Isolation and divalent-metal activation of a ß-xylosidase, RUM630-BX

Authors: Jordan, Douglas; Braker, Jay; Wagschal, Kurt; Stoller, Jeanette; Lee, Charles

Submitted to: Enzyme and Microbial Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/27/2015
Publication Date: 11/26/2015
Citation: Jordan, D.B., Braker, J.D., Wagschal, K.C., Stoller, J.R., Lee, C.C. 2015. Isolation and divalent-metal activation of a ß-xylosidase, RUM630-BX. Enzyme and Microbial Technology. 82:158-163. doi: 10.1016/j.enzmictec.2015.10.001.

Interpretive Summary: Complete enzymatic hydrolysis of xylan hemicellulose is a critical component to the success of a lignocellulosic biorefinery. Many different enzymes are required to hydrolyze the xylan to the simple sugar xylose; however, the ß -xylosidase enzyme is responsible for cleaving more bonds than any others. We have used a metagenomic DNA approach to discover a new ß-xylosidase enzyme with the highest reported activity against a natural substrate (xylotetraose) ever reported.

Technical Abstract: The gene encoding RUM630-BX, a ß-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (kcat) by divalent metals Mg2+, Mn2+ and Co2+ but not by Ca2+, Ni2+, and Zn2+. Activation of RUM630-BX by Mg2+ (t0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (kcat/Km) of 299 s-1mM-1 on xylotetraose being the highest reported.