Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of the catalytic domain of a hyperthermostable endo-1,4-B-D-mannanase from Thermotoga petrophila RKU-1

Abstract

Endo-1,4-[beta]-D-mannanases play key roles in seed germination and fruit ripening and have recently received much attention owing to their potential applications in the food, detergent and kraft pulp industries. In order to delineate their structural determinants for specificity and stability, X-ray crystallographic investigations combined with detailed functional studies are being performed. In this work, crystals of the catalytic domain of a hyperthermostable endo-1,4-[beta]-D-mannanase from Thermotoga petrophila RKU-1 were obtained from three different conditions, resulting in two crystalline forms. Crystals from conditions with phosphate or citrate salts as precipitant (CryP) belonged to space group P212121, with unit-cell parameters a = 58.76, b = 87.99, c = 97.34 A, while a crystal from a condition with ethanol as precipitant (CryE) belonged to space group I212121, with unit-cell parameters a = 91.03, b = 89.97, c = 97.89 A. CryP and CryE diffracted to resolutions of 1.40 and 1.45 A, respectively.