Study Of Lactobacillus Acidophilus ?-Glucosidase And Its Interaction With Quercetin Glycosidase Substrate

Lactobacillus acidophilus is an important probiotic,and its function development and application are concern.Previous studies had found that Lactobacillus acidophilus GIM 1.208 has good fermentation quality characteristics for Rosa roxburghii Tratt Juice,especially significant the activity of?-glucosidase,and has obvious release function for flavonoid aglycone-quercetin,but the relevant mechanism is still unclear.Therefore,Lactobacillus acidophilus GIM 1.208 was selected as the fermentation strain in this study.Firstly,the culture medium and fermentation conditions of Lactobacillus acidophilus were optimized,and then the physical and chemical properties of the protein and its primary and Secondary structure were explored by molecular biology.At the same time,gene cloning technology was used to amplify the target gene fragment for heterologous expression and purification,and its secondary structure was explored by Circular Dichroism.In addition,the enzymatic properties of reombinant?-glucosidase and?-glucosidase produced by Lactobacillus acidophilus GIM 1.208 were compared and analyzed.Finally,the binding modes of rutin?Rut?and isoquercetin?Iso?in quercetin glycoside substrate with reombinant enzyme were studied by Fluorescence Spectrum,Ultraviolet Absorption Spectrum and synchronous Fluorescence Spectroscopy.The quenching mechanism of?-glucosidase by Rut or Iso and their effects on the conformational change of?-glucosidase.The details and results were as follows:1.Study on fermentation conditions of?-glucosidase production by Lactobacillus acidophilus GIM1.208.By single factor test,carbon source,inducer and initial pH was studied,then maked L₉?3⁴?orthogonal test to optimized the culture medium of Lactobacillus acidophilus fermentation.The results showed that the main order of?-glucosidase production by Lactobacillus acidophilus GIM1.208 was as follows:inducer>carbon source>initial pH value,in which carbon source and inducer had significant effects on?-glucosidase production of Lactobacillus acidophilus?P<0.05?,the initial pH value had no significant effect on the enzyme production by Lactobacillus acidophilus?P>0.05?.The optimal fermentation enzyme production medium was:glucose of 3%?v/w?,sodium carboxymethyl cellulose of 0.4%?v/w?,initial pH value of 5.5,the obtained enzyme activity was 11.684?U/mL.min?,which was 17.20%higher than before optimization.Through the single factor test of fermentation temperature,fermentation time,ratio of material to liquid and inoculating quantity,the significant factors affecting the enzyme production of Lactobacillus acidophilus were screened,and the fermentation conditions were optimized by Box-Behnken test.The results showed that the fermentation temperature,solid-liquid ratio and inoculum amount had significant effects on the production of?-glucosidase by Lactobacillus acidophilus?P<0.05?.The optimal fermentation conditions were:Sodium carboxymethyl cellulose of 0.4%?v/v?,initial pH of 5.5,fermentation temperature of 31?,fermentation time of 24 h,the inoculum of 2.5%?v/w?,and solid-liquid ratio of 1:4?v/v?.The activity of the enzyme obtained by the fermentation was 16.8446 U/mL.min,which was 12.06%higher than that before the optimization.By validating the prediction model,the difference between the measured value and the theoretical value was 0.08%.The model can truly reflect the case of Lactobacillus acidophilus producing?-glucosidase.2.Bioinformatics method to explore the structural characteristics of?-glucosidase?1?Sequence analysis of the coding gene of?-glucosidaseThe coding gene sequence of?-glucosidase was obtained and the sequence of?-glucosidase was analyzed.The results showed that the coding sequence of?