Simultaneous degradation of aflatoxin B1 and zearalenone by Porin and Peroxiredoxin enzymes cloned from Acinetobacter nosocomialis Y1.

Mycotoxin contamination can cause severe health issues for both humans and animals. This study examined the potential of enzymes derived from Acinetobacter nosocomialis Y1 to simultaneously degrade aflatoxin B1 (AFB1) and zearalenone (ZEN), which could have significant implications in reducing mycotoxin contamination. Two enzymes, Porin and Peroxiredoxin, were identified with molecular weights of 27.8 and 20.8 kDa, respectively. Porin could completely degrade 2 µg/mL of AFB1 and ZEN within 24 h at 80 °C and 60 °C, respectively. Peroxiredoxin could completely degrade 2 µg/mL of AFB1 and reduce ZEN by 91.12% within 24 h. The addition of Na+, Cu2+, and K+ ions enhanced the degradation activities of both enzymes. LC-MS/MS analysis revealed that the molar masses of the degradation products of AFB1 and ZEN were 286 g/mol and 322.06 g/mol, and the products were identified as AFD1 and α or ß-ZAL, respectively. Vibrio fischeri bioluminescence assays further confirmed that the cytotoxicity of the two degradation products was significantly lower than that of AFB1 and ZEN. Based on these results, it can be inferred that the degradation product of ZEN is ß-ZAL. These findings suggest that both enzymes have the potential to be utilized as detoxification enzymes in food and feed.

SakA Regulates Morphological Development, Ochratoxin A Biosynthesis and Pathogenicity of Aspergillus westerdijkiae and the Response to Different Environmental Stresses.

Ochratoxin A (OTA), as a common mycotoxin, has seriously harmful effects on agricultural products, livestock and humans. There are reports on the regulation of SakA in the MAPK pathway, which regulates the production of mycotoxins. However, the role of SakA in the regulation of Aspergillus westerdijkiae and OTA production is not clear. In this study, a SakA deletion mutant (ΔAwSakA) was constructed. The effects of different concentrations of D-sorbitol, NaCl, Congo red and H2O2 on the mycelia growth, conidia production and biosynthesis of OTA were investigated in A. westerdijkiae WT and ΔAwSakA. The results showed that 100 g/L NaCl and 3.6 M D-sorbitol significantly inhibited mycelium growth and that a concentration of 0.1% Congo red was sufficient to inhibit the mycelium growth. A reduction in mycelium development was observed in ΔAwSakA, especially in high concentrations of osmotic stress. A lack of AwSakA dramatically reduced OTA production by downregulating the expression of the biosynthetic genes otaA, otaY, otaB and otaD. However, otaC and the transcription factor otaR1 were slightly upregulated by 80 g/L NaCl and 2.4 M D-sorbitol, whereas they were downregulated by 0.1% Congo red and 2 mM H2O2. Furthermore, ΔAwSakA showed degenerative infection ability toward pears and grapes. These results suggest that AwSakA is involved in the regulation of fungal growth, OTA biosynthesis and the pathogenicity of A. westerdijkiae and could be influenced by specific environmental stresses.

Microbial Enzymes Involved in the Biotransformation of Major Mycotoxins.

Mycotoxins, the most researched biological toxins, can contaminate food and feed, resulting in severe health implications for humans and animals. Physical, chemical, and biological techniques are used to mitigate mycotoxin contamination. The biotransformation method using whole microbial cells or isolated enzymes is the best choice to mitigate mycotoxins. Using specific enzymes may avoid the disadvantages of utilizing a full microbe, such as accidental harm to the product's organoleptic characteristics and hazardous safety features. Moreover, the degradation rates of the isolated enzymes are higher than those of the whole-cell reactions, and they are substrate-specific. Their specificity is comprehensive and is shown at the positional and/or chiral center in many circumstances. Currently, only a few enzymes of microbial origin are commercially available. Therefore, there is a need to identify more novel enzymes of microbial origin that can mitigate mycotoxins. In this review, we conducted an in-depth summary of the microbial enzymes involved in the biotransformation of mycotoxins.

AwAreA Regulates Morphological Development, Ochratoxin A Production, and Fungal Pathogenicity of Food Spoilage Fungus Aspergillus westerdijkiae Revealed by an Efficient Gene Targeting System.

Aspergillus westerdijkiae, the producer of ochratoxin A (OTA), which is of worldwide concern, is an import fungal species in agriculture, food, and industry. Here, we got the uridine auxotrophic mutant of A. westerdijkiae by deleting AwpyrG. The ΔAwpyrG could be used for bio-transformation with exogenous AfpyrG expression cassette as a selection marker. In order to enhance the efficiency of gene targeting, Awku70 and Awlig4 were homologously deleted from ΔAwpyrG. The efficiencies of homologous replacement for ΔAwku70 and ΔAwlig4 were 95.7 and 87.0% in the deletion of AwAreA, respectively, demonstrating a drastic increase from 4.3% of the wild type (WT) strain. Furthermore, the function of AwAreA was identified with AwAreA deletion mutant and the control strain ΔAwku70. AwAreA regulated the growth and conidiation of A. westerdijkiae in response to nitrogen sources. The concentration of OTA for ΔAwku70 was in the range of 19.4 to 186.9 ng/cm2 on all kinds of nitrogen sources. The OTA production influenced by the deletion of AwAreA was different based on nitrogen sources. Pathogenicity assays on pears, grapes, salted meat, and cheese showed that AwAreA acted as a negative regulator in the infection of food substrates. Therefore, the genetic methods and engineered strains enable us to substantially expand the use of A. westerdijkiae, one of more than twenty OTA-producing fungi, in the study of mycotoxin biosynthesis and regulation, and consequently to aim at providing new ways for controlling this pathogen.