Spectrophotometric and Fluorimetric High‐Throughput Assays for Phenolic Acid Decarboxylase

• Published in: Chembiochem : a European journal of chemical biology Vol. 24; no. 16; pp. e202300207 - n/a
• Main Authors: Terholsen, Henrik, Myrtollari, Kamela, Larva, Mirna, Möller, Christina, Taden, Andreas, Kourist, Robert, Bornscheuer, Uwe T., Kracher, Daniel
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 15.08.2023
• Subjects: activity assays; Assaying; Bacillus subtilis; Caffeic acid; Carbon dioxide; Decarboxylation; Epoxy coatings; Ferulic acid; Fluorimetry; Gas chromatography; High performance liquid chromatography; Hydroxycinnamic acid; Liquid chromatography; Lysates; Mass spectrometry; Mass spectroscopy; NMR; Nuclear magnetic resonance; Phenolic acid decarboxylase; Phenolic acids; Phenols; Photometry; Spectrophotometry; spectroscopy; Styrenes; Substrate inhibition; fluorimetry; Bacillus subtilis; spectroscopy; activity assays; phenolic acid decarboxylase
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202300207

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. The enzymatic decarboxylation of biomass‐derived hydroxycinnamic acids was measured in two independent assays that follow fluorescence and absorbance changes during the reaction. Both assays are suitable for high‐throughput measurements in cell lysates or with whole cells and greatly extend the currently available toolbox to quantify decarboxylation reactions.