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Cloning, Expression, and Biochemical Characterization of a GH16 β-Agarase AgaH71 from Pseudoalteromonas hodoensis H7

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Abstract

An agarase gene (agaH71) was identified from Pseudoalteromonas hodoensis, an agar utilizing marine bacterium. The nucleotide sequence revealed that AgaH71 had significant homology to glycosyl hydrolase (GH) 16 agarases. agaH71 encodes a primary translation product (32.7 kDa) of 290 amino acids, including a 21-amino-acid signal peptide. The entire AgaH71 was expressed in a fused protein with glutathione-S-transferase (GST) at its N-terminal (GST-AgaH71) in Escherichia coli. Purified GST-AgaH71 had an apparent molecular weight of 59 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was consistent with the calculated molecular weight (58.7 kDa). Agarase activity of the purified protein was confirmed by zymogram assay. GST-AgaH71 could hydrolyze p-nitrophenyl-β-d-galactopyranoside, but not p-nitrophenyl-α-d-galactopyranoside. The optimum pH and temperature for GST-AgaH71 were 6.0 and 45 °C, respectively. GST-AgaH71 retained more than 95 and 90 % of its initial activity at 40 and 45 °C after heat treatment for 60 min, respectively. The K m and V max for agarose were 28.33 mg/ml and 88.25 U/mg, respectively. GST-AgaH71 did not require metal ions for its activity, but severe inhibition by divalent metal ions was observed. Thin-layer chromatography (TLC) analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectrometry of the GST-AgaH71 hydrolysis products revealed that GST-AgaH71 is an endo-type β-agarase that hydrolyzes agarose into predominantly neoagarotetraose and small proportions of neoagarobiose and neoagarohexaose.

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Acknowledgments

This work was supported by grants PJ009536 from the Next-Generation Bio Green 21 Program, Rural Development Administration, Republic of Korea.

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Authors and Affiliations

  1. Division of Bioscience and Bioinformatics, Myongji University, San 38-2, Namdong, Yongin, Gyeonggi-do, 449-728, South Korea

    Da Yeon Park, Jae-Seon Park & Soon-Kwang Hong

  2. Life Science R&D Center, Sinil Pharmaceutical Co. Ltd., San5-1, Bonpyong-ri, Angsung-myun, Chungju, Chungcheongbuk-do, South Korea

    Won-Jae Chi

  3. Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yusung-gu, Daejon, 305-701, South Korea

    Yong-Keun Chang

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  1. Da Yeon Park
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Correspondence to Soon-Kwang Hong.

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Park, D.Y., Chi, WJ., Park, JS. et al. Cloning, Expression, and Biochemical Characterization of a GH16 β-Agarase AgaH71 from Pseudoalteromonas hodoensis H7. Appl Biochem Biotechnol 175, 733–747 (2015). https://doi.org/10.1007/s12010-014-1294-3

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