Enzyme and Microbial Technology 33 (2003) 729–737 Purification and characterization of a Fusarium oxysporum feruloyl esterase (FoFAE-I) catalysing transesterification of phenolic acid esters Evangelos Topakas a , Haralambos Stamatis b , Maria Mastihubova c , Peter Biely c , Dimitris Kekos a , Basil J. Macris a , Paul Christakopoulos a,∗ a Biotechnology Laboratory, Chemical Engineering Department, National Technical University of Athens, 5 Iroon Polytechniou Str, Zografou Campus, Athens 15700, Greece b Biotechnology Laboratory, Biochemical Applications and Technologies Department, University of Ioannina, University Campus, Ioannina 45110, Greece c Institute of Chemistry, Slovak Academy of Sciences, Bratislava 84238, Slovak Republic Received 20 March 2003; accepted 29 June 2003 Abstract An extracellular feruloyl esterase (FoFAE-I) from the culture filtrates of Fusarium oxysporum F3 was purified to homogeneity by ion-exchange, hydrophobic interaction and gel filtration chromatographies. The protein corresponded to molecular mass and pI values of 31kDa and 9.5, respectively. The enzyme was optimally active at pH 7.0 and 55 ◦ C. The purified esterase was fully stable at pH 7.0–9.0 and temperature up to 30 ◦ C. Determination of k cat /K m revealed that the enzyme hydrolysed methyl p-coumarate (MpCA) 4.5, 9, and 239 times more efficiently than methyl caffeate (MCA), methyl ferulate (MFA) and methyl sinapinate (MSA), respectively. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose but showed preference for the ester at position 2. 4-Nitrophenyl-2-O-trans-feruloyl--l-arabinofuranoside (NPh-5-Fe-Araf) was hydrolysed 100 times more efficiently than 4-nitrophenyl-5-O-trans-feruloyl--l-arabinofuranoside (NPh-2-Fe-Araf). Ferulic acid (FA) was efficiently released from destarched wheat bran (DSWB) when the esterase was incubated together with xylanase from Sporotrichum thermophile (a maximum of 92% total ferulic acid released after 4h incubation). FoFAE-I by itself could release FA but at a level almost five-fold lower than that obtained in the presence of xylanase. The potential of FAE-I for the synthesis of various phenolic acid esters was tested using as a reaction system a surfactantless microemulsions formed in ternary mixture consisting of n-hexane, 1-butanol and water. © 2003 Elsevier Inc. All rights reserved. Keywords: Fusarium oxysporum; Feruloyl esterase; Phenolic acid esters; Transesterification; Surfactantless microemulsions 1. Introduction Biodegradation of plant cell wall polysaccharides is of economical importance in many aspects. The digestibility by microbial enzymes is strongly influences by cross-linked nature of plant cell wall. The main role in the cross-linking Abbreviations: CTAB, cetyltrimethylammoniumbromide; DSWB, destarched wheat bran; FA, ferulic acid; FAE, feruloyl esterase; IEF– PAGE, isoelectric focusing polyacrylamide gel electrophoresis; MCA, methyl caffeate; MpCA, methyl p-coumarate; MFA, methyl ferulate; MSA, methyl sinapinate; NPh-2-Fe-Araf, 4-nitrophenyl-2-O-trans-feruloyl--l- arabinofuranoside; NPh-5-Fe-Araf, 4-nitrophenyl-5-O-trans-feruloyl--l- arabinofuranoside; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis ∗ Corresponding author. Present address: Center for Process Biotechnol- ogy, Biocentrum-DTU, Building 223, Technical University of Denmark, Lyngby 2800, Denmark. Tel.: +45-45252656; fax: +45-45160455. E-mail address: pc@biocentrum.dtu.dk (P. Christakopoulos). is played by polymer-bound hydroxycinnamic acids, which in vivo undergo an oxidative coupling reactions to form de- hydrodimers of the acids or take part in the lignin poly- merization [1–3]. This is why the hydroxycinnamic acids associated with plant hemicelluloses play an important role in cell wall integrity and protection of the plant tissues against digestion by plant-invading microorganisms [4–6]. The most abundant hydroxycinnamic acid is trans-ferulic acid, (E)-4-hydroxy-3-methoxycinnamic acid. FA is usually esterified at position C-5 to -l-arabinofuranosyl side chains in arabinoxylans, at position C-2 to -l-arabinofuranosyl residues in arabinans, at position C-6 to -galactopyranosyl residues in pectic substances and galactans [7] and at po- sition C-4 to -d-xylopyranosyl residues in xyloglucans [8]. The FA esterified with carbohydrates is found either non-substituted or linked to another esterified ferulic acid to form several types of di-feruloyl bridges connecting two polysaccharide chains [2,3,6,9]. Carbohydrate esters of FA 0141-0229/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0141-0229(03)00213-8