淀粉蔗糖酶AcAS的工作机理研究与稳定性改造

目的:对新发现的一种新型淀粉蔗糖酶AcAS的结构功能进行深入讨论。方法:用同源模建方法构建AcAS的三维结构;用高斯网络模型和各项异性网络模型,对其功能型运动和工作机理进行预测;利用迭代高斯网络模型方法对其去折叠路径进行预测;根据去折叠路径预测及折叠自由能计算结果设计突变体。结果:模建结果表明,AcAS结构与淀粉蔗糖酶NpAS的结构更相似;AcAS有扭转运动的趋势,其中AcAS的N/C结构域运动性较强,而催化核心的运动性较弱;根据去折叠路径预测,发现N、B和C结构域较易去折叠;通过自由能计算,针对上述3个结构域设计了5株突变体。结论:构建了AcAS的三维结构模型并根据模型探讨了其工作机理;根据去折叠路径预测及折叠自由能计算结果,对AcAS的稳定性改造提出了有益的建议。

Study on the Working Mechanism of Arthrobacter chlorophenolicus Amylosucrase and Stability Engineering

Objective： The structure and functions of the newly identified Arthrobacter chlorophenolicus amylosucrase（AcAS） was thoroughly investigated. Methods： The structural model of AcAS was predicted by homology modeling; Gaussian network model and anisotropic network model were used to predict its functional motions amt the working mechanism： the unfolding pathway of AcAS was predicled by using the Iterative Gaussian network model; mutants were designed for better stability according to results of unfolding pathway and folding free energy predictions. Results： The result of the modeling shows that the structure of AcAS is more similar with that of NpAS： AcAS shows intrinsic twisting motions, wherein the motions of N/C-domains are stronger, while the movement of the catalytic： core is weak; unfolding pathway prediction shows N, B, and C domains of AcAS are easier to fold than the other domains; on the basis of free energy calculation, five nutants were designed against the above three domains. Conclusion： The structural model of AcAS was constructed, and the working mechanism of AcAS was explored according to the model; several mutants were designed fur better stability according to results of unfolding pathway and fohting free energy predictions, which might lay the foundation for the stability engineering of AcAS.