Characterization And Molecular Modification Of Sucrose Isomerase

Isomaltulose is an isomer of sucrose,which has excellent processing characteristics and pHysiological functions.It has been widely used as a substitute of sucrose in many fields such as food and medicine fields.At present,the production of isomaltulose in industry is mostly catalyzed by sucrose isomerase with a certain concentration of sucrose.Different sources of sucrose isomerase have different catalytic effects.Sucrose isomerase inevitably has many by-products and low expression level of recombinant protein,so it is of great significance to explore high-efficiency isomaltulose producing strains and modify sucrose isomerase.In this study,we identified a sucrose isomerase from Raoultella Terrigena,optimized the induction conditions of the recombinant strain and explored the enzymatic properties of the recombinant Pal-2.Site-directed mutagenesis was performed to improve the enzymatic activity and isomaltulose conversion rate.The sucrose isomerase gene sequence from Raoultella Terrigena can be retrieved from the NCBI database,and its entry number in Gen Bank is VUC84579.1.The gene fragment was synthesized and inserted into the vector pET-28a(+)to construct the recombinant plasmid.The recombinant plasmid verified by double enzyme digestion was recovered by gel.The recombinant plasmid was transformed into E.coli BL21(DE3)strain for overexpression and purified using a Ni²⁺affinity column.The molecular weight of the recombinant Pal-2 was 70k Da,which was consistent with the theoretical value.The induction conditions of recombinant Pal-2 were optimized and the activity of recombinant Pal-2 was improved.The optimized conditions:the initial cell concentration OD₆₀₀ was 0.6,the induction temperature was 25?,the IPTG concentration was 0.4 mmol/L,and the culture time after induction was 12 h.The enzyme activity of Pal-2 crude enzyme solution was 48.52 U/m L under optimized induction conditions,which was 39.2%higher than that before.The optimum temperature and pH value of recombinant Pal-2 were 40?and 5.5,respectively.The concentration of Mg²⁺,Mn²⁺,Ca²⁺,Al³⁺at 10mmol/L increased the enzyme activity.Ca²⁺increased the enzyme activity by 28.5%and Al³⁺increased the enzyme activity by 44.3%.K_m and V_max of recombinant Pal-2 were 62.89 mmol/L and 286.35 U/mg,respectively.Finally,the synthesis conditions of isomaltulose were optimized.The conversion rate of isomaltulose reached the maximum of 81.7%after 6 h with 400 g/L sucrose as substrate and 25 U/g sucrose of Pal-2.Eight site-directed variants were designed and generated by molecular modification.The enzyme activities,thermostability and product specificity of these variants were measured.Among them,the enzyme activity of N498P and Q275R mutant was increased by 89.2%and42.2%,respectively.The isomaltulose conversion rates of Y246L,H287R and H481P were increased to 89.1%,90.7%and 92.4%,respectively.