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

Abstract

An alkaline mannanase (EC 3.2.1.78) from the alkaliphilic Bacillus sp. strain JAMB-602 was cloned and sequenced. The deduced aminoacid 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 A Ê . The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 70.7, b = 79.5, c = 80.4 A Ê . The asymmetric unit contains one protein molecule, with a corresponding V M of 2.26 A Ê 3 Da À1 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 suf®cient phases to build the unknown domain