Abstract：
Biocatalytic decarboxylation of hydroxycinnamic acids yields phenolic styrenes, which are important precursors for antioxidants, epoxy coatings, adhesives and other polymeric materials. Bacillus subtilis decarboxylase (BsPAD) is a cofactor-independent enzyme that catalyzes the cleavage of carbon dioxide from p-coumaric-, caffeic-, and ferulic acid with high catalytic efficiency. Real-time spectroscopic assays for decarboxylase reactions remove the necessity of extensive sample workup, which is required for HPLC, mass spectrometry, gas chromatography, or NMR methods. This work presents two robust and sensitive assays based on photometry and fluorimetry that allow decarboxylation reactions to be followed with high sensitivity while avoiding product extraction and long analysis times. Optimized assay procedures were used to measure BsPAD activity in cell lysates and to determine the kinetic constants (KM and Vmax ) of the purified enzyme for p-coumaric-, caffeic- and ferulic acid. Substrate inhibition was shown for caffeic acid.
摘要：
羟基肉桂酸的生物催化脱羧产生酚醛苯乙烯，它们是抗氧化剂的重要前体，环氧涂料,粘合剂和其他聚合物材料。枯草芽孢杆菌脱羧酶（BsPAD）是一种不依赖辅因子的酶，可催化二氧化碳从对香豆酸，caffeic-,和阿魏酸具有较高的催化效率。脱羧酶反应的实时光谱测定消除了大量样品后处理的必要性，这是HPLC所需要的，质谱,气相色谱法,或NMR方法。这项工作提出了两种基于光度法和荧光法的稳健而灵敏的测定法，可以高灵敏度地进行脱羧反应，同时避免了产品提取和分析时间长。优化的测定方法用于测量细胞裂解物中的BsPAD活性，并确定纯化酶对香豆酸的动力学常数（KM和Vmax）。咖啡酸和阿魏酸。咖啡酸显示底物抑制。
