© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 197 Biotechnol. J. 2006, 1, 197–202 DOI 10.1002/biot.200500024 www.biotechnology-journal.com 1 Introduction Xylan is the major constituent of hemicellulosic polysac- charide in cell walls of land plants, representing up to 30–35% of the total dry weight. It is the second most abun- dant renewable resource with a high potential for degra- dation to useful end products [1]. Most xylans occur as heteropolysaccharides, containing different substituent groups in both the backbone chain and side chains [2]. They comprise a backbone of xylose residues linked by β- 1,4-glycosidic bonds. Due to their complex chemical na- ture, xylans require the participation of several hydrolytic enzymes with diverse specificity and modes of action for complete breakdown [3]. The enzymatic system responsi- ble for the xylan hydrolysis is usually composed of a reper- toire of hydrolytic enzymes, including β-1,4-endoxy- lanase, β-xylosidase, α-L-arabinofuranosidase (Abf), α- glucuronidase, acetyl xylan esterase, and phenolic acid (ferulic and p-coumaric acid) esterase. With overlapping but different specificities, these enzymes act coopera- tively to convert xylan into its constituent sugars. Among them, xylanase and β-xylosidase have particularly at- tracted a great deal of attention in the last decade. Xy- lanase catalyzes the hydrolysis of xylo-polysaccharides to form oligosaccharides, which then become inhibitors of xylanase. β-Xylosidase is responsible for degrading short Research Article Rapid and selective isolation of β-xylosidase through an activity- based chemical approach Lee-Chiang Lo 1 , Chi-Yuan Chu 1 , Yen-Ru Pan 2 , Chin-Feng Wan 3 , Yaw-Kuen Li 3 and Jing-Jer Lin 2 1 Department of Chemistry, National Taiwan University, Taipei, Taiwan 2 Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan 3 Department of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan β-Xylosidase is a key enzyme in the xylanolytic system with a great potential in many biotechno- logical applications, especially in the food as well as the pulp and paper industries. We have de- veloped a chemical approach for the rapid screening and isolation of β-xylosidase. Activity probe LCL-6X targeting β-xylosidase was utilized in this study. It carries a β-xylopyranosyl recognition head, a latent trapping device consisting of a 2-fluoromethylphenoxyl group, and a biotin reporter group. The biotin reporter group serves both as a readout device and as a tool for enriching the la- beled proteins. LCL-6X could selectively label a model β-xylosidase from Trichoderma koningii. All other bystander proteins used in this study, including phosphorylase b, BSA, ovalbumin, carbon- ic anhydrase, and trypsin inhibitor, gave negligible cross-labeling effect. With the assistance of streptavidin agarose beads and mass spectrophotometry for the recovery and identification of the biotinylated proteins, we demonstrated that LCL-6X could be successfully applied to identify a bi- functional enzyme with α-L-arabinofuranosidase/β-xylosidase activity from the total protein extract of a Pichia expressing system and a prospective β-xylosidase in the culture medium of Aspergillus fumigatus. The β-xylosidase activities from numerous microbes were also screened using the LCL- 6X probe. Preliminary results showed significant differences among these microbial sources and some distinct protein bands were observed. Thus, we have successfully developed a novel chem- ical probe that has potential applications in xylan-related research. Keywords: Activity probe · Mechanism-based · Quinone methide · Xylan · β-Xylosidases Correspondence: Dr. Lee-Chiang Lo, Department of Chemistry, National Taiwan University, Taipei 106, Taiwan E-mail: lclo@ntu.edu.tw Fax: +886-2-23636359 Abbreviations: Abf, α-L-arabinofuranosidase ; PTP, protein tyrosine phos- phatase ; Q, quadrupole; TTBS, TBS with Tween-20 Received 10 November 2005 Revised 28 December 2005 Accepted 4 January 2006