Abstract
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-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for rAgaH92 were 6.0 and 45°C, respectively. It was thermostable and retained more than 85% of its initial activity after heat treatment at 50°C for 1 h. rAgaH92 required Fe(2+) for agarase activity and inhibition by EDTA was compensated by Fe(2+). TLC analysis, mass spectrometry and NMR spectrometry of the GST-AgaH71 hydrolysis products revealed that rAgaH92 is an endo-type β-agarase, hydrolyzing agarose into neoagarotetraose and neoagarohexaose.
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