Elisavet Vekiru

Degradation of fumonisin B1 by the consecutive action of two bacterial enzymes

Detoxification of the mycotoxin fumonisin B(1) comprises at least two enzymatic steps, an initial deesterification reaction, followed by deamination of the resulting hydrolyzed fumonisin B(1). In this study, two genes that are responsible for degradation of fumonisin B(1) by the bacterium Sphingopyxis sp. MTA144 were identified within a gene cluster, assumed to be associated with fumonisin degradation. The first gene encodes a protein which shows similarity to carboxylesterases, type B. The second gene encodes a polypeptide homologous to aminotransferases, class III. The two genes were isolated and expressed heterologously. The effect of the recombinant enzymes on fumonisin B(1) and hydrolyzed fumonisin B(1) was determined. The recombinant carboxylesterase was shown to catalyze the deesterification of fumonisin B(1) to hydrolyzed fumonisin B(1). The heterologously expressed aminotransferase was shown to deaminate hydrolyzed fumonisin B(1) in the presence of pyruvate and pyridoxal phosphate. We propose that the consecutive action of these two enzymes is sufficient for fumonisin B(1) detoxification. The results of this work provide a basis for the development of an enzymatic detoxification process for fumonisin B(1) in food and animal feed, especially under oxygen limited conditions, as they are found, e.g. in ensilaged forage or in the intestinal tract of animals.

Cleavage of zearalenone by Trichosporon mycotoxinivorans to a novel nonestrogenic metabolite.

ABSTRACT Zearalenone (ZON) is a potent estrogenic mycotoxin produced by several Fusarium species most frequently on maize and therefore can be found in food and animal feed. Since animal production performance is negatively affected by the presence of ZON, its detoxification in contaminated plant material or by-products of bioethanol production would be advantageous. Microbial biotransformation into nontoxic metabolites is one promising approach. In this study the main transformation product of ZON formed by the yeast Trichosporon mycotoxinivorans was identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-diode array detector (DAD) analysis. The metabolite, named ZOM-1, was purified, and its molecular formula, C18H24O7, was established by time of flight MS (TOF MS) from the ions observed at m/z 351.1445 [M-H]− and at m/z 375.1416 [M+Na]+. Employing nuclear magnetic resonance (NMR) spectroscopy, the novel ZON metabolite was finally identified as (5S)-5-({2,4-dihydroxy-6-[(1E)-5-hydroxypent-1-en-1-yl]benzoyl}oxy)hexanoic acid. The structure of ZOM-1 is characterized by an opening of the macrocyclic ring of ZON at the ketone group at C6′. ZOM-1 did not show estrogenic activity in a sensitive yeast bioassay, even at a concentration 1,000-fold higher than that of ZON and did not interact with the human estrogen receptor in an in vitro competitive binding assay.

Investigation of various adsorbents for their ability to bind aflatoxin B1

The contamination of animal feed with mycotoxins represents a worldwide problem for the animal industry. The most applied method for protecting animals against aflatoxicosis is the utilization of clay minerals. In the course of a research project adsorption experiments were performed in buffer solutions in order to evaluate the ability to bind Aflatoxin B1 (AfB1) at various pH-values. In order to investigate the strength of binding, the chemisorption index was calculated. Isothermal analysis was used to determine the values for the maximum adsorption capacity. Adsorption experiments in simulated gastrointestinal fluid and real gastric juice were carried out. Furthermore binding capability of the materials regarding selected vitamins was examined. Special attention was paid to the formation of AfB2a during experimental conditions. Based on the obtainedin vitro results, highly promising sorbent materials were ranked for furtherin vivo studies. Some adsorbing bentonites were also analysed mineralogically, but the results did not indicate which smectite property influences the adsorption process for AfB1

Determination of fumonisins and hydrolyzed fumonisin B1 in microbial culture media by LC/ESI-MS.

In order to analyze liquid growth media for fumonisin B1, B2 and hydrolyzed fumonisin B1 (HFB1) after incubation with microorganisms for degradation studies, a liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) method was developed. The use of phytosphingosine hydrochloride (PSH) and T-2-toxin-d3 (T2d3) as internal standard has been tested. The detection limit established with PSH as an internal standard was about 20 ng/mL for FB1 and FB2 and about 50 ng/mL for HFB1. The developed method allows the rapid, simultaneous and quantitative determination of these analytes in microbial culture media without any cleanup.

Isolation and characterisation of enzymatic zearalenone hydrolysis reaction products

Zearalenone (ZEA) is an oestrogenic mycotoxin produced by several Fusarium species, and it frequently contaminates cereals used for food or animal feed. A ZEA-lactonase of Gliocladium roseum was previously described to hydrolyse ZEA to an unstable intermediate, which spontaneously decarboxylates to non-oestrogenic, decarboxylated hydrolysed ZEA (DHZEN). We expressed a codon-optimised version of the ZEA-lactonase (ZHD101) gene of G. roseum MA 918 with a secretion leader in Pichia pastoris and purified the recombinant enzyme from culture supernatant by His-tag mediated affinity chromatography. After incubation of the enzyme with ZEA, we detected the previously elusive primary reaction product hydrolysed ZEA (HZEN) by liquid chromatography tandem mass spectrometry, purified it by preparative high-performance liquid chromatography, and confirmed its postulated structure ((E)-2,4-dihydroxy-6-(10-hydroxy-6-oxo-1-undecen-1-yl)benzoic acid) by nuclear magnetic resonance techniques. Spontaneous decarboxylation to ...

Toxicity of ochratoxin A in aBrevibacillus brevis - Growth inhibition assay.

Ochratoxin A (OTA) is a nephrotoxic, carcinogenic and immunosuppressive mycotoxin. It can be detoxified by various microorganisms, e.g. different yeast strains, via metabolisation into ochratoxin α (OTα). Within this study a growth inhibition assay was developed to compare the toxicity of OTA and its degradation product OTα. As an indicator organismBrevibacillus brevis was used. The assay was performed in microtiterplates. Growth inhibition was determined by comparing the optical density values ofBrevibacillus brevis cultures grown in medium supplemented with OTA/OTα and OTA/OTα-free medium, respectively.It could be shown thatB. brevis is sensitive to OTA (EC100=0.5 mg/L±0.03 mg/L), which is not the case for its metabolite OTα. Therefore this bioassay is a useful tool to show the detoxification of OTA to OTα by microbial degradation.