基于酶活指纹分析的酰胺酶底物特异性与对映选择性规律研究

Amidases are promising synthesis tools for chiral carboxylic acids and amide derivatives. Due to the lack of deep understanding of substrate specificity and enantioselectivity law, the development, application and reconstruction of amidase are severely restricted. This project focused on the new perspective of activity fingerprint analysis, to reveal the substrate specificity and enantioselectivity laws of amidase. By establishing typical amidase library, structure library and varied substituent group library, together with enzyme activity fingerprint analysis system with high resolution, the fingerprint map of substrate specificity and enantioselectivity can be obtained. The homologous modeling and molecular docking technologies will be further applied to study the effects of key amino acid sites of amidase structure on the activity and enantioselectivity to different substrates, to accurately position the amino acid residues and functional domains that affect the amidases' substrate recognition, stereoselective recognition and the acyl intermediate state formation, which can finally reveal the molecular mechanisms of its substrate specificity and enantioselectivity. The whole research plays an important role in the field of amidases' rational selection, design and reconstruction, which is benefit to the development of enantioselective biocatalytic synthesis methods of amidase.

酰胺酶是合成手性羧酸和酰胺衍生物的重要工具酶，但由于对其底物特异性和对映选择性规律认识的匮乏，酰胺酶开发、应用和改造受到严重制约。本项目尝试从酶活指纹分析的全新视角，揭示酰胺酶的底物特异性和对映选择性规律。通过构建典型的酰胺酶酶库和结构、取代基多样的底物文库，建立高分辨率酰胺酶酶活力指纹分析体系，获得酰胺酶底物特异性和对映选择性指纹图谱。以特征指纹谱为依据，进一步运用同源模建和分子对接技术，研究各酰胺酶结构对不同底物活性和对映选择性改变的关键位点，准确定位影响酰胺酶底物识别、酰基中间态形成、立体选择性识别的氨基酸残基和结构功能域，揭示酰胺酶底物特异性和对映选择性规律。本项目研究对于实现酰胺酶的理性选择、设计和改造，发展酰胺酶对映选择性生物催化合成方法学均具有重要意义。

酰胺酶能够催化酰胺水解生成相应的羧酸和氨，且在羟胺存在的情况下能催化酰基转移生成相应的氧肟酸和氨。酰胺酶的作用底物谱广，立体选择性严格，可水解非天然脂肪族酰胺，芳香族及杂环类酰胺。本研究利用酰胺酶的高度保守序列，通过基因组挖掘、HiTAIL-PCR等方法，从Brevibacterium epidermidis ZJB-07021、Klebsiella pneumoniae和Delftia tsuruhatensis ZJB-05174等基因组中克隆、表达了16个酰胺酶，其中6个为首次报道的新酰胺酶，构建了新颖的酰胺酶文库。筛选了32种具有代表性的脂肪族、芳香族和杂环族酰胺化合物，其中外消旋化合物8种，构建了特征性的酰胺酶底物文库，并建立相应的实时酶活指纹分析方法。考察了腈水解酶家族和酰胺酶标签家族酰胺酶的底物特异性、催化动力学和立体选择性，对酰胺酶底物特异性和立体选择性差异产生的分子机制有了更为深入的认识和理解。

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