-glucosidase was 1278 bp,among which A accounted for 34.06%,T 33.26%,G 20.27%and C 12.52%.The sequence was composed of 426amino acids,among which Asp and Lys had the highest frequency?8.5%and8%?,followed by Iie?7.7%?and Gly?6.6%?.The used frequency of His was the lowest?0.7%?.?2?Analysis of physicochemical properties of?-glucosidaseThe physicochemical properties of the nucleic acid sequence encoding?-glucosidase were analyzed by on-line analysis tool of NCBI database.The results showed that the total hydrophilicity of?-glucosidase was-0.455 and the pI value was 5.43,which was an acidic protein.The protein has a negatively charged amino acid residue number?Asp+Glu?of 60 and a positively charged amino acid residue number?Arg+Lys?of 54.The amino acid sequence has an instability coefficient of 39.85?less than its threshold of 40?,and the stability of the protein was determined.?3?Primary structure analysis of?-glucosidaseThe hydrophilic/hydrophobic,transmembrane domainproperties,signal peptide information,phosphorylation site of?-glucosidase were predicted and analyzed by online analysis tools.The hydrophilicity of?-glucosidase was stronger and it belonged to soluble protein.The maximum value of C-score at the 29th cleavage site of amino acid sequence was 0.118,and the S value was lower than the threshold value of secretory protein 0.5,which was non-secretory protein.At the same time,the protein contained 39 phosphorylation sites,including 15 Thr phosphorylation sites,13 Tyr phosphorylation sites and 11 Ser phosphorylation sites.The subcellular localization of protein was predicted by Cell-PLoc 2.0,and the enzyme was located in the cell membrane.Therefore,it was preliminarily speculated that the enzyme was located in the cell membrane,that was a soluble non-secretory protein,has strong hydrophilicity and has no signal peptide,transmembrane domain,and has multiple phosphorylation sites.?4?Analysis of homologous sequence alignment of?-glucosidase and construction and analysis of phylogenetic treeThe encoded amino acid sequence of Lactobacillus acidophilus?-glucosidase was introduced into the NCBI database,and homologous sequence alignment analysis was performed using BLAST.The results showed that the amino acid sequence of Lactobacillus acidophilus?-glucosidase was considered to be important for the recognition and binding of substrates.The conserved regions of amino acid residues were located at positions 210²14,which were Tyr?Y?and Gln?Q?,Ser?S?,Gly?G?,His?H?.By constructing a phylogenetic tree of?-glucosidase and performing homology alignment analysis.The results showed that:Lactobacillus acidophilus?-glucosidase had the highest homology with the hydrolase HMPREF0492?accession number EEJ75268.1?from Lactobacillus acidophilus ATCC 4796,and had the highest affinity with the sequence;followed by Lactic acid bacteria DSM 20249?accession number KRK76876.1?was 100%identity with its amino acid sequence;It was the farthest related to bacillus polymyxis?accession number SUA71531.1?and bacillus polymyxis sqr-21?accession number AHM67667.1?.?5?Secondary structure analysis of?-glucosidaseThe secondary structure of the protein was predicted using PSIPRED and WOLF SOPMA on-line analysis software.The results showed that PSIPRED predicted 24.88%of?-helix,15.26%of?-turn in the secondary structure of the protein,and the Random coil element penetrated the entire amino acid sequence.The WOLF SOPMA predicted protein secondary structure of the protein was mainly composed 46.95%of Random coil,29.58%of?-helix,17.37%of?-Prarallel,and 6.10%of?-turn.Therefore,it was known that the secondary structure of the protein mainly consists of an?-helix structure,?-turn structure,and Random coil.3.Cloning,expression,purification and circular dichroism analysis of?-glucosidase geneThe target gene of?-glucosidase was obtained by PCR in vitro amplification technology,and the target gene fragment and the vector PET-28a?