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 A. 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. The asymmetric unit contains one protein molecule, with a corresponding VM of 2.26 A3 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.
Akita
Masatake
M
Japan Agency for Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka 237-0061. akitam@jamstec.go.jp
Takeda
Nobuhiro
N
Hirasawa
Kazumichi
K
Sakai
Hisanobu
H
Kawamoto
Masahide
M
Yamamoto
Masaki
M
Grant
William D
WD
Hatada
Yuji
Y
Ito
Susumu
S
Horikoshi
Koki
K
D001407:Bacillus / Q000201:enzymology*; D002853:Chromatography, Liquid; D003460:Crystallization; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D044902:beta-Mannosidase / Q000737:chemistry* / Q000302:isolation & purification
D044902:beta-Mannosidase