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

A beta-agarase was identified from Pseudoalteromonas sp. Q3OF and heterologously expressed in Bacillus subtilis WB800n. The beta-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 degrees 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 degrees C for 3 h. The agarase exhibited a K-m value of 14.15 mg/mL toward agarose and a V-max 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. (C) 2018 Elsevier B.V. All rights reserved.