Rapid and selective isolation of beta-xylosidase through an activity-based chemical approach.
beta-Xylosidase is a key enzyme in the xylanolytic system with a great potential in many biotechnological applications, especially in the food as well as the pulp and paper industries. We have developed a chemical approach for the rapid screening and isolation of beta-xylosidase. Activity probe LCL-6X targeting beta-xylosidase was utilized in this study. It carries a beta-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 labeled proteins. LCL-6X could selectively label a model beta-xylosidase from Trichoderma koningii. All other bystander proteins used in this study, including phosphorylase b, BSA, ovalbumin, carbonic 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 alpha-L-arabinofuranosidase/beta-xylosidase activity from the total protein extract of a Pichia expressing system and a prospective beta-xylosidase in the culture medium of Aspergillus fumigatus. The beta-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 chemical probe that has potential applications in xylan-related research.
Lo
Lee-Chiang
LC
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan. lclo@ntu.edu.tw
Chu
Chi-Yuan
CY
Pan
Yen-Ru
YR
Wan
Chin-Feng
CF
Li
Yaw-Kuen
YK
Lin
Jing-Jer
JJ
D005591:Chemical Fractionation / Q000379:methods*; D004789:Enzyme Activation; D015336:Molecular Probe Techniques; D012680:Sensitivity and Specificity; D014242:Trichoderma / Q000201:enzymology*; D014995:Xylosidases / Q000737:chemistry* / Q000302:isolation & purification*
D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase