短小芽孢杆菌漆酶基因的克隆表达及重组漆酶降解黄曲霉毒素M_1研究  

Cloning and Expression of Laccase Gene from Bacillus pumilus and Degradation of A?atoxin M_1 by Recombinant Laccase

作　　者：霍超[1] 卢海强[1] 刘晓宇[1] 李琪 高洁 桑亚新[1] HUO Chao;LU Haiqiang;LIU Xiaoyu;LI Qi;GAO Jie;SANG Yaxin(College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China)

机构地区：[1]河北农业大学食品科技学院,河北保定071000

出　　处：《食品科学》2019年第10期92-98,共7页Food Science

基　　金："十三五"国家重点研发计划重点专项(2017YFC160089);河北省科技厅科技支撑计划项目(130600067)

摘　　要：为明确短小芽孢杆菌Cot A漆酶的酶学性质,探究其在黄曲霉毒素M_1降解中的应用潜力。经分析,短小芽孢杆菌E-1-1-1中cotA基因全长1 486 bp,编码495个氨基酸,无信号肽序列,分子质量约为58 kDa。该重组漆酶最适反应温度为40℃,最适pH 4.0;K_m为3.01 mmol/L,V_(max)为0.795 mmol/(L·min);金属离子和化学试剂会对酶活力造成不同程度影响,K^+、Na^+、乙醇和十二烷基硫酸钠对该酶有明显的抑制作用,抑制率分别为31.0%、13.6%、64.5%和100%,而Mn^(2+)和Zn^(2+)则表现出明显的促进作用,酶活力分别提高了33.0%和23.9%;该酶与牛奶中的黄曲霉毒素M_1反应72 h后,黄曲霉毒素M_1降解率为54.3%,毒性降低9.1%。结果表明,短小芽孢杆菌CotA漆酶作为黄曲霉毒素M_1降解酶具有巨大的应用潜力。This study aimed at determining the enzymatic properties of recombinant CotA laccase and evaluating its potential for the degradation of aflatoxin M1(AFM1). Our analysis demonstrated that the cotA gene of Bacillus pumilus E-1-1-1 was 1 486 bp long, encoding 495 amino acids and having no signal peptide, and its putative protein molecular mass was 58 kDa. The optimum temperature for the recombinant laccase was 40 ℃, and the optimum pH was 4.0. The Km value was 3.01 mmol/L, and the Vmax value was 0.795 mmol/(L·min). Metal ions and chemical reagents had different effects on its enzymatic activity. K^+, Na^+, ethanol and SDS inhibited the enzymatic activity obviously with percentage inhibition of 31.0%,13.6%, 64.5% and 100%, respectively. In contrast, Mn^2+ and Zn^2+ promoted the enzymatic activity by 33.0% and 23.9%,respectively. After 72 hours of reaction with this enzyme, the content of AFM1 in milk decreased by 54.3%, accompanied by a decrease in the toxicity of 9.1%. From this study, we concluded that CotA laccase from B. pumilus can have a great potential as an AFM1-degrading enzyme.

关 键 词：CotA蛋白 漆酶 黄曲霉毒素M1 生物脱毒 短小芽孢杆菌 

分 类 号：Q939.99[生物学—微生物学]