Protein Expression and PuriWcation 37 (2004) 126–133 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.05.019 Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme Anthony Levasseur, ¤ Isabelle Benoit, Michèle Asther, Marcel Asther, and Eric Record UMR 1163 INRA de Biotechnologie des Champignons Filamenteux, IFR-BAIM, Universités de Provence et de la Méditerranée, ESIL, 163 avenue de Luminy, Case Postale 925, 13288 Marseille cedex 09, France Received 2 March 2004, and in revised form 28 May 2004 Available online 8 July 2004 Abstract The faeB gene encoding the feruloyl esterase B (FAEB) was isolated from Aspergillus niger BRFM131 genomic DNA. The faeB gene, with additional sequence coding for a C-terminal histidine tag, was inserted into an expression vector under the control of the gpd promoter and trpC terminator and expressed in a protease deWcient A. niger strain. Homologous overproduction allows to reach an esterase activity of 18 nkat mL ¡1 against MCA as substrate. The improvement factor was 16-fold higher as compared to the pro- duction level obtained with non-transformed A. niger strain induced by sugar beet pulp. The corresponding secretion yield was esti- mated to be around 100 mg L ¡1 . Recombinant FAEB was puriWed 14.6-fold to homogeneity from an 8-day-old culture by a single aYnity chromatographic step with a recovery of 64%. SDS–PAGE revealed a single band with a molecular mass of 75 kDa, while under non-denatured conditions, native enzyme has a molecular mass of around 150 kDa conWrming that the recombinant FAEB is a homodimer. The recombinant and native FAEB have the same characteristics concerning temperature and pH optima, i.e., 50 °C and 6, respectively. In addition, the recombinant FAEB was determined to be quite stable up to 50 °C for 120 min. Kinetic constants for MCA, MpCA, and chlorogenic acid (5-O-caVeoyl quinic acid) were as follows: K m : 0.13, 0.029, and 0.16 mM and V max : 1101, 527.6, and 28.3 nkat mg ¡1 , respectively. This is the Wrst report on the homologous overproduction of feruloyl esterase B in A. niger.  2004 Elsevier Inc. All rights reserved. Plant cell wall is composed of polysaccharides and lignin embedded in a complex and organized structure. This physical biobarrier constitutes an important protec- tion against microbial invasion. This structure is strengthened by a close interaction between cellulose microWbrills and hemicellulose or pectin polysaccha- rides. Additional cross-linkages such as diferulic acid bridges between hemicellulose adjacent chains [1] or between lignin and hemicellulose [2] increase the com- plexity of the cell wall structure and contribute to improve its resistance to hydrolysis. Ferulic acid and other aromatic compounds are present as terminal side groups in xylan and pectin but with diVerent polysaccha- ride sites of attachment. In xylan, ferulic acid is attached to O 5 of terminal arabinose residue [3], whereas in the pectic hairy regions, this aromatic compound is linked to O 2 of arabinose residues or O 6 of galactose residues [4]. Plant saprophytic micro-organisms produce a pleth- ora of enzymes to degrade plant cell wall and use the cell wall components as nutrients. Some micro-organisms have evolved enzymes such as esterases to release aro- matic acids and to allow a facilitated accessibility of main-chain degrading enzymes to the polysaccharide backbone (for a review, see [5,6]). In Aspergillus niger, two diVerent feruloyl esterases were puriWed from the liquid culture when the strains were grown on oat spelts xylan or sugar beet pulp [7,8]. Their corresponding genes (faeA and faeB) were cloned and their regulation was studied using inducers such as a range of aromatic com- pounds, polysaccharides or diVerent carbon sources [9– 12]. Expression proWles of both genes indicated distinct activating mechanisms because diVerent aromatic com- pounds were able to induce preferentially a particular gene. However, the two esterase genes were commonly ¤ Corresponding author. Fax: +33-4-91-82-86-01. E-mail address: anthony.levasseur@esil.univ-mrs.fr (A. Levasseur).