Crystallization and preliminary X-ray study of alkaline mannanase from an alkaliphilic Bacillus isolate

An alkaline mannanase (EC 3.2.1.78) from the alkaliphilic Bacillus sp. strain JAMB-602 was cloned and sequenced. The deduced amino-acid sequence of the enzyme suggested that the enzyme consists of a catalytic and unknown additional domains. The recombinant enzyme expressed by B. subtilis was crystallized using the hanging-drop vapour-diffusion method at 277 K. X-ray diffraction data were collected to 1.65 Å. The crystal belongs to space group P2[subscript: 1]2[subscript: 1]2[subscript: 1], with unit-cell parameters a = 70.7, b = 79.5, c = 80.4 Å. The asymmetric unit contains one protein molecule, with a corresponding V[subscript: M] of 2.26 Å[superscript: 3] Da[superscript: -1] and a solvent content of 45.6%. Molecular replacement for initial phasing was carried out using the three-dimensional structure of a mannanase from Thermomonospora fusca as a search model, which corresponds to the catalytic domain of the alkaline mannanase. It gave sufficient phases to build the unknown domain.