Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize alpha-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure-function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 degrees C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce alpha-D-glucosylglycerol (alpha GG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 degrees C for 60 h and achieved the product concentration of 193.2 +/- 12.9 g/L and 195.8 +/- 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 +/- 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme.

Automated summary

In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was successfully synthesized and overexpressed in Escherichia coli with optimized codons. A semi-rational design strategy and molecular dynamic simulations were used to improve the thermostability of LmSPase, resulting in single-point mutation T219L and combination mutation I31F/T219L/T263L/S360A (Mut4) with enhanced thermostability. These variants showed approximately two-fold increase in half-lives at 50 degrees C compared to wild-type LmSPase, and also led to higher product concentrations when used for enzymatic synthesis of alpha-D-glycosides.