Carbohydrate Binding Module 74 is a novel starch binding domain associated with large and multi-domain α-amylase enzymes

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C-terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore we propose that this novel starch binding domain is a new carbohydrate binding module (CBM), the first representative of family CBM74, that assists MaAmyA in efficient pore formation in starch granules. Protein-sequence based BLAST searches revealed that CBM74 occurs widespread, but in Bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches. Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut associated Bifidobacteria where they may assist in raw starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of resistant starch digestion in the mammalian gastrointestinal tract. This article is protected by copyright. All rights reserved.