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

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-beta-d-galactopyranoside, but not p-nitrophenyl-alpha-d-galactopyranoside. The optimum pH and temperature for GST-AgaH71 were 6.0 and 45 A degrees C, respectively. GST-AgaH71 retained more than 95 and 90 % of its initial activity at 40 and 45 A degrees 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 beta-agarase that hydrolyzes agarose into predominantly neoagarotetraose and small proportions of neoagarobiose and neoagarohexaose.