Targeted Mining Of The Hyperthermostable Levansucrase And The Modification Of Product Distribution

At present,most of the studies about fructans are focused on the levan and inulin,which are linked byβ-（2,6）andβ-（2,1）-type bonds,respectively.Due to its excellent physicochemical properties and physiological functions,levan is widely used in various fields.For example,levan can be used as antibacterial nanomaterials in the materials field,prebiotic and structural modifier in the food field,and anti-inflammatory and anticancer adjuvant in the medical field.Unlike inulin,levan is rare in nature,and its mainly producer is levansucrase（LS,EC 2.4.1.10）.The main contents and conclusions are as follows:（1）Targeted mining and expression of hyper-thermostable LS.A computer-aided enzyme screening method combined with the Enzyme Miner online server was employed to screen out potential thermostable LSs in this study.The obtained LSs were from C.diazotrophica and P.orientalis,respectively.Sequence alignment and phylogenetic tree analysis indicated that they were potential LSs.The recombinant plasmid was constructed and enzyme heterologous express in the Escherichia coli BL21.The purification of target protein was carried out via Ni²⁺-affinity chromatography.（2）Enzymatic properties of recombinant LSs.The C.diazotropica and P.orientalis LS both showed the maximum activity at 65°C,and their optimal p H was 5.5 and 6.5,respectively.The specific enzyme activity against sucrose（total）of C.diazotropica and P.orientalis LS was 1003 and 1219 U/mg,respectively.Meanwhile,C.diazotropica LS remained more than 90%activity when incubated at 45°C for 72 h,and its half-life at 55°C was 97 h,exhibiting an excellent thermostability.（3）Product optimization and identification of recombinant LS.The production conditions of levan by C.diazotropica and P.orientalis LS were optimized.The optimized conditions of C.diazotropica LS was conducted used 20μg/m L enzyme and 30%sucrose under p H 5.5 and 65°C.The optimal production of P.orientalis LS was conducted at 30%sucrose and 25μg/m L enzyme at p H 6.5 and 65°C.The conversion ratio of sucrose to levan of two LSs can reach 30.2%and 29.2%,respectively.The products of two recombinant LSs were determined by NMR,and it was proved that the polysaccharide produced by two LSs wasβ-（2,6）type fructan.（4）The effect of temperature on the product distribution of recombinant LSs.At 65℃,The C.diazotropica LS produced FOS,LMW,and HMW levan simultaneously in the reaction,while the P.orientalis LS would specifically produce FOS and a small amount of HMW levan.The molecular weight of levan synthesized by C.diazotropica LS was increased when the temperature was decreased.Meanwhile,the production of HMW levan was also increased.On the contrary,P.orientalis LS prefered to generate FOS rather than HMW levan at 35°C.（5）The change on product distribution through semi-rational design based on structure.A series of E.amovora LS mutants were built based on the corresponding oligosaccharide binding site in B.subtilis LS.The mutants with different product spectrum were successfully obtained.The mutants of G98E,V151F,and N200T around the loop 3 and loop 4 all showed a significant increase in both the molecular mass and production of HMW levan,suggesting that the polymerization of E.amovora LS was successfully strengthened.