Identification and biochemical characterization of a novel exo-type β-agarase Aga3463 from an Antarctic Pseudoalteromonas sp. strain

An agar-degrading Antarctic bacterium was isolated and identified as Pseudoalteromonas sp. NJ21. This strain showed strong agarolytic activity and could use agar as the sole carbon source for growth. A novel β-agarase gene aga3463 was cloned and identified from the genomic library of Pseudoalteromonas sp. NJ21. It was predicted to encode a peptide of 366 amino acids, including a 21-amino-acid signal peptide. BLAST analysis revealed the deduced amino acid sequence of aga3463 shared less than 36% similarity with any characterized agarase, and phylogenetic analysis showed it belonged to the glycoside hydrolase GH86 family. The recombinant Aga3463 protein had optimal temperature and pH of 50 °C and 7.0, respectively, and retained more than 60% activity from 10 to 50 °C. The metal ions Cd2+, Ca2+, Fe3+, and Mn2+ increased Aga3463 activity, whereas Cu2+, Si2+, Fe2+, and Ni2+ decreased Aga3463 activity. Notably, Aga3463 exhibited good thermostability in the presence of Ca2+; however, Ca2+ was not necessary for its catalytic activity. The Km and Vmax values for Aga3463 were 6.17 mg/ml and 557 U/mg, respectively. Thin layer chromatography, liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and enzyme assay using p-nitrophenyl-α/β-d-galactopyranoside revealed Aga3463 as an exo-type β-agarase with the ability to degrade agarose to neoagarobiose as the major end product.