A thermophilic glycoside hydrolase family 16 (GH16)beta-1,3-1,4-glucanase from Clostridium thermocellum (CtLic16A) holds great potentials in industrial applications due to its high specific activity and outstanding thermostability. In order to understand its molecular machinery, the crystal structure of CtLic16A was determined to 1.95 angstrom resolution. The enzyme folds into a classic GH16 beta-jellyroll architecture which consists of two beta-sheets atop each other, with the substrate-binding cleft lying on the concave side of the inner beta-sheet. Two Bis-Tris propane molecules were found in the positive and negative substrate binding sites. Structural analysis suggests that the major differences between the CtLic16A and other GH16 beta-1,3-1,4-glucanase structures occur at the protein exterior. Furthermore, the high catalytic efficacy and thermal profile of the CtLic16A are preserved in the enzyme produced in Pichia pastoris, encouraging its further commercial applications. (C) 2015 Elsevier Inc. All rights reserved.