Engineering Modification And Application Of Recombinant Sucrose Phosphorylase

Sucrose phosphorylase（SPase）,part of the glycoside hydrolase 13 family,catalyzes the reversible phosphorylation of sucrose.Based on its extensive substrate promiscuity,SPase can move the glucosyl group to glycerol,D-glucose and D-galactose,and the synthesized products2-O-α-D-glucosylglycerol（αGG）,kojibiose and melibiose can be widely used in food,medicine and cosmetics.These reaction products can be generally applied in food,medicine and cosmetics.Compounds catalyzed by SPase are widely used in industry,so the enzyme has attracted more and more attention.At present,the industrial application of sucrose phosphorylase from Leuconostoc mesenteroides（Lm SP）is still limited by many limitations,such as the low thermostability of this enzyme and the weak transglycosylation activity of D-glucose and D-galactose.Therefore,in this study,the wild-type Lm SP strain was used as the initial strain,and the thermostability of Lm SP was modified through semi-rational design,and the synthesis of 2-O-α-D-glucosylglycerol was catalyzed by SPase,and the transglycosylation activity of Lm SP to D-glucose and D-galactose was improved through molecular modification.The main results are as follows:1.Two mutants with improved thermostability and activity were obtained by semi-rational modification of wild-type SPase.A three-dimensional structure of sucrose phosphorylase derived from Leuconostoc mesenteroides was constructed by homology modeling.Then,PROSS and Fire Prot were used to predict the thermostability of the simulated structure,and a small mutant library was constructed.Two mutants were screened out for the first time and a combined mutant was carried out.The enzyme activity of V23L,S424R and V23L/S424R was1.1,1.5 and 1.75 times that of the wild type,respectively.The results of enzyme parameters showed that the half-lives of V23L and V23L/S424R at 50℃were 57.5 min and 27.1 min,respectively,and the T₅₀ values of V23L and V23L/S424R were 7℃higher than those of the wild type.2.2-O-α-D-glucosylglycerol was synthesized by wild-type SPase and two improved mutants,and the reaction conditions were optimized.Under the same reaction conditions,theαGG production of V23L and V23L/S424R was 1.27 times that of the wild type.The optimized reaction conditions ofαGG were as follows:glycerol concentration of 3.2 mol·L^-1,sucrose concentration of 1.2 mol·L^-1,enzyme concentration of 40 U·m L^-1,temperature of 37℃,and reaction time of 60 h,the yield ofαGG reached the maximum.At this moment,theαGG concentrations of the wild type,V23L and V23L/S424R were 190.6,206.6 and 217.2 g·L^-1,respectively.The sucrose conversion rate of V23L and V23L/S424R was 70.7%and 76.3%,respectively,which were higher than that of the wild type.3.Wild-type SPase（WT）was modified by virtual screening,sequence alignment,molecular docking and other methods to expand the substrate spectrum and obtained mutants with enhanced glycosylation activity.When D-glucose and D-galactose were used as receptors,two mutants G161M and G161F,with higher sucrose conversion than the wild type,were screened based on SPase simulation structure using virtual saturation mutation.At the same time,the mutant Q341S was constructed by homologous sequence alignment.When using D-glucose as receptor,Q341S showed 18%higher transglycosylation activity than WT,and the yield of the product was twice as high as that of WT.In addition,two mutants Q341A and I297L with enhanced glycosylation activity were obtained by docking the receptor substrate with the covalent glycosyl-enzyme intermediate,alanine mutation at the interacting amino acid sites and the design and screening of degenerate primers.Using D-glucose as substrate,the two mutants produced 2.6 and 1.5 times more kojibiose than the wild type,respectively.