枯草芽孢杆菌YQ-1降解玉米赤霉烯酮膜蛋白酶的提取及性质分析

Membrane Protease Studies of the Degradation of Zearalenone by Bacillus subtilis

摘要:

玉米赤霉烯酮（Zearalenone，ZEN）是一类常见于发霉谷物、饲料中的真菌毒素，具有生殖、遗传和致癌性等危害，在世界各地的谷物中常超标检出。本课题组前期筛选得到一株高效降解ZEN的枯草芽孢杆菌Bacillus subtilis YQ-1，全细胞催化12 h后ZEN（20 μg/mL）的降解率达98.36%。根据降解酶的细胞定位，推测ZEN降解酶定位于细胞膜表面。本实验以ZEN降解率为指标，提取YQ-1细胞膜表面ZEN降解酶蛋白，结合氨基酸序列分析，推测可能的降解酶。结果显示，以辛基-β-葡萄糖苷（OG）、十二烷基-β-D-麦芽糖苷（DDM）、Triton X-100和Triton X-114作为膜蛋白提取剂比NaCl和尿素提取的蛋白量显著增加，但提取的膜蛋白酶ZEN降解活性较差，以丁醇为提取剂提取得到的水相蛋白粗酶液在12 h内对20 μg/mL ZEN的降解率为64.09%，粗酶液蛋白含量为9.86 μg/mL，酶活为0.43 U。该降解酶的提取为后续的ZEN降解酶挖掘奠定了实验基础，枯草芽孢杆菌及其ZEN降解酶的研究为霉菌毒素的体外脱毒提供支撑。

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.