Characterization And Molecular Modification Of Inulosucrase

There are two types of fructan in nature based on differences in sugar linkage,namely,inulin and levan-type fructan.Inulin is abundant in some plants and possesses many versatile properties and physiological functions.Inulin is a soluble dietary fiber that has been used in many industries including in food and medicine.In industrial settings,inulin is mostly extracted from chicory.In contrast,levan-type fructan is not abundant and is mainly obtained from microorganisms rather than plants.There are no reports of microbial inulin in China,but some microorganisms can biosynthesize inulin using inulosucrase?EC:2.1.4.9?and sucrose as a sole substrate.It is well known that the molecular weight of a polysaccharide plays a pivotal role in determining its specific properties and functions.Generally,the molecular weight of microbial inulin is higher than 10⁶ g/mol,which is hundreds of times greater than that of vegetal inulin.However,little attention has been focused on microbial inulin and inulosucrase.In this work,inulosucrase from L.gasseri DSM 20604 was characterized to determine the enzymatic properties and the glycosidic bonding patterns of the biosynthesized polysaccharide.The conditions for biosynthesizing inulin were also optimized,and site-directed mutagenesis was performed to improve the enzymatic activity and thermostability.In present study,different methods of N-terminal truncation were used to construct recombinant inulosucrase.The corresponding truncated genes were inserted into pET-22b?+?to construct recombinant plasmids,which were transformed into E.coli BL21?DE3?and induced with IPTG for protein overexpression.The activities of the two truncated inulosucrases were determined at an optimal pH and temperature.We determined that different methods of N-terminal truncation changed the optimal pH and temperature of the recombinant inulosucrases,but had little influence on enzyme activity.The enzymatic properties of Laga-ISase?the signal peptide and 101 amino acids at the N-terminal and C-terminal were truncated?functioned optimally at pH 5.5 and 35?.Most of the tested bivalent metal ions improved the total activity of Laga-ISase.Remarkably,Mn²⁺increased the total activity to 157%.Compared to reported inulosucrases,Laga-ISase was the most thermostable,and the relative activity was maintained at 84%at 45? for 3 h.The denaturation temperature?T_m?of Laga-ISase was determined to be 55?.The kinetic behavior of the total and transfructosylation reactions did not follow Michaelis-Menten kinetics,but could be described by the Hill equation.However,the hydrolysis reaction obeyed Michaelis-Menten kinetics because the hydrolysis activity could be saturated by sucrose,and the K_m of hydrolysis reaction was calculated to be 44.12 mmol/L.The type of glycosidic bond was determined by FTIR and NMR,and the produced polysaccharide was determined to be inulin with a?-?2,1?fructosyl linkage.The reaction conditions for inulin biosynthesis were optimized using 30%sucrose as the sole substrate.The optimal enzyme dosage and reaction time were determined to be 4.5 U/g sucrose and 1.5 h,respectively.The average molecular weight of inulin produced under optimal conditions was5.86?10⁶ g/mol.Thirteen site-directed variants were then designed and generated by combining molecular biological methods with computational simulation.The activities and denaturation temperatures of these variants were measured,and residues with important roles on activity and stability were determined.Compared to the wild-type enzyme,the total activity and T_m of variant Q196E decreased to 37.8%and 54.2?,respectively,which implies that Gln196 is significant for activity and structural stability.In contrast,variant D288E enhanced the total activity to 131.5%.The T_m of other variants improved by varying degrees:variants A310E,S346A,I478M and A491S increased by 1.38,1.39,1.83 and 1.31?,respectively.