Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from the novel marine isolate, JAMB-A94

Abstract

A gene, agaA, for a novel β-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 β-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60°C. The optimal pH and temperature for activity were around 7.0 and 55°C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).