PDF(Pubmed)
Abstract：
Aflatoxin B1 (AFB1) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from Bacillus licheniformis ANSB821 previously reported by our laboratory showed great potential to degrade AFB1 without redox mediators. However, the use of this CotA-laccase to remove AFB1 in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB1, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB1, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (kcat/Km) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into Pichia pastoris GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in P. pastoris GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB1 detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.
摘要：
黄曲霉毒素B1(AFB1)沾染严重威逼营养平安和公共卫生。我们实验室先前报道的地衣芽孢杆菌ANSB821的CotA漆酶显示出在没有氧化还原介质的情况下降解AFB1的巨大潜力。然而,由于其催化效率低和表达水平低，因此使用这种CotA-漆酶去除动物饲料中的AFB1受到限制。为了更好地利用这种优异的酶有效降解AFB1，通过定点诱变构建了12个CotA-漆酶突变体。在这些突变体中，E186A和E186R对AFB1的降解能力最好,12h内降解率分别为82.2%和91.8%,比野生型CotA漆酶高1.6倍和1.8倍，分别。E186A和E186R的催化效率（kcat/Km）分别为1.8和3.2倍，分别,比野生型CotA漆酶的那些。然后构建具有优化信号肽的表达载体pPICZαA-N-E186A和pPICZαA-N-E186R，并将其转化到毕赤酵母GS115中。优化的信号肽改善了毕赤酵母GS115中E186A和E186R的分泌表达。总的来说，本研究为食品和动物饲料中AFB1解毒提供了理想的候选CotA-漆酶突变体，并提供了可行的方案，这是工业生产CotA漆酶迫切需要的。
