Journal of Biotechnology 127 (2007) 235–243 Substrate and positional specificity of feruloyl esterases for monoferuloylated and monoacetylated 4-nitrophenyl glycosides Vladim´ ır Puchart a , M´ aria Vrˇ sansk´ a a , M´ aria Mastihubov´ a a , Evangelos Topakas b , Christina Vafiadi b , Craig B. Faulds c , Maija Tenkanen d , Paul Christakopoulos b , Peter Biely a,∗ a Institute of Chemistry, Slovak Academy of Sciences, D´ ubravsk´ a cesta 9, SK-845 38 Bratislava, Slovak Republic b National Technical University of Athens, Department of Chemical Engineering, Zografou Campus, Athens 15780, Greece c Institute of Food Research, Sustainability of the Food Chain Exploitation Platform, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom d University of Helsinki, Department of Applied Chemistry and Microbiology, P.O. Box 27, FIN-00014 Helsinki, Finland Received 15 March 2006; received in revised form 20 June 2006; accepted 26 June 2006 Abstract 4-Nitrophenyl glycosides of 2-, 3-, and 5-O-(E)-feruloyl- and 2- and 5-O-acetyl--l-arabinofuranosides and of 2-, 3-, and 4-O- (E)-feruloyl- and 2-, 3- and 4-O-acetyl--d-xylopyranosides, compounds mimicking natural substrates, were used to investigate substrate and positional specificity of type-A, -B, and -C feruloyl esterases. All the feruloyl esterases behave as true feruloyl esterases showing negligible activity on sugar acetates. Type-A enzymes, represented by AnFaeA from Aspergillus niger and FoFaeII from Fusarium oxysporum, are specialized for deferuloylation of primary hydroxyl groups, with a very strong preference for hydrolyzing 5-O-feruloyl--l-arabinofuranoside. On the contrary, type-B and -C feruloyl esterases, represented by FoFaeI from F. oxysporum and TsFaeC from Talaromyces stipitatus, acted on almost all ferulates with exception of 4- and 3-O-feruloyl- -d-xylopyranoside. 5-O-Feruloyl--l-arabinofuranoside was the best substrate for both TsFaeC and FoFaeI, although catalytic efficiency of the latter enzyme toward 2-O-feruloyl--l-arabinofuranoside was comparable. In comparison with acetates, the corresponding ferulates served as poor substrates for the carbohydrate esterase family 1 feruloyl esterase from Aspergillus oryzae. The enzyme hydrolyzed all -l-arabinofuranoside and -d-xylopyranoside acetates. It behaved as a non-specific acetyl esterase rather than a feruloyl esterase, with a preference for 2-O-acetyl--d-xylopyranoside. © 2006 Elsevier B.V. All rights reserved. Keywords: Feruloyl/acetyl esterase; Substrate and positional specificity; -l-Arabinofuranoside and -d-xylopyranoside acetates and ferulates ∗ Corresponding author. Tel.: +42 1 2 59410 275; fax: +42 1 2 59410 222. E-mail address: chempbsa@savba.sk (P. Biely). 1. Introduction Ferulic acid esters have been found in several plant cell wall hemicellulosic polysaccharides. In dicotyle- 0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.06.020