Purification and properties of a feruloyl esterase involved in lignocellulose degradation by Aureobasidium pullulans.
The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60 degrees C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 micro mol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-[5-O-[(E)-feruloyl]-alpha-L-arabinofuranosyl]-(1,3)-O-beta-D-xylopyranosyl-(1,4)-D-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (k(cat)/K(m)) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.
Rumbold
Karl
K
Department of Microbiology, University of Stellenbosch, 7602 Matieland, South Africa.
Biely
Peter
P
Mastihubová
Maria
M
Gudelj
Marinka
M
Gübitz
Georg
G
Robra
Karl-Heinz
KH
Prior
Bernard A
BA
D001203:Ascomycota / Q000201:enzymology* / Q000254:growth & development; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D005285:Fermentation; D007700:Kinetics; D008031:Lignin / Q000378:metabolism*; D013379:Substrate Specificity; D014462:Ultrafiltration
C036909:lignocellulose; D002482:Cellulose; D008031:Lignin; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase