Molecular Cloning and Enzymatic Characterization of GH16 beta-agarase from Marine Bacterium

Abstract

A novel β-agarase gene was identified in a Saccharophagus sp. AG21 isolate from the Jeju Island coastal evironment, designated as agy1. Subsequent cloning and expression of the recombinant β-agarase(rAgy1) was analyzed to detemine its biochemical properties. The predicted sequence has 1908 bp ORF encoding 636 amino acids, and includes a glycosyl hydrolase 16 β-agarase module and two carbohydrate binding modules of family 6. The deduced amino acid seqeunce showed 93.7% similarity to beta-agarase of Saccharophagus degradans. The mature agy1 was cloned and overexpressed as a His-tagged recombinant beta-agarase (rAgy1) in Escherichia coli, and had a predicted molecular weight of 69 kDa and an Isoelectric point of 4.5. rAgy1 showed optimum activity at 55℃ and pH 7.5, and had a specific activity of 85U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%) and NaCl (34%). The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose and neoagarohexaose and may be useful for applications in the cosmetics, food and reagent industries. Supported by KIOST and MLTM (PE98931, PM57210, PE98932). biochemical properties. The predicted sequence has 1908 bp ORF encoding 636 amino acids, and includes a glycosyl hydrolase 16 β-agarase module and two carbohydrate binding modules of family 6. The deduced amino acid seqeunce showed 93.7% similarity to beta-agarase of Saccharophagus degradans. The mature agy1 was cloned and overexpressed as a His-tagged recombinant beta-agarase (rAgy1) in Escherichia coli, and had a predicted molecular weight of 69 kDa and an Isoelectric point of 4.5. rAgy1 showed optimum activity at 55℃ and pH 7.5, and had a specific activity of 85U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%) and NaCl (34%). The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose and neoagarohexaose and may be useful for applications in the cosmetics, food and reagent industries. Supported by KIOST and MLTM (PE98931, PM57210, PE98932).