Membrane Protease Studies of the Degradation of Zearalenone by Bacillus subtilis
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Abstract:
Zearalenone (ZEN) is a fungal toxin commonly found in moldy cereals and feedstuffs. It has reproductive, mutagenic, and carcinogenic effects and is often detected at levels exceeding the standard levels in cereals worldwide. Bacillus subtilis YQ-1 efficiently degrades ZEN (20 μg/mL) at a rate of 98.36%. Based on its cellular localization, it was speculated that ZEN was localized to the cell membrane surface. In this experiment, the ZEN degradation rate was used as an indicator of the successful extraction of ZEN from the cell membrane surface of YQ-1, and combined with amino acid sequence analysis, possible degradation enzymes were speculated. The results showed that the amount of protein extracted with octyl-β-glucoside (β-OG), dodecyl-β-D-maltoside (DDM), Triton X-100, and Triton X-114 as membrane protein extractants was significantly higher than the amount extracted with NaCl and urea, but the ZEN degradation activity of the extracted membrane proteases was notably lower. Moreover, the aqueous phase crude enzyme solution obtained when using butanol as the extractant was less effective at 12 h. The degradation rate of 20 μg/mL ZEN within 12 h was 64.09%, the protein content of the crude enzyme solution was 9.86 μg/mL, and the enzyme activity was measured to be 0.43 U. The amino acid sequence comparison revealed a possible α/β hydrolase in the crude enzyme solution. The extraction of this degradation enzyme laid the experimental foundation for subsequent ZEN degradation enzyme extraction and further investigation of B. subtilis as well as its ZEN degradation enzyme in in vitro mycotoxin studies. The study of B. subtilis and its ZEN-degrading enzyme offers support to the prospect of in vitro detoxification of mycotoxins.
