同源模建结合分子对接指导的新酶发现策略

For sustainable development of chemical industry, improving or replacing the traditional chemical process by biocatalysis has become the inevitable trend. Discovery of the ideal and novel enzymes possessing high catalytic activity, high substrate specificity and good stability, will be the fundamental premise of biocatalysis. The public databases hold a large number of the uncharacterized genes, the proteins with uncertain, unknown, or incorrectly annotated functions, and the unfunctional structures. Retrieving and mining the novel and ideal biocatalysts from these unexploited resources, not only has great significance to theory research, but also possesses great potential for industrial application. To date, three strategies have been developed for discovery of novel enzymes or functional assignment of uncharacterized enzymes: direct cloning from genome, key motifs for function guides in silico enzyme identification, and structure-guided discovery of novel enzymes. The existing methods have some shortcomings, such as strong blindness, large experimental workload, and large difficulty. In this project, aiming to mine the novel enzymes with high catalytic activity for interesting substrates targetly, effectively and accurately, we will rationally select the interesting enzymes from the structure level by introducing the technologies of homology modeling and molecular docking, and we will finally develop a new approach for discovery of novel enzymes guided by homology modeling combined with molecular docking. We will choose the α-L-rhamnosidases as the research model, and aid to find novel α-L-rhamnosidases possessing high catalytic activity, wide substrate spectrum, and independent intellectual property right.

利用生物催化技术改造或取代传统化工工艺已成为化学工业可持续发展的必然趋势。开发催化活性高、专一性强以及稳定性高的理想酶制剂是生物催化的基本前提。针对公共数据库中存在大量未表征的新基因序列、功能被错误注释的蛋白序列以及催化功能未知的蛋白结构，从中挖掘开发出新型酶制剂，不仅具有重要的理论意义，也具有良好的工业应用前景。目前新酶发现策略大致有三种：数据库检索直接克隆法，关键基序指导的新酶发现以及三级结构指导的新酶发现。现有方法存在盲目性强、实验工作量大、实施难度大等缺点，本研究立足于高效、精确、靶向挖掘对特定底物（酶促反应）催化活性高的新型酶资源，引入同源模建与分子对接技术在三级结构层面指导优选目标酶基因，旨在建立同源模建结合分子对接指导的新酶发现策略。具体选择α-L-鼠李糖苷酶作为研究模型，力求开发出催化活性高、底物谱广、具有自主知识产权的新型α-L-鼠李糖苷酶，并对生物催化应用进行初步研究。

开发催化活性高、专一性强以及稳定性高的理想酶制剂是生物催化的基本前提。现有新酶发现方法存在盲目性强、实验工作量大、实施难度大等缺点，本项目立足于高效、精确、靶向挖掘对特定底物催化活性高的新型酶资源，创新性引入同源模建与分子对接方法在三级结构层面指导优选目标酶，旨在建立同源模建结合分子对接指导的新酶发现策略。本项目具体选择α-L-鼠李糖苷酶作为研究模型，从CAZy数据库检索α-L-鼠李糖苷酶Rha78序列，依据目标酶来源菌株的生长环境和生理代谢特性以及目标酶氨基酸序列特点初选了250条α-L-鼠李糖苷酶Rha78序列，随后利用同源模建成功构建232个高可信度三级结构模型，最后对目标酶与特定底物（合成底物pNPR与天然底物芦丁）进行分子对接，基于结合自由能排序并优选目标酶，进行异源表达酶学性质验证，本项目已部分建立同源模建结合分子对接指导的新酶发现策略。此外，本项目已建立了基因组信息挖掘新酶发现策略与保守氨基酸基序驱动的宏基因组学新酶发现策略，将有助于快速、靶向挖掘细菌源新型酶资源；本项目亦获得了一系列催化活性高、底物谱广、稳定性高的新型细菌源α-L-鼠李糖苷酶资源，具有广泛的生物催化应用潜力。

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