Differentiation of feruloyl esterases on synthetic substrates in alpha-arabinofuranosidase-coupled and ultraviolet-spectrophotometric assays.
4-Nitrophenyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-alpha-L-arabinofuranoside, synthesized by our group (M. Mastihubová, J. Szemesová, and P. Biely), were found to be suitable substrates for determination of activity of feruloyl esterases (FeEs) exhibiting affinity for 5-O- and 2-O-feruloylated alpha-L-arabinofuranosyl residues. One assay is based on coupling the FeE-catalyzed formation of 4-nitrophenyl alpha-L-arabinofuranoside with its efficient hydrolysis by alpha-L-arabinofuranosidase to release 4-nitrophenol. An alternative assay explores the difference in the molar absorbances at 340 nm of the substrate (ferulic acid esters) and the reaction products, which are (1) free ferulic acid and 4-nitrophenyl alpha-L-arabinofuranoside in samples free of alpha-L-arabinofuranosidase and (2) ferulic acid, 4-nitrophenyl alpha-L-arabinofuranoside, and/or 4-nitrophenol in samples containing alpha-L-arabinofuranosidase. The new substrates represent convenient tools to differentiate FeEs on the basis of substrate specificity.
Biely
Peter
P
Institute of Chemistry, Slovak Academy of Sciences, Dúbravska Cesta 9, 84238 Bratislava, Slovakia. chempbsa@savba.sk
Mastihubová
Maria
M
van Zyl
Willem H
WH
Prior
Bernard A
BA
D001089:Arabinose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001234:Aspergillus niger / Q000201:enzymology; D001236:Aspergillus oryzae / Q000201:enzymology; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002855:Chromatography, Thin Layer; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009596:Nitrophenols / Q000032:analysis / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013379:Substrate Specificity
D009596:Nitrophenols; D001089:Arabinose; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase; C024836:4-nitrophenol