Study On The Synthesis Of Fructo-oligosaccharide (FOS) By Fructansucrase From Lactobacillus Reuteri

Fructooligosaccharides?FOS?,a natural active substance,is famous for numerous health function,especially on the regulation of intestinal flora,and the proliferation of bifidobacteria.At present,bio-enzymatic engineering technology has become the main method to produce FOS.Therefore screening,optimization and application of high-efficiency fructosyltransferases have caught much interesting.Sequence analysis of the full-length levansucrase gene in Lactobacillus reuteri FS002,which was isolated from feces of giant panda,revealed a 2523 bp gene and encodes a protein of 806 amino acid residues.The protein includes a signal peptide of38 amino acid residues at the N-terminus,and the C-terminal 60 amino acids contain a LPQTG cell wall anchoring region,which suggests that this enzyme is a cell surface protein.lev gene was amplified by PCR and the recombinant Escherichia coli BL21?DE3?/pET-lev expressing the corresponding protein were constructed for recombinant Lev expression.SDS-PAGE showed that 100 kDa protein was expressed in E.coli under induction of IPTG.The optimal expression conditions of the recombinant strain BL21?DE3?/pET-lev were studied,which found that when the OD₆₀₀ was 0.6,the inducer IPTG was added at a final concentration of 0.4 mmol/L,the recombinant protein Lev reached the maximum expression level after 4 hours of induction under 37^oC.Soluble pure Lev was obtained after affinity chromatography followed by gel filtration.Analysis of genes and proteins of 4 levansuacrase in L.reuteri showed that levansucrase genes was founded in 3 L.reuteri strains.however with 2 of them having mutation.These mutation could cause the deletion of the enzyme protein.The levansucrase gene in L.reuteri FS002 isolated from the panda faeces can only encoded a complete enzyme.Western blotting showed that a 100 kDa protein was only detected in FS002strain.The enzyme activity assay also showed that FS002 showed higher sucrose hydrolysis activity than the other strains.This indicates that mutations in the levansucrase have occurred in different sources of L.reuteri for some unkown reason,which could be related to the adaption of microorganisms in different environments.The enzymatic properties of Lev were studied by measuring in the production of reducing sugars after enzymatic reactions.The results showed that Lev had a maximum enzyme activity of 425 U/m L at pH 5.5 and 55°C,also had good stability at45°C.The Michaelis constant K_m for sucrose was 22.83 mmol/L,and the maximum reaction rate V_max was 0.86?mol/?mL.min?.FOS synthesis was studied using sucrose,maltose,lactose,glucose,fructose,and galactose as substrate,respectively,which revealed that only sucrose can be used as a substrate.As reported levansucrase,Lev also need Ca²⁺to develop activity.However our study showed that Ca²⁺could be substituted by Mn²⁺and Lev showed higher activity with the later ion existing.These results show the difference between this enzyme and the other reported levansucrase.Recombinant Lev was expressed to synthesize FOS in vitro with sucrose as substrate.Thin-layer chromatography?TLC?showed that the recombinant Lev had strong hydrolytic activity on sucrose and showed a certain activity of FOS synthesis.The synthetic oligosaccharide bands were estimited by their relative migration rate to be kestose and iso-kestose.The production of FOS by the recombinant Lev was optimized by conventional reaction conditions such as temperature,pH,and metal ions.The results showed that the highest synthesis of FOS was achieved when using177?g/m L enzyme,400 g/L sucrose,100 mmol/L Mn²⁺under pH 6.5,45°C and reacted for 30 min.However,site-directed mutagenesis of M262L and N384D mutations in the Lev significantly reduced Lev hydrolysis activity to less than50U/mL,and the synthesis activity of FOS was not improved.