Constructing arabinofuranosidases for dual arabinoxylan debranching activity (Biotechnology and Bioengineering)

Enzymatic conversion of arabinoxylan requires ?-L-arabinofuranosidases able to remove ?-L-arabinofuranosyl residues (?-L-Araf) from both mono- and double-substituted D-xylopyranosyl residues (Xylp) in xylan (i.e., AXH-m and AXH-d activity). Herein, SthAbf62A (a family GH62 ?-L-arabinofuranosidase with AXH-m activity) and BadAbf43A (a family GH43 ?-L-arabinofuranosidase with AXH-d3 activity), were fused to create SthAbf62A-BadAbf43A and BadAbf43A-SthAbf62A. Both fusion enzymes displayed dual AXH-m,d and synergistic activity towards native, highly branched wheat arabinoxylan (WAX). When using a customized arabinoxylan substrate comprising mainly ?-(1->3)-L-Araf and ?-(1->2)-L-Araf substituents attached to disubstituted Xylp (d-2,3-WAX), the specific activity of the fusion enzymes was twice that of enzymes added as separate proteins. Moreover, the SthAbf62A-BadAbf43A fusion removed 83% of all ?-L-Araf from WAX after a 20 h treatment. 1H NMR analyses further revealed differences in SthAbf62A-BadAbf43 rate of removal of specific ?-L-Araf substituents from WAX, where 9.4 times higher activity was observed towards d-?-(1->3)-L-Araf compared to m-?-(1->3)- L-Araf positions. This article is protected by copyright. All rights reserved