+?was double-digested,ligated,transformed,and the positively cloned screening to obtain the recombinant vector PET-28a?+?-bgl was transformed into competent E.coli to induce expression.The cell was broken by ultrasound,subjected to nickel affinity chromatography,overnight dialysis by dialysis bag,concentrated by PEG 20000,concentrated by a microfiltration membrane,and verified for protein purity by SDS-PAGE and Western Blotting.The purified target protein was subjected to circular dichroism to determine its secondary structure.The results showed that the reombinant enzyme was successfully expressed and the purity of the reombinant enzyme reached electrophoretic purity.The concentration of?-glucosidase was 1.88 mg/mL.In the far ultraviolet region of 190²60 nm,the?-helix structure of the?-glucosidase secondary structure accounted for 15.9%,the?-Parallel accounted for 44.1%,the?-turn angle accounted for 18.1%,and the Random coil component accounted for 27.2%.4.Study on the enzymatic properties of?-glucosidase?1?Optimum reaction temperature and thermal stability of?-glucosidaseTo explore the optimum reaction temperature of wild-type enzyme and reombinant enzyme at20⁹0°C;incubate it at different temperatures to study the thermal stability of wild-type enzyme and reombinant enzyme.The results showed that the optimal reaction temperature of wild-type enzyme and reombinant enzyme was 47°C.Both wild type?-glucosidase and reombinant enzyme have good thermal stability at 20⁵0°C.The enzyme has good heat resistance and thermal stability.?2?Optimum reaction pH and pH stability of?-glucosidaseTo explore the optimal reaction pH of wild-type enzyme and reombinant enzyme in the catalytic reaction system at pH 2.2-8.0,and let it sit at different pH conditions for 4 h,study pH stability of wild-type enzyme and reombinant enzyme.The results showed that the optimum pH difference between wild-type enzyme and reombinant enzyme was small,and the optimal pH value of wild-type enzyme was 5.0.The optimal reaction pH of reombinant enzyme was 5.6.Both wild-type enzyme and reombinant enzyme had certain pH stability at pH 2.2⁶.0.The enzyme not only has a wide range of pH stability,but also has a certain acid resistance.?3?Effect of organic solvents on?-glucosidaseThe effects of four kinds of organic solvents such as absolute ethanol,methanol,ethyl acetate and acetone on the activity of wild-type enzyme and reombinant enzyme were investigated.The results showed that methanol,ethyl acetate and acetone had stronger inhibitory effect on?-glucosidase;anhydrous ethanol inhibited wild-type enzyme and reombinant enzyme relatively weakly.Anhydrous ethanol,methanol,ethyl acetate and acetone had different degrees of inhibition on wild-type and reombinant enzyme.?4?Effect of metal ions on?-glucosidaseThe effects of metal ions such as Na⁺,Ka⁺and Li+on wild-type enzyme and reombinant enzyme were investigated.The results showed that Na⁺,Ka⁺,Li⁺,Mg²⁺,Zn²⁺and Al³⁺inhibited wild-type enzyme and reombinant enzyme to different extents.The inhibitory effects of Ka⁺,Li⁺,Mg²⁺and Zn²⁺on?-glucosidase were relatively small.However,Hg²⁺,Ca²⁺,Cu²⁺,Ba²⁺,Fe³⁺and Fe²⁺increased the enzyme activity Significant.The promotion of wild-type enzyme and reombinant enzyme by Fe³⁺and Fe²⁺was more prominent.Na⁺and Al³⁺had stronger inhibitory effects on wild-type enzyme and reombinant enzyme.The inhibitory effects of Ka⁺and Mg²⁺on wild-type enzyme were not significant compared with other metal ions,and the inhibitory effects of Ka⁺,Li⁺and Zn²⁺on reombinant enzyme were not significant.?5?tolerance of?