Biochemical characterization of a novel iron-dependent GH16 β-agarase, AgaH92, from an agarolytic bacterium Pseudoalteromonas sp H9

A putative agarase gene (agaH92) encoding a primary translation product (50.1 kDa) of 445 amino acids with a 19-amino-acid signal peptide and glycoside hydrolase 16 and RICIN superfamily domains was identified in an agarolytic marine bacterium, Pseudoalteromonas sp. H9 (= KCTC23887). The heterologously expressed protein rAgaH92 in Escherichia coli had an apparent molecular weight of 51 kDa on SDS-PAGE, consistent with the calculated molecular weight. Agarase activity of rAgaH92 was confirmed by a zymogram assay. rAgaH92 hydrolyzed p-nitrophenyl-beta-D-galactopyranoside, but not p-nitrophenyl-alpha-D-galactopyranoside. The optimum pH and temperature for rAgaH92 were 6.0 and 45 degrees C, respectively. It was thermostable and retained more than 85% of its initial activity after heat treatment at 50 degrees C for 1 h. rAgaH92 required Fe2+ for agarase activity and inhibition by EDTA was compensated by Fe2+. TLC analysis, mass spectrometry and NMR spectrometry of the GST-AgaH71 hydrolysis products revealed that rAgaH92 is an endo-type beta-agarase, hydrolyzing agarose into neoagarotetraose and neoagarohexaose.