Structural analyses and yeast production of the ß-1,3-1,4-glucanase catalytic module encoded by the licB gene of Clostridium thermocellum.

A thermophilic glycoside hydrolase family 16 (GH16) ß-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 1.95Å resolution. The enzyme folds into a classic GH16 ß-jellyroll architecture which consists of two ß-sheets atop each other, with the substrate-binding cleft lying on the concave side of the inner ß-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 ß-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.

Preliminary X-ray diffraction analysis of a thermophilic ß-1,3-1,4-glucanase from Clostridium thermocellum.

ß-1,3-1,4-Glucanases catalyze the specific hydrolysis of internal ß-1,4-glycosidic bonds adjacent to the 3-O-substituted glucose residues in mixed-linked ß-glucans. The thermophilic glycoside hydrolase CtGlu16A from Clostridium thermocellum exhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed in Escherichia coli, purified and crystallized in the trigonal space group P3121, with unit-cell parameters a=b=74.5, c=182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Šresolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress.