Characterization,Molecular Modification,Product Extension Mechanism And Application Of Inulosucrase

Inulin and levan are two types of fructans in nature.Their fructosyls are connected byβ-（2,1）andβ-（2,6）glycosidic bonds,respectively.Inulin is a solvable dietary fiber occurring in many Compositae plants widely.Inulin has many exceptional physiological functions,such as promoting mineral absorption,regulating the immune system,anti-obesity,anti-diabetes,promoting the proliferation of intestinal probiotics,etc.Inulin also has many good physicochemical properties,such as solubility,stability,thickening,gelation,etc.Therefore,inulin has promising application potential in food,medicine,cosmetics,and other fields.Microbial inulosucrase（IS,EC 2.4.1.9）and levansucrase（LS,EC 2.4.1.10）can directly synthesize inulin and levan,respectively,through a one-step reaction using sucrose as the sole substrate.The two fructansucrases have many common properties in the crystal structure,catalytic mechanism,reaction process,and enzymatic properties,especially IS and LS from Lactobacillus.At present,domestic studies about the two fructansucrases mainly focus on LS,while few reports focus on IS,which may result from the limited microbial sources of IS.The reported ISs are mainly from Lactobacillus,a probiotic.Therefore,this study selected ISs from Lactobacillus as the research object,constructed recombinant plasmids,carried out heterologous expression and enzymatic properties identification,and ISs with excellent properties were screened out.The rational design was used to modify IS,and the activity and thermostability were improved.The product chain length of IS was modulated by semi-rational design,and the product specificity of IS was altered,making IS directionally accumulate 1-kestose.The crystal structure of IS was resolved to reveal the key residues controlling the sugar chain elongation of IS and LS.The sugar-chain extension mechanism of IS catalyzing the synthesis of polysaccharides and oligosaccharides was proposed.The specific research contents and conclusions are as follows:（1）Characterization of novel IS.The accession numbers of ISs from L.jensenii JV-V16and L.psittaci DSM 15354 in the NCBI database are EFH30007.1 and KRL62549.1,respectively.Laje-IS△93-683 and Laps-IS△94-657 were constructed according to the reported truncation ways of ISs from L.johnsonii NCC533 and L.gasseri DSM 20604 by partially truncating N-and C-terminal sequences.The corresponding gene sequences of the truncated enzymes were respectively introduced into the vector p ET-22b（+）to construct the recombinant plasmids.The recombinant plasmid was transferred into E.coli BL21（DE3）competent cells for heterologous expression,isolation,purification,and characterization.The molecular mass of Laje-IS△93-683 and Laps-IS△94-657 were 65.6 k Da and 63.4 k Da,respectively,consistent with the theoretical results.The optimum p H and temperature of Laje-IS△93-683 are p H 6.0 and 45 ^oC,respectively,and the T_mvalue is 64.01 ^oC.The optimum p H and temperature of Laps-IS△94-657 are p H 5.5 and 45 ^oC,respectively,and the T_mvalue is53.91 ^oC.The activities of both enzymes were inhibited by most metal ions.Laje-IS△93-683exhibits high thermostability with a half-life of 14.5 h at 60 ^oC.The inulin production of Laje-IS△93-68 reached approximately 287.4 g/L at p H 6.0 and 45 ^oC after 6 h,and the conversion rate of sucrose to inulin was about 46%,with 600 g/L sucrose as substrate and 18.2 U/g sucrose enzyme dosage.Laje-IS△93-683 also shows a high transfructosylation capacity,and the ratio of transfructosylation activity and hydrolysis activity（T/H）was the highest compared to all identified ISs.Prominent transfructosylation products were produced using D-xylose,D-maltose,and D-trehalose as the acceptors of the fructosyl moiety of sucrose.（2）Food-grade expression of IS and physicochemical characterization of microbial inulin.The gene of IS（Laga-ISΔ138-702）from L.gasseri DSM 20604 was inserted into the vector of Bacillus subtilis to construct the plasmid p UB-P43-IS-DAL.The recombinant plasmid was transferred into B.subtilis 1A751（dal-）whose gene of D-alanine racemase was deleted.The D-alanine screening way was used instead of antibiotic screening to express the recombinant enzyme.The produced inulin was separated and purified,and its physicochemical properties were identified.Scanning electron microscopy demonstrated that the inulin had a structure analogous to that of Arabic gum,and atom force microscopy identified a long-chain structure without branches.Thermal gravimetric analysis showed three weight loss stages,and the melting enthalpy and peak temperature were determined to be 145.4 J/g and 131.6°C,respectively,by differential scanning calorimetric analysis,which was similar to vegetal inulin.The small-angle X-ray diffraction pattern exhibited weak peaks at 2?