A novel iron-dependent GH16 β-agarase, AgaH92, from Pseudoalteromonas sp. H9 newly isolated from the West Sea of South Korea

An agarolytic marine bacterium (H9) was isolated from coastal seawater. The cells were gram-negative and rod-shaped with smooth surfaces and polar flagella. Cells grew at 20–30°C, between pH 5.0 and 9.0, and in media containing 1–5% (w/v) NaCl. The G+C content was 41.56 mol%. Based on 16S rRNA sequence and biochemical and chemotaxonomic characteristics, we designated it as Pseudoalteromonas sp. H9 (=KCTC23887). 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 H9. 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 thermo-stable and retained more than 85% of its initial activity at 50°C after heat treatment for 1 h. rAgaH92 required Fe²⁺ for agarase activity and inhibition by EDTA was compensated by Fe²⁺. A thin-layer chromatography analysis of the rAgaH92 hydrolysis products and the kinematic viscosity of the agarase revealed that rAgaH92 is an endo-type β-agarase belonging to GH16 and hydrolyzes agarose into neoagarotetraose and neoagarohexaose. [Supported by Basic Science Research Program (NRF-2012R1A1B3002174) through the National Research Foundation of Korea]