-glucosidase to Glucose and NaClTo explore the effects of different concentrations of glucose and NaCl on wild-type enzyme and reombinant enzyme.The results showed that 1.5 mol/L glucose promoted the maximum activity of wild-type enzyme and reombinant enzyme,and the relative enzyme activity increased by 84.30%and 39.01%.reombinant enzyme had better glucose tolerance than wild-type enzyme and was suitable for subsequent application development.2.0 mol/L NaCl had a certain activation effect on the activity of wild-type and reombinant enzyme.The enzyme activity was increased by79.11%and 51.40%.NaCl was more resistant to reombinant enzyme than wild-type enzyme.?6??-glucosidase kinetic parametersUsing p-NPG,rutin,isoquercetin as substrates to interact with reombinant enzyme and wild-type enzyme to explore the kinetic parameters of different substrates and?-glucosidase.Michaelis constant K_m and maximum reaction rate V_max.The results showed that when the recombinant and wild-type enzyme reacted with p-NPG,the K_m values were 0.204 mmol/L and0.684 mmol/L,and the V_maxax values were 0.133 mmol/L.min and 0.662 mmol/L.min.When wild-type enzyme reacts with Rut and Iso,the K_m value was 1.000 mmol/L,0.601 mmol/L,and the V_maxax value was 1.340 mmol/L.min and 1.157 mmol/L.min;when recombinant?-When glucosidase reacts with Rut and Iso,the K_m value is 0.357 mmol/L,0.326 mmol/L,and the V_maxax value was 0.822 mmol/L.min and 1.030 mmol/L.min.Therefore,it is known that the affinity between the reombinant enzyme and p-NPG is stronger than that of the wild-type enzyme,and K_m?Rut?<K_m?Iso?indicates that Rut interacts was better ability with?-glucosidase.5.Fluorescence spectroscopy analysis of the interaction between?-glucosidase and quercetin glycoside substrateFluorescencespectroscopy,ultravioletabsorptionspectroscopyandsimultaneous fluorescence spectroscopy were used to investigate the binding mode of Rut and Iso to?-glucosidase,the quenching mechanism of Rut and Iso to?-glucosidase,and their effects on the conformational changes of?-glucosidase.The results show that:?1?The binding distances of?-glucosidase to Rut and Iso was 4.52×10^-33 nm and 3.65×10^-3nm,both less than 7 nm and r_?Rut?>r_?Iso?.It can be concluded that there was non-radiative energy transfer between Rut or Iso and?-glucosidase,and the binding distance of Iso and?-glucosidase was closer than that of Rut,which was more conducive to the interaction of Iso and?-glucosidase.Due to the non-radiative energy transfer between the?-glucosidase and the quercetin glycoside substrate,the endogenous fluorescence intensity of the?-glucosidase was reduced,resulting in fluorescence quenching.?2?The fluorescence quenching effected of Rut and Iso on?-glucosidase accords with the static quenching mechanism.The quenching constant K_SVV of Rut was 6.13×10⁵ L/mol,and the rate constant K_q was 6.13×10¹33 L/mol;The K_SVV of Iso was 3.13×10⁵ L/mol and its K_q was 3.13x10¹³L/mol.The quenching constant of Rut for?-glucosidase was greater than Iso,indicating that the fluorescence quenching effect of Iso on?-glucosidase was stronger than that of Rut.It also affected the conformation of?-glucosidase to some extent.?3?When??=15 nm,the maximum emission wavelength of Rut-?-glucosidase system was blue-shifted?284?279 nm?;Iso promotes blue shift of the maximum emission wavelength of?-glucosidase catalytic reaction system?278?277 nm?,which indicated that the hydrophobicity of tyrosine residues in the microenvironment was enhanced.When??=60 nm,the effects of Rut and Iso on the microenvironment of Trpptophan residues were not obvious.At the same time,under the same fluorescence quenching condition,the synchronous fluorescence quenching effected of Iso on Trpptophan residues was stronger than Rut,but Rut had a greater influence on the hydrophobicity of the microenvironment of tyrosine residue.?4?when Rut and Iso interacted with?-glucosidase,the binding constants K_a of Rut and Iso with?-glucosidase was 0.50×10⁷ L/mol,0.31×10⁷ L/mol,respectively.It is indicating that the interaction between Rut and?-glucosidase was stronger than Iso.