Measuring β-1,3-Glucan Synthase Activity

β-1,3-glucan synthase activity was measured referring to the method of Belewa et al. (2017) (link). Microsomal proteins were obtained by crushing mycelia and pulverizing by ultracentrifugation. The microsomal proteins were resuspended in 500 μl buffer (1 mmol/l EDTA; 1 mmol/l DTT; 33% (v/v) glycerol; 50 mmol/l Tris–HCl, pH 7.5) and stored at −80°C until use. A 50 μl reaction system was established consisting of 50 mmol/l Tris–HCl (pH 7.5), 20 μmol/l GTP, 4 mmol/l EDTA, 0.5% Brij-35, 6.6% glycerol, 2 mmol/l UDP-Glc, and 100 μg microsomal protein. After incubation at 25°C for 30 min, the reaction was stopped by adding 10 μl of 6 mol/l NaOH. The dextran produced by the previous process was dissolved at 80°C for 30 min. The glucan content was measured by the aniline blue method. One unit of enzymatic activity (U/mg protein) is defined as 1 mg protein catalyzed to produce 1 μg of glucan.