耐盐氨基酸脱氢酶的抗逆机制研究与元件优化组装

Oxidoreductases which can efficiently catalyze asymmetric reduction in non-aqueous media for chiral compounds are significant for both scientific research and biomanufacturing. Organic solvent tolerant amino acid dehydrogenases are challenge for the short of natural resource and difficult to be obtained by protein modification. This project offers a novel strategy for studying the organic solvent tolerance of halotolerant amino acid dehydrogenase based on defining of resistant structure motifs, and mechanism study from the aspects of dynamic interaction networks and energy transfer analysis. First, the combination of structure alignment and analysis of amino acid residues energy response to media will be applied for the definition of resistant motifs. Structural foundation for organic solvent tolerance will be revealed. Second, key amino acids and the component of dynamic modules will be investigated based on dynamic amino acid interaction networks and dynamic simulation. The assembly disciplinary and dynamic cooperation of motifs will be revealed. Third, optimization of resistant motifs and follows with designing of spatial arrangement and linkers for the assemble of motifs to new enzyme. Enzymes will be evaluated from the aspects of structural rationality, interaction network compatibility, and dynamic harmony. Novel amino acid dehydrogenases will be obtained by experimental verification of catalytic efficiency and organic solvent tolerance. This project result will provide access to reveal the resistance mechanism of halotolerant amino acid dehydrogenase by the new methodologies. From characterization and recognition to design and innovation will also provide a new strategy for deep mining of resistant motifs resource from extremozymes and lay foundation for designing and assembling of robust oxidoreductases.

可在非水相体系高效催化不对称还原反应制备手性化合物的氧化还原酶具有重要科学意义与工业应用前景。针对耐有机溶剂的氨基酸脱氢酶难以从自然界筛选或通过局部改造获得的难题,提出解构耐盐氨基酸脱氢酶获得抗逆元件，基于氨基酸残基动态相互作用网络和能量传递分析揭示其抗逆机制，进而优化设计抗逆元件，组装获得抗逆酶的新思路。首先，基于结构分析和氨基酸残基对溶剂体系的能量响应，界定抗逆元件，揭示抗逆机制的结构基础；第二，基于动态相互作用网络和分子动力学模拟分析能量传递路径，界定关键氨基酸节点和动态模块组成，解析元件组装与动态协同机制；第三,优化抗逆元件，设计元件空间排布和组装接头，考察新酶结构合理性、网络相容性和动态协调性，实验验证催化效率与抗逆性能，获得新型抗逆酶。本研究成果将从“认识表征”到“设计创新”，揭示耐盐氨基酸脱氢酶的抗逆机制，为挖掘极端酶抗逆元件提供借鉴，为新型抗逆氧化还原酶的设计组装奠定基础。

可在非水相体系高效催化不对称还原反应制备手性化合物的氧化还原酶具有重要科学意义与工业应用前景。项目从“认识表征”到“设计创新”，揭示耐盐氨基酸脱氢酶的抗逆机制，为挖掘极端酶抗逆元件提供借鉴，为新型抗逆氧化还原酶的设计组装奠定基础。.基于极端微生物的基因组挖掘与分析，筛选获得了17种嗜热、嗜盐的氨基酸脱氢酶(AaDH)。来源于Natranaerobius thermophilus的AaDH催化还原胺化制备L-高苯丙氨酸的体系，最适温度为70 ℃。在含30%的二甲亚砜反应体系中，催化活性是原始活性的99.2%。研究表明，该耐盐酶具有较好的耐热、耐有机溶剂等抗逆性能。.基于嗜盐苹果酸脱氢酶(MDH)和嗜热谷氨酸脱氢酶的模块组装获得新型AaDH。通过结构划分和氨基酸残基对溶剂体系的能量响应，界定获得抗逆元件库。对组装酶的结构相容性和动态协同性进行评估和优化，筛选获得新型氨基酸脱氢酶AaDH05。分析得知不同盐浓度下AaDH05的氢键和盐桥数量均大于亲本氨基酸脱氢酶。在90 ℃高温下孵育6h仍可保持47%的活性。.通过抗逆模块组装并提升元件的结构相容性，优化相互作用网络，获得新型抗逆苹果酸脱氢酶。实验结果表明MDH03在甲苯、丙酮等多种有机溶剂中，酶活显著提高。在含离子液体体系中，酶活性是水相的1.78倍。氨基酸相互作用网络分析表明，MDH03具有高密度盐桥网络。.基于静电相互作用、氢键等多层次作用力固定化酶，构建了氧化石墨烯-聚乙烯亚胺(GO-PEI)和纳米无机材料(Cu3(PO4)2)固定化酶体系。利用离子液体修饰碳基复合材料和有机-无机杂化材料构建载体，用于固定化抗逆氨基酸脱氢酶，可进一步用于有机相体系生物催化和生物电催化。