氨基酸转氨/脱羧双功能酶克隆表达、功能鉴定及其机理研究

Clone,expression,functional characterization,and mechanism of amino acid transamination/decarboxylation bifunctional enzymes

大肠杆菌来源的L-天冬氨酸转氨酶(L-AspAT)是一种具有严格立体选择性、高效一步合成特性的生物催化剂,研究该酶对于手性胺化合物的生物合成至关重要。该研究将野生大肠杆菌来源的L-天冬氨酸转氨酶基因克隆表达至大肠杆菌工程菌BL21(DE3)中高效表达、分离纯化出该酶,并对该酶催化的反应进行功能鉴定。结果表明该酶是一种具有催化氨基酸转氨和二羧酸底物非氧化脱羧2种催化能力的双功能酶,该研究对其催化的2种反应酶学性质分别进行了研究,同时对酶催化机理进行了探讨。检测反应液中底物和产物的浓度随时间的变化,发现反应体系中先进行转氨反应,待反应液中脱羧底物浓度足够多时才开始进行脱羧反应,脱羧反应同时又促进转氨反应,2种反应相互影响,互相促进。同时将L-AspAT分别与转氨和脱羧的底物小分子对接,结果显示,L-天冬氨酸转氨酶对于谷氨酸和酮戊二酸而言,对接获得的结合位点是不同的,该双功能酶存在2个催化活性中心用来催化不同的反应。实验发现转氨反应的最适温度为35℃,脱羧反应的最适温度为37℃。2种反应的最适pH均为8,5 mmol/L的Cu^(2+)和Mg^(2+)能够促进转氨反应,5 mmol/L的Ni^(2+)能够促进脱羧反应。

L-Aspartate transaminase(L-AspAT)from E.coli is an efficient enzyme,and its advantages include stereoselectivity and one-step synthesis,amongst others.The study of L-AspAT was essential for chiral amine biosynthesis,and in the wild E.coli L-AspAT was expressed in an E.coli engineering stain BL21(DE3)with high efficiency.The enzyme was purified to allow the identification of its catalytic function.The results showed that the enzyme was bifunctional with two catalytic abilities:the transamination of amino acids and a non-oxidative decarboxylation of dicarboxylic acid substrates.Transamination was first carried out in the reaction system during the reaction process.Then the decarboxylation reaction was started,and when the concentration of decarboxylation substrate in the reaction solution was sufficient,the two reactions influenced and promoted each other.With regard to the docking of L-AspAT with small molecules of the substrates of ammonia transfer and decarboxylation respectively,the molecular docking results showed a difference between the binding sites of L-AspAT for glutamate and ketoglutarate,and the bifunctional enzyme had two active catalytic centers that catalyze different reactions.The catalytic mechanism was also studied and it was found that the enzyme-catalyzed the enzymatic properties of the two reactions.The study also found that the optimum temperature of the transamination was 35℃and that of the decarboxylation was 37℃.The optimum pH of both reactions was 8.5 mmol/L at which level Cu^(2+) and Mg^(2+) can promote transamination,and 5 mmol/L Ni^(2+) can promote decarboxylation.