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http://dx.doi.org/10.4014/jmb.1209.09009

Molecular Cloning, Overexpression, and Enzymatic Characterization of Glycosyl Hydrolase Family 16 ${\beta}$-Agarase from Marine Bacterium Saccharophagus sp. AG21 in Escherichia coli  

Lee, Youngdeuk (Korea Institute of Ocean Science Technology)
Oh, Chulhong (Korea Institute of Ocean Science Technology)
Zoysa, Mahanama De (College of Veterinary Medicine, Chungnam National University)
Kim, Hyowon (Department of Marine Life Sciences, Jeju National University)
Wickramaarachchi, Wickramaarachchige Don Niroshana (Department of Marine Life Sciences, Jeju National University)
Whang, Ilson (Department of Marine Life Sciences, Jeju National University)
Kang, Do-Hyung (Korea Institute of Ocean Science Technology)
Lee, Jehee (Department of Marine Life Sciences, Jeju National University)
Publication Information
Journal of Microbiology and Biotechnology / v.23, no.7, 2013 , pp. 913-922 More about this Journal
Abstract
An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The ${\beta}$-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) ${\beta}$-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to ${\beta}$-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant ${\beta}$-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at $55^{\circ}C$ and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by $FeSO_4$ (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a ${\beta}$-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.
Keywords
Saccharophagus sp. AG21; beta-agarase; GH16; neoagaro-oligosaccharide;
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