Cloning, expression, and characterization of a glycoside hydrolase family 86 beta-agarase from a deep-sea Microbulbifer-like isolate.
The gene for a novel beta-agarase from a deep-sea Microbulbifer-like isolate was cloned and sequenced. It encoded a mature protein of 126,921 Da (1146 amino acids), which was a modular protein including two tandem carbohydrate-binding module (CBM)-like sequences and a catalytic module. The catalytic module resembled a glycoside hydrolase family 86 beta-agarase, AgrA, from Pseudoalteromonas atlantica T6c with 31% amino acid identity. Its recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host and purified to homogeneity. The activity and stability were strongly enhanced by CaCl2. The maximal enzyme activity was observed at 45 degrees C and pH 7.5 in the presence of 10 mM CaCl2. The enzyme was an endo-type beta-agarase and degraded agarose and agarose oligosaccharides more polymerized than hexamers to yield neoagarohexaose as the main product. This is the first glycoside hydrolase family 86 enzyme to be homogeneously purified and characterized.
Ohta
Yukari
Y
Japan Agency for Marine-Earth Science and Technology (JAMSTEC, formerly Japan Marine Science and Technology Center), 2-15 Natsushima, Yokosuka, 237-0061, Japan.
Hatada
Yuji
Y
Nogi
Yuichi
Y
Li
Zhijun
Z
Ito
Susumu
S
Horikoshi
Koki
K
D034241:Alteromonadaceae / Q000201:enzymology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019624:agarase