An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum Marcela Colombres Æ Jose ´ A. Garate Æ Carlos F. Lagos Æ Rau ´ l Araya-Secchi Æ Patricia Norambuena Æ Soledad Quiroz Æ Luis Larrondo Æ Tomas Pe ´rez-Acle Æ Jaime Eyzaguirre Received: 5 July 2007 / Accepted: 12 November 2007 Ó Springer Science+Business Media B.V. 2007 Abstract The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 A ˚ resolution (PDB 1G66). The enzyme possesses the a/b hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three- dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a com- parative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop’s resi- dues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters. Keywords Acetyl xylan esterase Á Cutinase Á Comparative modeling Á Mutagenesis Á Penicillium purpurogenum Introduction Hemicelluloses are a set of amorphous polysaccharides present in plant cell walls in association with cellulose and lignin, and constitute about 30% of lignocellulose [1]. The main component of the hemicelluloses is xylan. This gly- can is composed of a linear chain of xylose residues linked b (1?4) and presents a variety of substituents in carbons 2 and 3 of the xyloses, their type and amount depending on its source. Joined by glycosidic linkages are L-arabinoses and methyl glucuronate, while acetate is bound by ester linkages. In addition, hydroxycinnamic acids (mainly ferulic and p-coumaric acids) are found in ester linkages to the arabinose substituents [2]. The biodegradation of xylan is performed by a number of bacteria and fungi. It is a complex process requiring the concerted action of several glycanases and esterases, mainly extracellular, which attack the main chain and the substituent linkages [3]. Among them are the acetyl xylan Marcela Colombres and Jose ´ A. Garate have equally contributed to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10822-007-9149-4) contains supplementary material, which is available to authorized users. M. Colombres Á P. Norambuena Á S. Quiroz Á L. Larrondo Á J. Eyzaguirre Departamento de Gene ´tica Molecular y Microbiologı ´a, Pontificia Universidad Cato ´lica de Chile, Casilla 114-D, Santiago, Chile J. A. Garate Á C. F. Lagos Á R. Araya-Secchi Á T. Pe ´rez-Acle Centre for Bioinformatics (CBUC), Pontificia Universidad Cato ´lica de Chile, Casilla 114-D, Santiago, Chile J. Eyzaguirre (&) Universidad Andre ´s Bello, Repu ´blica, 217 Santiago, Chile e-mail: jeyzaguirre@unab.cl 123 J Comput Aided Mol Des DOI 10.1007/s10822-007-9149-4