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Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1

Applied Microbiology and Biotechnology volume 98, pages 4545–4555 (2014)Cite this article

Abstract

Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene (agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-β-d-galactopyranoside but not p-nitrophenyl-α-d-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K m and V max for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and 13C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type β-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.

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Acknowledgments

This work was supported by grant no. 2012-R1A1B3002174 from the Basic Research Program of the National Research Foundation (KRF) of Korea.

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  1. Division of Bioscience and Bioinformatics, Myongji University, Yongin, Gyeonggi-do, 449-728, South Korea

    Won-Jae Chi, Da Yeon Park & Soon-Kwang Hong

  2. Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, Yusung-gu, Taejon, 305-701, South Korea

    Young Bin Seo & Yong Keun Chang

  3. Department of Biotechnology, Hankyong National University, Anseong, Gyeonggi-do, 456-749, South Korea

    Soon-Youl Lee

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  1. Won-Jae Chi
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  2. Da Yeon Park
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Correspondence to Soon-Kwang Hong.

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Chi, WJ., Park, D.Y., Seo, Y.B. et al. Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1. Appl Microbiol Biotechnol 98, 4545–4555 (2014). https://doi.org/10.1007/s00253-014-5510-4

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  • DOI: https://doi.org/10.1007/s00253-014-5510-4

Keywords

  • Alteromonas sp. GNUM-1
  • β-Agarase
  • Neoagarobiose
  • AgaG1