Open AccessCrystallization and preliminary X‐ray study of alkaline mannanase from an alkaliphilic Bacillus isolate
Author(s) -
Akita Masatake,
Takeda Nobuhiro,
Hirasawa Kazumichi,
Sakai Hisanobu,
Kawamoto Masahide,
Yamamoto Masaki,
Grant William D.,
Hatada Yuji,
Ito Susumu,
Horikoshi Koki
Publication year - 2004
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
ISSN - 1399-0047
DOI - 10.1107/s0907444904014313
Subject(s) - bacillus subtilis , crystallization , molecular replacement , enzyme , recombinant dna , chemistry , strain (injury) , crystallography , molecule , crystal structure , stereochemistry , biochemistry , biology , bacteria , organic chemistry , genetics , anatomy , gene
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 P 2 1 2 1 2 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 M of 2.26 Å 3 Da −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.