Heterologous expression of an agarase gene in Bacillus subtilis, and characterization of the agarase.

A β-agarase was identified from Pseudoalteromonas sp. Q30F and heterologously expressed in Bacillus subtilis WB800n. The β-agarase, Aga862 encoded by aga862 gene in an open reading frame of 1338 bp is 445 amino acids in length, and has a calculated molecular mass of 50.1 kDa and an estimated isoelectric point of 4.81. Protein sequence analysis showed that Aga862 belongs to family 16 of glycoside hydrolases (GH16) and carbohydrate-binding module family 13 (CBM13). The agarase was expressed in B. subtilis WB800n and purified by precipitation, anion exchange and gel filtration for a specific activity of 4.6 U/mg, a 27.8-fold improvement over the activity of the crude enzyme. Aga862 exhibited optimal activity at 45 °C and pH 6.5, and showed excellent pH stability with retention of over 80% relative activities after preincubation for the pH range of 3.0-10.0 at 4 °C for 3 h. The agarase exhibited a Km value of 14.15 mg/mL toward agarose and a Vmax of 256.41 U/mg. The mass spectrometry analysis revealed that the end products of agar degradation were neoagarotetraose and neoagarohexaose. Recombinant Aga862 has great potential for the manufacture of agaro-oligosaccharides for the non-pathogenic nature and safety of the B. subtilis WB800n.