枯草芽孢杆菌漆酶的理性设计及在霉菌毒素降解的应用

Rational design of Bacillus subtilis laccase and its application in degradation of mycotoxins

为解决目前制约食品等领域发展的霉菌毒素污染问题,选取了枯草芽孢杆菌来源的BsCotA漆酶并且通过理性设计提高酶活力、稳定性,从而实现对霉菌毒素降解效率的提高。利用理性设计改造BsCotA漆酶,获得了4个突变体漆酶,包括3个点突变体CotA^(A344K)、CotA^(A317T)、CotA^(T36Y),和突变整合体CotAgold,使用毕赤酵母进行异源表达,测定其酶学性质,并检测其对霉菌毒素的降解效率。基于蛋白质结构进行设计的3个单点突变,均从不同程度上提高了漆酶的稳定性。而将3个突变位点同时整合到1个漆酶突变体上时,3个突变位点间未发生显著拮抗作用,使得突变整合体CotAgold展示出最好的稳定性以及催化能力;CotAgold比酶活力为99.4 U/mg,相比BsCotA的比酶活力提高了10倍,对于ABTS的k_(cat)/K_(M)达到了7.12×10^(5)L/(mol·s),相比BsCotA提高了1.9倍;突变体CotAgold的最适反应pH值为4,最适反应温度均为70℃,与野生型BsCotA催化条件保持一致;CotAgold在pH 2~12,孵育30 min,剩余酶活力均在70%以上,相比野生型BsCotA在酸性条件下的稳定性有明显提高;CotAgold在40~90℃,孵育30 min,残留酶活力均在90%以上,相比野生型BsCotA在高温条件下的稳定性有明显提高;并在ABTS阳离子自由基作为介体的条件下,漆酶CotAgold能完全降解黄曲霉毒素B1和玉米赤霉烯酮;相比之下,野生型漆酶BsCotA的降解率仅为5%。通过蛋白质的理性设计得到了漆酶突变体CotAgold,表现出显著提高的pH稳定性以及热稳定性,蛋白表达量也有一定的提高,在霉菌毒素降解等领域具有良好的应用前景。

To address the issue of mycotoxin contamination that currently constrains the development in fields such as food,a laccase BsCotA,sourced from Bacillus subtilis was selected.Through rational design,the activity and stability of the enzyme were enhanced,thereby increasing the degradation efficiency of the mycotoxins.Based on the protein structure,four mutant laccase enzymes derived from BsCotA were obtained,including three single-point mutants(CotA^(A344K),CotA^(A317T),CotA^(T36Y)),and a mutant integrase named CotAgold.These were heterologous expressed in Pichia pastoris.Their enzymatic properties were characterized,and their efficiency in degrading mycotoxins was evaluated.The design of three single-point mutations based on the protein structure improved the stability of the laccase to varying degrees.When the three mutations were integrated into one laccase mutant,no significant antagonism effects occurred between the mutation sites,and this mutant,CotAgold,demonstrated the best stability and catalytic ability.The specific enzyme activity of CotAgold reached 99.4 U/mg,which was 10-fold higher than that of BsCotA,and the k_(cat)/K_(M)for ABTS cationic radicals was 7.12×10^(5)L/(mol·s),which was 1.9-fold times that of BsCotA.The optimal pH of mutant CotAgold was 4,and the optimum reaction temperature was consistently 70℃,in line with the catalytic conditions of the wild-type BsCotA.CotAgold maintained over 70%of its enzyme activity after incubation for 30 min across a pH range of 2-12,displaying a noticeable improvement in stability under acidic conditions compared to the wild-type BsCotA.CotAgold retained more than 90%of its residual activity after incubation at temperatures ranging from 40-90℃for 30 min,indicating a significant enhancement in stability at high temperatures compared to the wild-type.When using ABTS+as the mediator,the laccase CotAgold completely degraded both aflatoxin B1 and zearalenone,in contrast,the degradation rate by the wild-type laccase BsCotA was only 5%.Through rational design,a laccase mutant CotAgold was obtained,which exhibited significantly enhanced pH stability as well as thermal stability.There was also an increase in protein expression.This mutant has promising application prospects in fields such as mycotoxin degradation.