=8.21°,16.19°,and18.52°.A 3%microbial inulin solution showed the characteristics of a Newtonian fluid,and the6%,9%,and 12%solutions showed the shear-thinning property of a non-Newtonian pseudoplastic fluid.A 3%microbial inulin solution showed gelation,and the gel strength increased with the increase in concentration.Additionally,microbial inulin exhibited better storage stability than vegetal inulin.（3）The activity of IS was improved by truncation.IS and LS from L.reuteri 121 were used to construct Lare121-ISΔ121-701 and Lare121-LSΔ103-686,respectively,according to the most widely studied truncation method.The effects of N-and C-terminal regions on enzyme activity and stability were analyzed based on Lare121-ISΔ121-701 and Lare121-LSΔ103-686,Lare121-ISΔ177-701 and Lare121-LSΔ154-686（truncating N-terminal flexible region）,Lare121-ISΔ207-701 and Lare121-LSΔ186-686（further truncating N-terminal flexible region）,Lare121-ISΔ121-676 and Lare121-LSΔ103-654（truncating C-terminal flexible region）,Lare121-ISΔ177-676 and Lare121-LSΔ154-654（truncating N-and C-terminal flexible region simultaneously）.It was found that Lare121-ISΔ177-701 and Lare121-LSΔ154-686,whose flexible N-terminal regions were truncated,showed the highest enzyme activity,but the product chain length distribution did not change a lot.The ISs and LSs from other Lactobacillus were constructed according to the truncation mode of Lare121-ISΔ177-701 and Lare121-LSΔ154-686,and the universal promoting effect of enzyme activity through N-terminal truncation was found.However,the effect of the N-terminus on the thermostability of IS and LS from Lactobacillus did not show a uniform rule and varied with the source of the enzyme.（4）Site-directed mutation was used to improve the thermostability of IS.Molecular modification of Laga-ISΔ138-702 was carried out to improve its thermostability by site optimization strategy based on phylogenetic analysis and regional modification strategy based on the unique region.Four variants,A466E,S482A,I614M,and A627S,whose T_m values increased by over 1°C,were screened from 11 sites.The quadruple variant M4^N-33with a 6.2°C improvement in T_m value and a 120 times improvement in half-life at 55°C was obtained by selecting specific sites,performing site-directed mutation,combining positive mutation,and truncating partial sequence.The optimum p H of M4^N-33was consistent with Laga-ISΔ138-702,and the optimum temperature of M4^N-33 increased by 5°C.The enzyme activity of M4^N-33 had a 52%increase compared to that of Laga-ISΔ138-702 at optimal conditions,and the inulin production of M4^N-33 was unchanged.Molecular dynamics simulations showed that the variant M4^N-33 reduced the flexibility of the loops on the surface and thus improved the thermostability.（5）Site-directed mutation was used to alter the product specificity of IS.The variants of Lare121-ISΔ121-701 were screened and constructed to control the product chain length and directionally synthesize 1-kestose by semi-rational design.Arg541 and Arg544 were determined to influence the chain length distribution of the products significantly.The variants of Arg541 showed severe loss of enzyme activity,and the saturated mutagenesis of Arg544could produce more 1-kestose than the wild-type enzyme.In particular,the variant R544W changed the product specificity of IS,and produced short-chain oligosaccharides（Sc FOS）with1-kestose as the main component.Molecular dynamics simulations confirmed that the binding free energy of R544W to 1-kestose was higher than that of the wild-type enzyme,making 1-kestose less likely to bind to the catalytic pocket and be further extended,and thus it was accumulated.The maximum production of 1-kestose and total Sc FOS reached 206 g/L and 307g/L,respectively,by optimizing the reaction conditions for 1-kestose production using variant R544W.（6）Crystal structure and product extension mechanism of IS were resolved.Taking Lare121-ISΔ121-701 and Lare5448-LSΔ145-698 as the research objects,the high-quality crystal structures of Lare121-ISΔ121-701 and its variant IS-R544W were obtained by X-ray diffraction.Nine sites were found in the same positions of IS and LS through molecular docking and structural alignment.Alanine and tryptophan substitution variants at these sites were designed and constructed.The 36 mutants had similar or different effects on the product distribution of IS and LS.By structural analysis and product chain length distribution analysis of these variants,four potential sugar-chain extension tunnels in IS were found and the mutational sites could be divided into two residue groups.Residue group 1 is located near tunnel2,which controls the polysaccharide synthesis of IS,while residue group 2 is situated near tunnel 4,which controls the oligosaccharide synthesis of IS.However,both residue groups control the oligosaccharide synthesis of LS.The double"U"mechanism for sugar-chain extension of IS in the synthesis of polysaccharides and oligosaccharides was further proposed.