Abstract
Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-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 °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 α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °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.Key points• Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design• Screened two positive variants with higher thermostability and higher activity• Synthesized α-D-glucosylglycerol to a high level by two screened positive variants
Highlights
Sucrose phosphorylase (SPase, EC 2.4.1.7) can catalyze the phosphorolysis of sucrose into α-D-glucose 1-phosphate (α-D-G1P) and D-fructose, and it can glycosylate a broad range of acceptors other than phosphate (Goedl et al 2010)
A semi-rational design strategy that combined the FireProt, a web server designed to predict thermostable mutants concerning structural and evolutionary information automatedly, structure–function analysis, and molecular dynamics (MD) simulations were used to improve the thermostability of LmSPase
The SPase gene of L. mesenteroides ATCC 12,291 was cloned into the pET28a vector after codon optimization and successfully overexpressed in E. coli BL21(DE3) (Figure S1A)
Summary
Sucrose phosphorylase (SPase, EC 2.4.1.7) can catalyze the phosphorolysis of sucrose into α-D-glucose 1-phosphate (α-D-G1P) and D-fructose, and it can glycosylate a broad range of acceptors other than phosphate (Goedl et al 2010). SPase from L. mesenteroides ATCC 12,291 (LmSPase) was reported to have high activity to synthesize α-arbutin but poor thermostability, restricting its industrial applications (Yao et al 2020). In the process of synthesizing αGG, due to the long reaction time, it is usually necessary to keep the temperature at 30 °C (Bolivar et al 2017; Goedl et al 2008). A semi-rational design strategy that combined the FireProt, a web server designed to predict thermostable mutants concerning structural and evolutionary information automatedly, structure–function analysis, and molecular dynamics (MD) simulations were used to improve the thermostability of LmSPase. To test whether the selected mutants can have advantages in industrial applications, we carried out the catalytic synthesis of αGG at a relatively high temperature
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