Differently activated agarose-based supports were evaluated for co-immobilization of a crude extract from Penicillium janczewskii containing xylanase, beta-xylosidase and alpha-L-arabinofuranosidase activities. Adequately selecting support and immobilization conditions (8 h, using agarose with 10% crosslinking) increased enzyme levels substantially, mainly in relation to the xylanase (2-fold). A coating with dextran aldehyde MW 6000 Da, partially oxidized, covalently attached the enzymes to the support. Optimum activity was verified in the pH range 2-4, and at 50, 65 and 80 degrees C for the xylanase, alpha-L-arabinofuranosidase and beta-xylosidase, respectively. The xylanase was highly thermostable retaining-more than 70% of activity even after 24 h incubation at 60 and 70 degrees C; and at 80 degrees C its half-life was 1.7 h. The half-lives of the beta-xylosidase and alpha-L-arabinofuranosidase at 50 degrees C were 2.3 and 3.8 h, respectively. The co-immobilization of the enzymes on a single support give raise to a functional multi-enzymatic biocatalyst acting in the complete hydrolysis of different and complex substrates such as oat spelt and wheat arabinoxylans, with xylose yield higher than 40%. The xylanase and the alpha-L-arabinofuranosidase presented high stability retaining 86.6 and 88.0% of activity after 10 reuse cycles. (C) 2016 Elsevier Ltd. All rights reserved.