Differentiation of feruloyl esterases on synthetic substrates in α-arabinofuranosidase-coupled and ultraviolet-spectrophotometric assays

Abstract

4-Nitrophenyl 5- O- trans-feruloyl-α- l-arabinofuranoside and 4-nitrophenyl 2- O- trans-feruloyl-α- 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 α- l-arabinofuranosyl residues. One assay is based on coupling the FeE-catalyzed formation of 4-nitrophenyl α- l-arabinofuranoside with its efficient hydrolysis by α- 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 α- l-arabinofuranoside in samples free of α- l-arabinofuranosidase and (2) ferulic acid, 4-nitrophenyl α- l-arabinofuranoside, and/or 4-nitrophenol in samples containing α- l-arabinofuranosidase. The new substrates represent convenient tools to differentiate FeEs on the basis of substrate specificity.