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Mar. Drugs 2014, 12(5), 2731-2747; doi:10.3390/md12052731
Article

Overexpression and Characterization of a Novel Thermostable β-Agarase YM01-3, from Marine Bacterium Catenovulum agarivorans YM01T

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Received: 26 November 2013 / Revised: 21 April 2014 / Accepted: 22 April 2014 / Published: 12 May 2014
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Abstract

Genome sequencing of Catenovulum agarivorans YM01T reveals 15 open-reading frames (ORFs) encoding various agarases. In this study, extracellular proteins of YM01T were precipitated by ammonium sulfate and separated by one-dimensional gel electrophoresis. The results of in-gel agarase activity assay and mass spectrometry analysis revealed that the protein, YM01-3, was an agarase with the most evident agarolytic activity. Agarase YM01-3, encoded by the YM01-3 gene, consisted of 420 amino acids with a calculated molecular mass of 46.9 kDa and contained a glycoside hydrolase family 16 β-agarase module followed by a RICIN superfamily in the C-terminal region. The YM01-3 gene was cloned and expressed in Escherichia coli. The recombinant agarase, YM01-3, showed optimum activity at pH 6.0 and 60 °C and had a Km of 3.78 mg mL−1 for agarose and a Vmax of 1.14 × 104 U mg−1. YM01-3 hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Notably, YM01-3 was stable below 50 °C and retained 13% activity after incubation at 80 °C for 1 h, characteristics much different from other agarases. The present study highlights a thermostable agarase with great potential application value in industrial production.
Keywords: Catenovulum agarivorans YM01T; expression; characterization; YM01-3; β-agarase; thermostable Catenovulum agarivorans YM01T; expression; characterization; YM01-3; β-agarase; thermostable
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Cui, F.; Dong, S.; Shi, X.; Zhao, X.; Zhang, X.-H. Overexpression and Characterization of a Novel Thermostable β-Agarase YM01-3, from Marine Bacterium Catenovulum agarivorans YM01T. Mar. Drugs 2014, 12, 2731-2747.

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