Ottó Bencsik

Characterization of a β-glucosidase with transgalactosylation capacity from the zygomycete Rhizomucor miehei

An extracellular β-glucosidase from the zygomycete Rhizomucor miehei NRRL 5282 cultivated in a wheat bran-based solid state fermentation system was characterized. The purified enzyme exhibited an optimum temperature of 68-70 °C and pH of 5.0. It efficiently hydrolyzed oligosaccharides having β-(1→4) glycosidic linkages and exhibited some β- and α-galactosidase activity. The V(max) for p-nitrophenyl-β-d-glucopyranoside and cellobiose was 468.2 and 115.5 U/mg, respectively, while the K(m) was 0.12 mM for both substrates. The enzyme had transglucosylation and transgalactosylation activities resulting in the formation of glycosides from cellobiose, lactose and ethanol. The enzyme increased the amounts of free phenolic antioxidants in sour cherry pomace indicating that its hydrolyzing activity could potentially be applicable to improve the bioavailability of these compounds.

Ophiobolin A from Bipolaris oryzae Perturbs Motility and Membrane Integrities of Porcine Sperm and Induces Cell Death on Mammalian Somatic Cell Lines

Bipolaris oryzae is a phytopathogenic fungus causing a brown spot disease in rice, and produces substance that strongly perturbs motility and membrane integrities of boar spermatozoa. The substance was isolated from the liquid culture of the fungal strain using extraction and a multi-step semi-preparative HPLC procedures. Based on the results of mass spectrometric and 2D NMR techniques, the bioactive molecule was identified as ophiobolin A, a previously described sesterterpene-type compound. The purified ophiobolin A exhibited strong motility inhibition and viability reduction on boar spermatozoa. Furthermore, it damaged the sperm mitochondria significantly at sublethal concentration by the dissipation of transmembrane potential in the mitochondrial inner membrane, while the plasma membrane permeability barrier remained intact. The study demonstrated that the cytotoxicity of ophiobolin A toward somatic cell lines is higher by 1–2 orders of magnitude compared to other mitochondriotoxic mycotoxins, and towards sperm cells unique by replacing the progressive motility by shivering tail beating at low exposure concentration.

Effect of the sesterterpene-type metabolites, ophiobolins A and B, on zygomycetes fungi

Ophiobolins are sesterterpene-type phytotoxins produced by fungi belonging mainly to the genus Bipolaris. In this study, the antifungal effect of ophiobolins A and B on different zygomycetes has been examined. Depending on the zygomycete tested, MIC values of 3.175-50 μg mL(-1) were found for ophiobolin A and 25-50 μg mL(-1) for ophiobolin B. Ophiobolin A inhibited sporangiospore germination of Mucor circinelloides and caused morphological changes; the fungus formed degenerated, thick or swollen cells with septa. Cytoplasm effusions from the damaged cells were also observed. Fluorescence microscopy after annexin and propidium iodide staining of the treated cells suggested that the drug induced an apoptosis-like cell death process in the fungus.

Species diversity and cytotoxic potency of airborne sterigmatocystin-producing Aspergilli from the section Versicolores

This study presents the distribution and species diversity of sterigmatocystin-producing Aspergilli from the section Versicolores in the indoor air of apartment-AP, basements-BS and grain mill-GM in Croatia, as well as the cytotoxic potency of isolates. The species comprised 0.7-20% of total airborne fungi detected in the AP, 11-55% in the BS, and 0-2% in the GM. Based on CaM sequences, seven species were identified; dominant were Aspergillus jensenii and Aspergillus creber, followed by Aspergillus protuberus, Aspergillus venenatus, Aspergillus tennesseensis, Aspergillus amoenus, Aspergillus griseoaurantiacus and three undescribed species. All of the identified species produced sterigmatocystin-STC (HPLC/UV-VIS); A. griseoaurantiacus (208.29μg/mL) and A. jensenii (1.192-133.63μg/mL) produced the highest levels, the lowest were detected in A. protuberus and A. tennesseensis (0.117-2.749μg/mL). Lower species diversity was obtained in the GM due to overgrowth with more propulsive fungi. Relatively high STC levels (0.06-2.35μg/g) detected in 52% of GM dust samples confirmed the presence of STC-producers, although this STC cannot be exclusively attributed to Aspergilli (Versicolores). STC and the majority of STC-producing Aspergilli were cytotoxic to human lung A549 cells (IC50 0.9-2.3μg/mL) and THP-1 macrophage-like cells (IC50 0.3-0.6μg/mL) in relatively low concentrations suggesting that humans can be at high risk during chronic exposure.

Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides

Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

Transcription of the three HMG-CoA reductase genes of Mucor circinelloides

BackgroundPrecursors of sterols, carotenoids, the prenyl groups of several proteins and other terpenoid compounds are synthesised via the acetate-mevalonate pathway. One of the key enzyme of this pathway is the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which catalyses the conversion of HMG-CoA to mevalonate. HMG-CoA reductase therefore affects many biological processes, such as morphogenesis, synthesis of different metabolites or adaptation to environmental changes. In this study, transcription of the three HMG-CoA reductase genes (designated as hmgR1, hmgR2 and hmgR3) of the β-carotene producing Mucor circinelloides has been analysed under various culturing conditions; effect of the elevation of their copy number on the carotenoid and ergosterol content as well as on the sensitivity to statins has also been examined.ResultsTranscripts of each gene were detected and their relative levels varied under the tested conditions. Transcripts of hmgR1 were detected only in the mycelium and its relative transcript level seems to be strongly controlled by the temperature and the oxygen level of the environment. Transcripts of hmgR2 and hmgR3 are already present in the germinating spores and the latter is also strongly regulated by oxygen. Overexpression of hmgR2 and hmgR3 by elevating their copy numbers increased the carotenoid content of the fungus and decreased their sensitivity to statins.ConclusionsThe three HMG-CoA reductase genes of M. circinelloides displayed different relative transcript levels under the tested conditions suggesting differences in their regulation. They seem to be especially involved in the adaptation to the changing oxygen tension and osmotic conditions of the environment as well as to statin treatment. Overexpression of hmgR2 and hmgR3 may be used to improve the carotenoid content.

