茶氨酸对映体的酶法拆分及对阿尔茨海默的调控

Theanine has important physiological and biochemical functions and health and pharmacological effects, and there have been L-theanine and D-theanine as enantiomers. In order to separate the enantiomers and study their effects on human health, based on our previously established micellar electrokinetic chromatography (MEKC) technique on quantitative analysis of theanine, a novel method is developed for chiral theaine separation by addition of chiral selector. Then, electrospinning technique is used to immobilize aminoacylase from Aspergillus oryzae, and obtain reactive enzymatic nanofibrous membrane with high effective-enzyme-loading, good stability and easy separation of the products. Immobilized-enzyme catalytic system is set up with its enzymatic properties and kinetic characteristics studied, and an ideal separation of theanine enantiomers is realized by immobilized aminoacylase. After that, prepared 293T cells and Drosophila Alzheimer's model are used to explore the effects and mechanisms of L-theanine and D-theanine on the Alzheimer disease, respectively, providing research foundation for the enrichment of chiral theanine enzymatic resolutions, the further revealing of health and pharmacological effects of theanine, and the development of our nation-characterized tea industry. At the same time, our project also has great significance in promoting deep processing of agricultural products and in exploring natural product use.

茶氨酸具有重要的生理生化功能和保健药理功效，同时存在L型和D型两种对映体。为拆分其对映体并分别研究它们与人体健康的关系，项目以前期建立的胶束电动毛细管电泳(MEKC)技术定性定量分析茶氨酸的方法为基础，通过添加手性试剂首先建立L/D-茶氨酸分离分析的新方法。然后利用静电纺丝技术，对米曲霉氨基酰化酶进行固定化，获得有效载酶量高、稳定性好，易于实现产物分离的纳米纤维活性酶膜。确立固定化酶催化反应体系，并探讨其酶学性质和动力学特性，实现固定化氨基酰化酶对茶氨酸对映体的高效拆分。再利用已有的293T细胞以及果蝇阿尔茨海默疾病模型，探讨L/D-茶氨酸对映体对阿尔茨海默的作用效果与机制，为丰富茶氨酸对映体酶法拆分理论，揭示茶氨酸的保健、药理功效，发展民族特色茶产业提供研究基础。同时对推动农产品深加工产业发展，开发天然产物的利用具有重大意义。

项目对源于米曲霉3042菌株的氨基酰化酶进行固定化，实现了茶氨酸L/D不同构型对映体的酶法拆分，并以果蝇、A549肺癌细胞为对象，研究了茶氨酸的生理活性功能及作用机制，对开发茶氨酸的保健活性和药理功效具有指导意义。实验首先以2,4-二硝基氟苯（DNFB）为衍生化试剂，以β-环糊精（β-CD）为手性拆分试剂，建立了毛细管电泳技术定性定量分析茶氨酸对映体的方法。进而对米曲霉氨基酰化酶进行静电纺丝固定化，获得以京尼平为交联剂，活力保留为77.8 %的聚乙烯醇（PVA）纳米复合酶膜，实现茶氨酸对映体的酶法拆分；同时为了克服固定化酶的空间位阻效应，采用3-氨丙基三乙氧基硅烷（APTES），对磁性四氧化三铁纳米颗粒进行氨基接枝，获得稳定性好、活力回收高的米曲霉氨基酰化酶固定化酶。最后以果蝇为模式动物，研究了茶氨酸对果蝇的运动能力、生育能力、饥饿耐受能力、脱水耐受能力和254nm紫外辐照耐受能力的显著影响效果；并采用MTT法以及流式细胞术，检测茶氨酸对A549肺癌细胞生长的影响，通过Western blot蛋白电泳法分析MAPK信号通路内关键蛋白的表达情况，揭示茶氨酸的作用机制可能与抑制p38、ERK、JNK信号通路有关。