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Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls
W S Borneman1, R D Hartley, D S Himmelsbach
1Richard B. Russell Agricultural Research Center, U.S. Department of Agriculture, Athens, Georgia 30613.
Analytical Biochemistry
|October 1, 1990
Summary
Researchers isolated a complex sugar (PAXX) from grass cell walls and used it to assay an enzyme from anaerobic fungi. This method accurately quanties trans-p-coumaric acid release, revealing Michaelis-Menten kinetics.
Area of Science:
- Biochemistry
- Plant Cell Wall Biology
- Enzymology
Background:
- Coastal Bermuda grass (Cynodon dactylon) cell walls contain complex polysaccharides.
- Polysaccharide hydrolases can release valuable compounds from plant cell walls.
- Anaerobic fungi possess unique enzymatic capabilities for degrading plant biomass.
Purpose of the Study:
- To isolate and characterize a specific oligosaccharide (PAXX) from Coastal Bermuda grass cell walls.
- To develop a sensitive assay for trans-p-coumaroyl esterase activity using the isolated PAXX.
- To investigate the enzymatic hydrolysis of PAXX by culture filtrates of the anaerobic fungus Neocallimastix MC-2.
Main Methods:
- Polysaccharide hydrolase treatment of grass cell walls.
- Isolation of O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-beta-D-xylopyranosyl-(1----4)-D-xylopyranose (PAXX) via liquid chromatography.
- Purity determination of PAXX using 1H NMR.
- Enzymatic hydrolysis of PAXX by Neocallimastix MC-2 culture filtrates.
- Quantification of released trans-p-coumaric acid using reverse-phase HPLC.
- Kinetic analysis (Michaelis-Menten) of enzyme activity.
Main Results:
- PAXX was successfully isolated and purified (>95%) from Coastal Bermuda grass.
- A sensitive assay for trans-p-coumaroyl esterase was established, detecting as little as 100 ng of trans-p-coumaric acid.
- Neocallimastix MC-2 culture filtrates hydrolyzed PAXX, releasing trans-p-coumaric acid.
- The enzymatic hydrolysis followed Michaelis-Menten kinetics with Vmax = 1.17 µmol min⁻¹ mg⁻¹ and Km = 13.2 µM at pH 7.5 and 30°C.
Conclusions:
- PAXX is a suitable substrate for sensitive trans-p-coumaroyl esterase assays.
- Neocallimastix MC-2 possesses trans-p-coumaroyl esterase activity capable of hydrolyzing PAXX.
- The characterized enzyme kinetics provide valuable data for understanding plant cell wall degradation by anaerobic fungi.