对香豆酰辅酶A酯生物合成及纯化方法

木质素是绿色植物生长发育所需的重要化合物之一。肉桂酰辅酶A还原酶(CCR)是在木质素合成过程中,催化苯丙烷类合成途径第1步的关键酶。本文以其底物之一——对香豆酰辅酶A酯为例,以现有的辅酶A酯合成纯化的文献报道为参考,介绍经过整合改进并不断试验而得到的CCR底物的合成与纯化方法。从大肠杆菌中提取出来的重组酶4-香豆酸辅酶A连接酶(4CL)用于合成CCR底物,这比从植物组织中提取更易获得。提纯后的4CL可以在-80 ℃下保存3、4个月。4CL反应后的产物在2个不同梯度下通过C18反相柱进一步纯化,并用高效液相色谱仪监测。在整个纯化过程中,所有用于装载对香豆酸和对香豆酰辅酶A酯的容器都用锡箔纸完全包裹起来。最后提纯出来的产物用质谱和核磁共振检测。检测的结果表明,提纯出来的辅酶A酯单一性非常好。 

Method of enzymatic synthesis and purification of p-coumaroyl-CoA

Lignin is one of the most important and abundant biomass compounds from green plants. Cinnamoyl-CoA Reductase( CCR) is a key enzyme which catalyzes the first step of the phenylpropanoid pathway in monolignol biosynthesis. Here we present the biosynthesis process of ρ-coumaryl-CoA as an example by the method, which we integrated and improved referring to existing biosynthesis and purification methods of CoA esters. The recombinant enzyme 4-coumarate:coenzyme A ligase (4CL) overexpressed in E. coli in vivo and extracted from E. coli for synthesis of the chemical, was easier to obtain than extract from plant tissues and was stable for several months when stored at 193 K. After being synthesized by 4CL, the product was purified by reverse phase C18 column and High Performance Liquid Chromatography ( HPLC) with two different gradients. During the entire purification process, from ρ-coumaric acid to ρ-coumaryl-CoA, the substance were all fully packed by foil. Structure was confirmed by Mass Spectrometry ( MS) and Nuclear Magnetic Resonance ( NMR) machine, and the result showed that the quality of this enzymatic uniform product was very good and could be applied to scientific researches.