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  • Title: Cloning, expression, and biochemical characterization of a GH16 β-agarase AgaH71 from Pseudoalteromonas hodoensis H7.
    Author: Park DY, Chi WJ, Park JS, Chang YK, Hong SK.
    Journal: Appl Biochem Biotechnol; 2015 Jan; 175(2):733-47. PubMed ID: 25342256.
    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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