Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls
✍ Scribed by William S. Borneman; Roy D. Hartley; David S. Himmelsbach; Lars G. Ljungdahl
- Publisher
- Elsevier Science
- Year
- 1990
- Tongue
- English
- Weight
- 458 KB
- Volume
- 190
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Cell walls of Coastal Bermuda grass (Cynodon ductylon) were treated with polysaccharide hydrolases to release O-[6-0-(truns-p-coumaroyl)-a-D-arabinofuranosyl]-(1+3)-0-B-D-xylopyranosyl-(1+4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 'H NMR and was used as substrate for a sensitive assay of tmns-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimaetix
MC-2. The tmns-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. V,, was determined to be 1.17 pmol min-' mg-' and K,,, 13.2 pM at pH 7.5 and 30°C.