hazardous alternative inulinase screening test developed in this work. Enzymatic activity of selected inulinase-producing strains was confirmed spectrophotometrically. Since some A. niger and A. welwitschiae strains are able to produce mycotoxins ochratoxin A (OTA) and fumonisins (FB), the toxigenic potential of selected inulinase producers was assessed analytically and genetically. Fungal enzyme producer can be considered safe for use in food industry only after comparing the results of both approaches for investigating toxic potential, the direct presence of mycotoxins in the enzyme preparation (analytically) and the presence of mycotoxin gene clusters (genetically). In some strains the absence of OTA and FB production capability was molecularly confirmed by the absence of complete or critical parts of biosynthetic gene clusters, respectively. The two best inulinase producers and mycotoxin non-producers (without mycotoxin production capability as additional safety) were selected as potential candidates for further development of enzyme production. Novelty and scientific contribution. The presented innovative approach for the selection of potential fungal enzyme producer shows that only non-toxigenic fungi could be considered as useful in food industry. Although this study was done on local isolates, the approach is applicable globally.

Duckweed (L. minor) is a cosmopolitan aquatic plant of simplified morphology and rapid vegetative reproduction. In this study, an H. paralvei bacterial strain and its influence on the antioxidative response of the duckweeds to phenol, a recalcitrant environmental pollutant, were investigated. Sterile duckweed cultures were inoculated with H. paralvei in vitro and cultivated in the presence or absence of phenol (500 mg L−1), in order to investigate bacterial effects on plant oxidative stress during 5 days. Total soluble proteins, guaiacol peroxidase expression, concentration of hydrogen peroxide and malondialdehyde as well as the total ascorbic acid of the plants were monitored. Moreover, bacterial production of indole-3-acetic acid (IAA) was measured in order to investigate H. paralvei’s influence on plant growth. In general, the addition of phenol elevated all biochemical parameters in L. minor except AsA and total soluble proteins. Phenol as well as bacteria influenced the expression of guaiacol peroxidase. Different isoforms were associated with phenol compared to isoforms expressed in phenol-free medium. Considering that duckweeds showed increased antioxidative parameters in the presence of phenol, it can be assumed that the measured parameters might be involved in the plant’s defense system. H. paralvei is an IAA producer and its presence in the rhizosphere of duckweeds decreased the oxidative stress of the plants, which can be taken as evidence that this bacterial strain acts protectively on the plants during phenol exposure.

Phenols containing halogens, which tend to deactivate the aromatic nuclei, constitute a significant category of highly toxic and difficult-to-degrade pollutants, which arise from a wide variety of industries. The main purpose of this study was to obtain an inexpensive immobilized enzyme for the removal of phenols. Partially purified potato polyphenol oxidase (PPO) was immobilized onto different commercial and laboratory produced carriers. Three of the obtained biocatalysts, with the highest PPO activities, namely Eupergit C250L–PPO; Celite–PPO and CelluloseM–PPO, were tested in a batch reactor for the removal of phenol, 4-chlorophenol and 4-bromophenol. In the case of 2.5 mM substrates with Eupergit C250L–PPO, an around 45 % removal of 4-bromophenol was achieved, while the removals 4-chlorophenol and phenol were 35 and 20 %, respectively. The reusability of Eupergit C250L–PPO for the removal of 4-chlorophenol was tested. After eight repeated tests, the efficiency of 4-chlorophenol removal by Eupergit C250L–PPO immobilisate had decreased to 55 %.