ASSAY FOR TRANS-PARA-COUMAROYL ESTERASE USING A SPECIFIC SUBSTRATE FROM PLANT-CELL WALLS

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-α-l-arabinofuranosyl]-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 μmol min-1 mg-1 and Km 13.2 μm at pH 7.5 and 30°C. © 1990.