Identification of Aspergillus species in Central Europe able to produce G-type aflatoxins

The occurrence of potential aflatoxin producing fungi was examined in various agricultural products and indoor air in Central European countries including Hungary, Serbia and Croatia. For species identification, both morphological and sequence based methods were applied. Aspergillus flavus was detected in several samples including maize, cheese, nuts, spices and indoor air, and several isolates were able to produce aflatoxins. Besides, three other species of Aspergillus section Flavi, A. nomius, A. pseudonomius and A. parasiticus were also isolated from cheese, maize and indoor air, respectively. This is the first report on the occurrence of A. nomius and A. pseudonomius in Central Europe. All A. nomius, A. pseudonomius and A. parasiticus isolates were able to produce aflatoxins B1, B2, G1 and G2. The A. nomius isolate came from cheese produced very high amounts of aflatoxins (above 1 mg ml⁻¹). All A. nomius, A. pseudonomius and A. parasiticus isolates produced much higher amounts of aflatoxin G1 then aflatoxin B1. Further studies are in progress to examine the occurrence of producers of these highly carcinogenic mycotoxins in agricultural products and indoor air in Central Europe.

New sterigmatocystin-producing species of Aspergillus section Versicolores from indoor air in Croatia

Multilocus DNA sequence-based identification methods raised the number of known species assigned to the Aspergillus section Versicolores. Currently, there are 16 species accepted in the section, including A. amoenus, A. austroafricanus, A. creber, A. cvjetkovicii, A. fructus, A. griseoaurantiacus, A. hongkongensis, A. jensenii, A. protuberus, A. puulaauensis, A. subversicolor, A. sydowii, A. tabacinus, A. tennesseensis, A. venenatus, and A. versicolor. Based on morphological identifications, most of these species were identified as either A. sydowii or A. versicolor, with the latter reported to have a world-wide distribution, growing in many habitats. Aspergillus versicolor has been implicated in health hazards including sick building syndrome, human and animal mycoses, and contamination of food and feed were assigned primarily to this species. A. versicolor is still commonly isolated from indoor surveys, even though species such as A. jensenii and A. creber seem more common. From indoor air samples collected at a grain mill in Croatia, we isolated an undescribed species assigned to the Aspergillus section Versicolores. A polyphasic approach, including sequence-based methods, morphological and physiological studies, was used for species characterization and in this paper is described as Aspergillus pepii. Additionally, sterigmatocystin producing abilities have been confirmed. Based on a combined phylogenetic tree, morphological features and sterigmatocystin producing abilities, A. pepii is closely related to A. versicolor. Further studies should explore the frequency of the species in indoor environments and its medical, industrial, and environmental significance.

The effect of acute ophiobolin: a treatment on HO-mediated inflammatory processes

Many microbial and plant-derived metabolites contribute to the production of inflammatory mediators and the expression of pro-inflammatory molecules. Ophiobolin A (OPA) is a fungal secondary metabolite produced by Bipolaris species. The aim of our study was to examine the acute effects of this compound on inflammatory processes. Male Wistar rats were treated with 5% ethanol, 0.01 mg/kg OPA, 0.1 mg/kg OPA and 1.0 mg/kg OPA per os. After 24 h of the administrations, inflammatory mediators such as interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α) and myeloperoxidase (MPO) enzyme as well as heme oxygenase (HO) activity were measured in both plasma and cardiac tissue, along with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). We found that OPA caused a significant elevation in the concentrations of IL-6 and TNF-α, increased MPO activity and decreased HO enzyme activity in the plasma. While OPA induces inflammation in the plasma, it did not change the level of inflammatory mediators in the cardiac tissue and the concentrations of serum ALT and AST. Our findings indicate that rapid release of inflammatory mediators by OPA promotes systemic inflammation. However, this acute OPA treatment does not show toxic effects on the cardiac tissue and the concentrations of liver enzymes.

Fumonisin Measurement from Maize Samples by High-Performance Liquid Chromatography Coupled with Corona Charged Aerosol Detector

Fumonisins are a class of mycotoxins produced mainly by Fusarium species, which is primary fungal contaminant of the maize and maize-derived products around the world. The B-series fumonisins (FB1, FB2 and FB3) are the most abundant and toxic constituent; thus, their levels are regulated generally worldwide. In this study, we developed a reliable method for the measurement of fumonisin FB1, FB2 and FB3 mycotoxins from maize samples without the time-consuming derivatization step using a high-performance liquid chromatograph coupled with corona charged aerosol detector. The detection and quantitation limit of the whole method were 0.02 and 0.04 mg/kg for each fumonisins, respectively. The detection linearity was tested in the calibration range of 2 orders of magnitude and the recoveries from the spiked samples were determined. The developed method proved to be sufficient to measure the maximum residue levels of fumonisins, which are specified in European Union and United States in maize and maize-based products.