Phage display based identification of novel stabilizing mutations in glycosyl hydrolase family 11 B. subtilis endoxylanase XynA Tim Belie ¨n * , Priscilla Verjans, Christophe M. Courtin, Jan A. Delcour Faculty of Applied Bioscience and Engineering, Laboratory of Food Chemistry and Biochemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium Received 4 January 2008 Available online 28 January 2008 Abstract Two combinatorial libraries of glycosyl hydrolase family 11 (GH11) Bacillus subtilis endoxylanase XynA were constructed and dis- played on phage. Both phage-displayed libraries were subjected to three consecutive biopanning rounds against immobilized endoxylan- ase inhibitor TAXI, each time preceded by an incubation step at elevated temperature. DNA sequence analysis of enriched phagemid panning isolates allowed identification of mutations conferring enhanced thermal stability. In particular, substitutions T44C, T44Y, F48C, T87D, and Y94C were retained, and their thermostabilizing effect was confirmed by testing site-directed XynA variants. None of these mutations was identified in earlier endoxylanase engineering studies. Each single mutation increased the half-inactivation tem- perature by 2–3 °C over that of the wild-type enzyme. Intriguingly, the three selected cysteine variants generated dimers by formation of intermolecular disulfide bridges. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Endoxylanase; Molecular evolution; Phage display; Thermostability Endo-b-1,4-xylanases (EC 3.2.1.8) (further referred to as endoxylanases) are crucial for (arabino)xylan depolymer- isation, as they break down the xylan backbone by catalyz- ing the hydrolysis of b-1,4-xylan linkages. Their substrate is the predominant hemicellulose in cell walls of plants and the second most abundant polysaccharide on earth. Endo- xylanases are widely used as improvers and processing aids in biotechnological processes such as industrial bread-mak- ing, feed production, biobleaching of pulps in paper man- ufacturing, and bioconversion of agricultural residues to fuel ethanol [1]. Apart from this, endoxylanases of micro- bial origin play an important role in phytopathogenesis, as they provide essential means to attacking microorgan- isms to break through the plant cell wall [2]. Based on amino acid sequence similarities and hydrophobic cluster analysis, endoxylanases have been classified mainly into glycosyl hydrolase families (GH) 10 and 11 [3]. GH11 endoxylanases adopt a b-jelly-roll fold, in which two large b-pleated sheets and one a-helix form a structure that resembles a partly-closed right hand [4]. An important factor governing endoxylanase function- ality in industrial applications is their stability under the often harsh conditions of biotechnological processes. Many endoxylanases used in industry today are of mesophilic ori- gin. As a consequence, numerous protein engineering efforts have been directed towards improvement of their thermostability. Among them, many studies focussed on the designed incorporation of intramolecular disulfide bridges [5–12]. Other rational design approaches include the introduction of surface charges [13,14], aromatic inter- actions [15] or a thermostabilizing N-terminal domain [16]. In addition, several directed evolution tactics were applied, in which the endoxylanase encoding gene was subjected to random mutagenesis over its full length [17–21]. We here report on the identification of thermostabilizing mutations in the B. subtilis GH11 endoxylanase by use of a newly developed phage display based selection system. In 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.047 * Corresponding author. Fax: +32 16 321997. E-mail address: tim.belien@biw.kuleuven.be (T. Belie ¨n). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 368 (2008) 74–80