Acyl-ACPs的规模化合成

脂酰-酰基载体蛋白(fatty acyl-acyl carrier protein, acyl-ACP)是多种生物合成途径中的酰基供体。因供给限制,体外研究常用类似物acyl-CoA替代,而CoA部分和ACP有较大差异,限制了相关酶对底物识别的认识。因此稳定获得大量acyl-ACP是体外研究相关酶的催化机制及其代谢途径的关键。研究以holo-ACP和C4~C18链长脂肪酸为底物,在哈氏弧菌acyl-ACP合成酶(Vibrio harveyi acyl-ACP synthetase, VhAasS)催化下合成不同碳链长度的acyl-ACP;通过高效液相色谱(HPLC)方法,确定不同碳链长度acyl-ACP的合成产率。结果表明:碳链为C4~C14的acyl-ACP产率均高于90.0%,16:0-ACP产率为85.9%,18:1-ACP产率仅为25.7%。通过加入Li ⁺优化反应体系,16:0-ACP、18:1-ACP的产率达90.0%。进一步优化扩大反应体系可稳定获得20mg以上acyl-ACP;最后,把合成的acyl-ACP应用到甘油-3-磷酸酰基转移酶催化的反应体系中。不同链长acyl-ACP的规模化合成研究,为体外研究相关酶的催化机制提供重要基础。

Large-Scale Synthesis of Acyl-ACPs

Acyl-acyl carrier proteins (acyl-ACPs) are substrates for many biosynthesis pathways. However, acyl-ACP is substituted by acyl-CoA for studies in vitro as a result of supply restriction, which causes many questions in enzymatic analysis. Thus, obtaining large scale of acyl-ACP steadily is very important to study the related enzymes and metabolic pathways in vitro. acyl-ACP synthetase catalyze the conversion of holo-ACP using fatty acid as acyl donor in vitro while no productivity has been reported before. Here an acyl-ACP synthetase from Vibrio harveyi was used to catalyze the synthesis of (C4~C18) with holo-ACP, and the yield of acyl-ACP was confirmed by high performance liquid chromatography (HPLC). The results indicated that the yields of medium chains (C4~C14) acyl-ACPs were more than 90.0% while the long chain yields of 16:0-ACP and 18:1-ACP were 85.9% and 25.7%, respectively. Via introducing Li ⁺ to the reaction system, the yield of long chain acyl-ACPs were elevated above 90.0%。Then the reaction parameters were optimized in the enlarged reaction system, and more than 20mg acyl-ACPs were steadily obtained. Additionally, two species of holo-ACP were used to validate the versatility of the reaction system. Finally the acyl-ACP activity was conformed using a glycerol-3-phosphate acyltransferase. The synthesis of different chain acyl-ACPs are of great significance for research of the catalysis mechanism of related enzymes.