Characterization and overexpression of a glycosyl hydrolase family 16 β-agarase Aga0917 from Pseudoalteromonas fuliginea YTW1-15-1

A gene (aga0917) encoding a putative β-agarase was identified from the genome of Pseudoalteromonas fuliginea YTW1-15-1. The nucleotide sequence analysis revealed that aga0917 had significant homology to the agarase genes of the GH16 family. aga0917 encodes a putative protein of 290 amino acids with an estimated molecular mass of 32.5 kDa, including a 21-amino acid signal peptide. A gene fragment encoding only the putative mature form of Aga0917 (269 amino acids) was overexpressed in Escherichia coli BL21 (DE3) pLysS as a 6 × histine-tagged fusion protein (rmAga0917). The Km, Vmax, and kcat for agarose of rmAga0917 were 39.6 mg/mL, 334 (U/mg) of protein, and 178 (1/s), respectively. According to the results of thin-layer chromatography and mass spectrometry analysis, the main end product from agarose with rmAga0917 was neoagarotetraose, in addition to a small amount of neoagarobiose. Notably, the recombinant protein rmAga0917 showed optimum activity at 60°C and retained approximately 100% agarolytic activity after being kept at 40°C for 1 h and 57% residual activity after incubation at 50°C for 1 h. The rmAga0917 exhibited maximum agarase activity at pH 6.0, and retained more than 80% of activity after incubation over a range of pH 4.0–9.0 for 1 h at 4°C.