Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate, JAMB-A94.
A gene, agaA, for a novel beta-agarase from the marine bacterium JAMB-A94 was cloned and sequenced. The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542(T). The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da. The deduced amino acid sequence showed 37-66% identity to those of known agarases in glycoside hydrolase family 16. A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region. The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The purified enzyme was an endo-type beta-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60 degrees C. The optimal pH and temperature for activity were around 7.0 and 55 degrees C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).
Ohta
Yukari
Y
Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Japan.
Nogi
Yuichi
Y
Miyazaki
Masayuki
M
Li
Zhijun
Z
Hatada
Yuji
Y
Ito
Susumu
S
Horikoshi
Koki
K
D034241:Alteromonadaceae / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012685:Sepharose / Q000378:metabolism; D013379:Substrate Specificity
D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase