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.