Enhancing thermostability and activity of sucrose phosphorylase for high-level production of 2-O-α-d-glucosylglycerol

Sucrose phosphorylase (SPase) can transfer the glucosyl group of sucrose to different compounds and has been widely used in industry. To overcome the low thermostability of the sucrose phosphorylase from Leuconostoc mesenteroides ATCC 12291 (LmSP), a method named PROSS was used to construct mutants with increased thermostability. All variants were screened by measuring their residual activities after heating at 50°C. Then, a single point mutant and a combined mutant with improved thermostability and activity were obtained. The half-lives of mutants at 50°C were approximately twice as high as those of the wild type. In addition, 2-O-α-d-glucosylglycerol (αGG) was synthesized by the wild type and the two improved variants, and the reaction conditions were optimized. Under the conditions of glycerol concentration of 3.2 mol/L, sucrose concentration of 1.2 mol/L, and enzyme concentration of 40 U/mL at 37°C for 60 h, the yield of αGG reached the maximum, and the sucrose conversion rate of the wild type, the mutant V23L and the combined mutant V23L/S424R were 62.3%, 70.7% and 76.3%, respectively. In this study, SPase mutants with higher activity and stability were obtained, and achieved high-level production of αGG.