Enhancement of catalytic performance of alginate lyase through combinational site-directed mutagenesis

Alginate oligosaccharides (AOS) produced through alginate biodegradation by alginate lyases have received extensive attention for their wide applications in the food, pharmaceutical, and agricultural industries. Thus, alginate lyases with good performance are urgently required for industrial production. In this study, combinational mutagenesis was employed for the alginate lyase from Pseudoalteromonas sp. Alg6B. After combinational mutation of key amino acids (for example, isoleucine and valine) in the catalytic residues, the specific activity of the mutant I62A/A99K/V132S/L157T (1124.1 U/mg) was increased 2.13 fold. The adaptability of the mutant enzyme to temperature (up to 60 degrees C), pH (4.0-10.0), and chemicals and metal ions was significantly improved. Kinetic analysis showed that the catalytic efficiency of the mutant enzyme was 4.67-fold higher than that of the wild-type enzyme. Our results indicate the feasibility of combinational mutagenesis for the modification of alginate lyases, which shows significant potential for the production of AOS from marine alginate.