Study On The Biosynthesis Mechanism Of Dextran From Leuconostoc Mesenteroides DRP105

Dextran is a kind of exopolysaccharide（EPS）produced by some lactic acid bacteria（LAB）during the growth and metabolism.Dextran has many biological functions,such as immunomodulation,anti-inflammation,anti-tumor,anti-oxidation and cholesterol lowering.It can be widely used in in food,chemical and pharmaceutical fields as tackifier,stabilizer,emulsifier and gelling agent.However,the research on the biosynthesis mechanism of LAB dextran is very limited.In this study,the high-yielding dextran-producing strain Leuconostoc mesenteroides DRP105isolated from fermented food was used as the starting strain.In order to elucidate the biosynthesis mechanism of Leu.mesenteroide dextran,the genes related to dextransucrase biosynthesis were identified,and the structure and function of dextransucrase were explored.First,the whole genome sequencing of Leu.mesenteroides DRP105 was performed using single-molecule Pac Bio sequencing technology to screen dextransucrase genes,and real-time quantitative fluorescent PCR（RT-PCR）technology was used to verify gene expression.The total length of the genemo was1815345 bp.It consisted of one complete chromosome and five complete plasmids,containing 12 r RNA,71 t RNA and 24 tandem repeats.A total of 140 carbohydrate active enzymes and 1,804 signal peptide sequences were predicted in the chromosome.Three dextransucrase genes dsr A,dsr B and dsr C were screened,and sucrose can significantly induce the expression of dsr C,which is the key gene in dextran synthesis.The prediction of the secondary structure of the enzyme protein indicated that DSR-C contains 17.07%α-helix,29.55%β-sheet,10.18%β-turn,43.20%random curl,and four basic structural regions,which were consistent with the structural characteristics of other dextransucrases.Second,a dsr C gene deletion mutant strain successfully constructed using homologous double exchange technique,and the difference in metabolite levels between the dsr C gene deletion mutant strain and the wild strain Leu.mesenteroides DRP105 was compared and explored.The results showed that the organic acid content of the mutant strain was higher than that of the wild strain,and the dextran yield and free amino acid content were lower than that of the wild strain when sucrose was used as the sole carbon source（P<0.05）.The types and content of metabolites were no significant difference between mutant and wild strains（P>0.05）when glucose was the only carbon source.It showed that the biosynthesis of Leu.mesenteroides DRP105 dextran was mainly regulated by dsr C.Third,the dsr C overexpression strain was successfully constructed using gene cloning technology to obtain recombinant dextransucrase protein.Sodium Dodcyl Sulfate Polyacrylamide Gel Electrophphoresis（SDS-PAGE）technology identified the molecular weight of the dextransucrase protein to be about 170 k Da.The optimal p H value of the enzyme was 5.5,and the optimal reaction temperature was 30℃.Hg²⁺,Pb²⁺,Cu²⁺,Cr³⁺,Al³⁺,Ni²⁺and Fe³⁺inhibited the enzyme activity,and Ca²⁺significantly promoted enzyme activity.Organic solvents,surfactants and inhibitors inhibited the enzyme activity,and the inhibition increased with the increase of concentration.The enzyme was also a dextransucrase whose main components wereβ-turns and random coils.The results of Circular Dichroism（CD）,Raman and Fourier Transform Infrared Spectroscopy（FT-IR）showed that the dextransucrase was an enzyme protein withβ-turn and random coil as the main components.Fourth,the natural dextransucrase from Leu.mesenteroides DRP105 was isolated and purified using ammonium sulfate precipitation,DEAE-Sepharose FF anion exchange chromatography and Sephadex G75 gel filtration chromatography.The structure and properties of dextransucrase were analyzed.Sodium dodcyl sulfate polyacrylamide gel electrophphoresis（SDS-PAGE）and liquid chromatograph-mass spectrometer/mass Spectrometer（LC-MS/MS）technology identified that the molecular weight of the enzyme was about 170 k Da,and the gene sequence had the highest similarity with the dsr C sequence（reached 92%）.The optimal p H value of the enzyme was 5.5,and the optimal reaction temperature was 30°C.Hg²⁺,Pb²⁺,Cu²⁺,Gr²⁺,Al³⁺,Ni²⁺and Fe³⁺had a strong inhibitory effect on the enzyme,low concentration of Na⁺,Li²⁺,Zn²⁺,Co²⁺,Sn²⁺,Mn²⁺and Mg²⁺had a promoting effect,and Ca²⁺significantly promoted the enzyme activity.Organic solvents,surfactants and inhibitors have an inhibitory effect on enzymes.K_m value was 0.61 m M,V_max was3.73 U/mg,and the enzyme has a high affinity for sucrose.The structure and properties of natural dextransucrase from Leu.mesenteroides DRP105 were similar to those of recombinant dextransucrase,and the enzyme was also a protein withβ-turn and irregular curl as its main components.Fifth,dextran with a molecular weight of 9.85×10⁷ Da was successfully synthesized in vitro using recombinant dextransucrase.The results of FT-IR and nuclear magnetic resonance（NMR）showed that the dextran was a highly linear glucan composed ofα-（1→6）glycosidic bonds and has no branched structure.It was different from natural dextran from Leu.mesenteroides DRP105.Enzymatically synthesized dextran has a sheet-like,compact structure and high thermal stability,water retention and solubility,it can enhance milk coagulation and promote the proliferation of probiotics.In summary,the structural and functional characteristics of dextransucrase and the mechanism of dextran biosynthesis of Leu.mesenteroides DRP105 were explored with a variety of biological techniques.The results showed that dsr C was the key gene of dextran synthesis.The dextransucrase expressed by dsr C could catalyze the production of dextran without branching structure,which was different from natural dextran.It may be that the biosynthesis of dextran in Leu.mesenteroides DRP105 was affected by the source of strain,fermentation conditions and other factors,as well as by the catalytic regulation of other enzymes in the process of sucrose metabolism.The results of this study provide a theoretical basis for further revealing the biosynthesis mechanism of LAB dextran and provide a scientific basis for studying the structure-activity relationship of dextran.