Marine algal polysaccharides are promising alternative resources to terrestrial biomass. Agarolytic enzymes degrade agarose to various kinds of oligosaccharides. In this study, we expressed miniCbpA, a recombinant scaffolding protein from Clostridium cellulovorans, in Escherichia coli. To assemble the agarolytic complex via cohesin-dockerin interaction, we constructed a chimeric agarase cAgaB from Zobellia galactanivorans containing the catalytic domain of AgaB fused with a dockerin domain from C. cellulovorans EngB. The assembly of functional agarolytic complexes increased the activity against the agar substrate approximately 1,4-fold compared with that for the corresponding enzymes alone. The carbohydrate-binding module (CBM) of miniCbpA was used as a tag for CBM-utilizing one-step purification using cellulose as a support. This is the first report on the formation of agarolytic complexes using the cohesin-dockerin interaction system. The assembly of agar-degrading complexes will lead to the commercial production of useful products from agar biomass at low costs. (C) 2012 Elsevier Ltd. All rights reserved.