Spectrophotometric and Fluorimetric High-Throughput Assays for Phenolic Acid Decarboxylase

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 (K_M and V_max ) of the purified enzyme for p-coumaric-, caffeic- and ferulic acid. Substrate inhibition was shown for caffeic acid.

Keywords: Bacillus subtilis; activity assays; fluorimetry; phenolic acid decarboxylase; spectroscopy.