Abstract: In order to clarify the effect of the non-catalytic carbohydrate-binding module (CBM) and F5/8 type C domains on the enzymatic properties of AlgL7, an alginate lyase from Microbulbifer sp. ALW1, the full-length enzyme AlgL7 and two truncated enzymes: CD1 (catalytic domain) and CD2 (containing F5/8 type C domain and catalytic domain) were constructed and characterized. The results showed that the truncated enzyme CD2 exhibited higher specific activity, thermostability, Michaelis constant (Km), and maximum reaction velocity (Vmax) compared to the full-length AlgL7, indicating that the CBM domain played an important role in maintaining the substrate affinity of the enzyme, but reduced the catalytic activity, thermostability, and Vmax value the enzyme. Compared to the truncated enzyme CD1, CD2 exhibited higher specific activity, optimal reaction temperature, thermostability, Vmax, and Km, indicating that the F5/8 type C domain contributed to improve the enzymatic activity, optimum reaction temperature, thermostability, and Vmax, but reduced the substrate affinity of the enzyme. Using sodium alginate as the substrate, the specific activity of CD2 was 183.9 U/mg. The optimal reaction temperature and pH were 40 ℃ and 7.0, respectively. The Km and Vmax were 39.80 mg/mL and 2 000 U/mg, respectively. The major enzymatic hydrolysates were disaccharides and trisaccharides. This study promotes the understanding of the structure-activity relationship between the non-catalytic domains and the properties of alginate lyase, and lays a theoretical basis for using the non-catalytic domains to improve the catalytic properties of alginate lysate.

Key words: alginate lyase; carbohydrate-binding module; F5/8 type C domain; enzymatic properties; enzymatic hydrolysate