Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2.
An extracellular p-coumaroyl esterase produced by the anaerobic fungus Neocallimastix strain MC-2 released p-coumaroyl groups from 0-[5-0-((E)-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-0-beta -D-xylopyranosyl-(1----4)-D-xylopyranose (PAXX). The esterase was purified 121-fold from culture medium in successive steps involving ultrafiltration column chromatography on S-sepharose and hydroxylapatite, isoelectric focusing, and gel filtration. The native enzyme had an apparent mass of 11 kDa under nondenaturing conditions and a mass of 5.8 kDa under denaturing conditions, suggesting that the enzyme may exist as a dimer. The isoelectric point was 4.7, and the pH optimum was 7.2. The purified esterase had 100 times more activity towards PAXX than towards the analogous feruloyl ester (FAXX). The apparent Km and Vmax of the purified p-coumaroyl esterase for PAXX at pH 7.2 and 40 degrees C were 19.4 microM and 5.1 microM min(-1), respectively. p-Coumaroyl tetrasaccharides isolated from plant cell walls were hydrolyzed at rates similar to that for PAXX, whereas a dimer of PAXX was hydrolyzed at a rate 20-fold lower, yielding 4,4'-dihydroxy-alpha-truxillic acid as an end product. Ethyl and methyl p-coumarates were hydrolyzed at very slow rates, if at all. The purified esterase released p-coumaroyl groups from finely, but not coarsely, ground plant cell walls, and this activity was enhanced by the addition of xylanase and other cell wall-degrading enzymes.
Borneman
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Richard B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Dept. of Agriculture, Athens, Georgia 30613.
Ljungdahl
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Hartley
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Akin
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D000693:Anaerobiosis; D000818:Animals; D002240:Carbohydrate Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008411:Chytridiomycota / Q000201:enzymology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D012417:Rumen / Q000382:microbiology; D013379:Substrate Specificity; D014312:Trisaccharides / Q000378:metabolism
D014312:Trisaccharides; C066379:O-(5-O-(4-coumaroyl)-alpha-arabinofuranosyl)-(1-3)-O-beta-xylopyranosyl-(1-4)-xylopyranose; C064239:O-(5-O-(feruloyl)-alpha-arabinofuranosyl)-(1-3)-O-beta-xylopyranosyl-(1-4)-xylopyranose; D002265:Carboxylic Ester Hydrolases; C067271:trans-4-coumaroyl esterase