Differentiation of feruloyl esterases on synthetic substrates in a-arabinofuranosidase-coupled and ultraviolet-spectrophotometric assays Peter Biely, a,b, * Maria Mastihubov a, a Willem H. van Zyl, b and Bernard A. Prior b a Institute of Chemistry, Slovak Academy of Sciences, D ubravska Cesta 9, 84238 Bratislava, Slovakia b Department of Microbiology, University of Stellenbosch, Private Bag X1, 7602 Matieland, South Africa Received 8 July 2002 Abstract 4-Nitrophenyl 5-O-trans-feruloyl-a-L -arabinofuranoside and 4-nitrophenyl 2-O-trans-feruloyl-a-L -arabinofuranoside, synthe- sizedbyourgroup(M.Mastihubov a,J.Szemesov a,andP.Biely),werefoundtobesuitablesubstratesfordeterminationofactivity of feruloyl esterases (FeEs) exhibiting affinity for 5-O-and2-O-feruloylated a-L -arabinofuranosyl residues. One assay is based on coupling the FeE-catalyzed formation of 4-nitrophenyl a-L -arabinofuranoside with its efficient hydrolysis by a-L -arabinofuranos- idase to release 4-nitrophenol. An alternative assay explores the difference in the molar absorbances at 340nm of the substrate (ferulicacidesters)andthereactionproducts,whichare(1)freeferulicacidand4-nitrophenyl a-L -arabinofuranosideinsamplesfree of a-L -arabinofuranosidaseand(2)ferulicacid,4-nitrophenyl a-L-arabinofuranoside,and/or4-nitrophenolinsamplescontaining a- L -arabinofuranosidase. The new substrates represent convenient tools to differentiate FeEs on the basis of substrate specificity. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Feruloyl esterase; Ferulic acid; a-L-Arabinofuranosidase; Enzyme assay; Chromogenic substrate; 4-Nitrophenyl-a-L-arabinofuranoside Hydroxycinnamic acids associated with plant hemi- celluloses play an important role in cell wall integrity and protection of plant tissues against digestion by plant-invading microorganisms [1–3]. The most abun- dant hydroxycinnamic acid is trans-ferulic acid, (E)-4- hydroxy-3-methoxycinnamicacid.Ferulicacid(FeA) 1 is usually esterified at position C-5 to a-L -arabinofurano- syl side chains in arabinoxylans, at position C-2 to a-L - arabinofuranosyl residues in arabinans, at position C-6 to b-galactopyranosyl residues in pectic substances and galactans [4], and at position C-4 to a-D -xylopyranosyl residues in xyloglucans [5]. The ferulic acid esterified with carbohydrates is found either nonsubstituted or linked to another esterified ferulic acid to form several types of diferuloyl bridges connecting two polysaccha- ride chains [3,6–8]. Carbohydrate esters of ferulic acid can also be involved in ether linkages with lignin com- ponents, thus providing the connection between lignin and hemicellulose [9]. Enzymes that can attack ester linkages between hy- droxycinnamic acids and carbohydrates in the process ofbiodegradationofplantcellwallshaveevolved.Since the first report on the occurrence of a Streptomyces olivochromogenes esterase releasing ferulic acid from wheat bran [10], esterases liberating hydroxycinnamic acids from plant cell walls or cleaving ester linkages of diferuloyl bridges have been recognized as common components of hemicellulolytic enzyme systems in a variety of microorganisms [11,12]. Feruloyl esterase (FeE) activity has also been reported to be endogenous to plants, e.g., in germinating barley [13], where it probably participates in the cell wall extension process. The growing interest in these esterases is due to their potentialapplicationsinbioconversionoflignocellulosic Analytical Biochemistry 311 (2002) 68–75 www.academicpress.com ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: +4212-5941-0222. E-mail address: chempbsa@savba.sk (P. Biely). 1 Abbreviations used: FeE, feruloyl esterase; FeA, ferulic acid; NPh- 5-Fe-Araf, 4-nitrophenyl 5-O-trans-feruloyl-a-L-arabinofuranoside; NPh-2-Fe-Araf, 4-nitrophenyl 2-O-trans-feruloyl-a-L-arabinofurano- side; NPh-Araf, 4-nitrophenyl a-L-arabinofuranoside; NPh-OH, 4- nitrophenol; DMSO, dimethyl sulfoxide. 0003-2697/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0003-2697(02)00401-3