Tc-AFO是本课题组从Trametes coccinea中鉴定的一个黄曲霉毒素B1(Aflatoxin B1，AFB1)降解酶，分子模拟结果表明Tc-AFO结构中存在一个影响活性中心空间的Bolt区，实验证实Bolt区中一些氨基酸对酶活的影响较大，但具体机制尚不清楚，为揭示Bolt区氨基酸影响酶催化性能的具体机制，本项目拟通过融合表达技术将Tc-AFO Bolt区序列全部或部分替换为同源蛋白的Bolt区序列，构建不同的Tc-AFO融合突变体；利用定点突变的方法在Bolt区不同氨基酸位点进行突变，构建不同的Tc-AFO突变体；应用AFB1核酸适配体交联的水凝胶可视化酶活检测技术，结合圆二色谱和分子模拟等手段，阐明各突变酶的酶学性质以及酶与底物结合能力的差异。全面揭示Bolt区影响酶催化性能的机制，为AFB1降解酶的定向改造和理性设计提供依据。

Previously, we have obtained an Aflatoxin B1(AFB1) degrading enzyme from Trametes coccinea, named Tc-AFO. A bolt region was indentified from 3D structure Tc-AFO and can influence the space of active site. We also confirmed amino acid variation at some site have great impact on Tc-AFO active site, but the mechanism were still unrealed. For explain details on amino acid influence catalytic preperties of Tc-AFO at the bolt region, the experimental designs will be performed by building of fusion recombinant and site-directed mutagenesis. A series of Tc-AFO fusion recombinant were used to found key area on bolt region by totally or partly replaced by different bolt regions origin from other family proteins. Key site of key area on bolt region and main type of amino acid at key site were confirmed using site-directed mutagenesis methods. A visual method basis of AFB1-aptamer cross-linked hydrogel was used to detect varies mutants enzymatic preperties and the mechanism of bolt region on substrate binding Tc-AFO was revealed by CD and molecular modeling software. The results of this study could help us to reveal the mechanism of bolt region on the catalytic preperties of Tc-AFO and provide theoretical and molecular basis for rational design.