The Screening And Application Of ?-L-rhamnosidase Production Strain

Flavonoids are distributed in natural plants in the form of free or glycosides.The structure of flavonoids is complex and diverse due to the different of glycosides and connection bonds.Flavonoids have the pharmacological activities of scavenging free radical,antioxidation,anti-tumor,hypolipidemic,anti-virus and other activities.However,flavonoids have poor water solubility,resulting in low utilization efficiency.In order to improve the water solubilization of flavonoids,the structures of flavonoids are mainly modified,and deglycosylation is a common method to modify the structure of flavonoids.Deglycosylation includes traditional chemical hydrolysis and bioenzyme catalysis.The traditional chemical method uses H₂SO₄ or HCl to hydrolyze flavonoids.This method is fiercely reactive,requires high equipment,and with by-products.The biocatalytic reaction of flavonoids was carried out by enzyme catalysis.Most of the flavonoids have rhamnose at the end,affecting their water solubility and pharmacological activity,?-L-rhamnosidase can be highly efficient and specific for hydrolysis of the terminal rhamnose of flavonoids,which in turn increasing water solubility and the utilization efficiency.Therefore,it is very important to screen the strains of?-L-rhamnosidase.In this study,the strains were firstly screened from mildewed grapefruit and soil nearby,and 20 strains producing?-L-rhamnosidase were initially screened.Among them No.5 strain had the highest enzyme activity,the enzyme activity of?-L-rhamnosidase up to 484.25 U/mL.Observing the colony morphology and microscopic structure of No.5 strain,the strain was initially identified as a strain of Aspergillus Niger.The 18S rRNA sequence of the strain was compared with that of the NCBI database to construct a phylogenetic tree.The 18S rRNA sequence has 99%similarity to Aspergillus Niger 513.88.Therefore,the strain can be identified as Aspergillus Niger and named as Aspergillus Niger NCU-317.The culture medium and fermentation conditions for the production of?-L-rhamnosidase by Aspergillus Niger NCU-317,with the enzyme activity as the optimization index.And result show that the optimum composition of the medium was:4 g/L sucrose,4 g/L yeast extract,0.2 g/L MgSO₄,2 g/L rutin;Optimum fermentation conditions for the optimal pH was 5,the optimum temperature was 28?,the best speed is 180 r/min,the best fluid volume was 100 ml,the best fermentation time was 72 h.After optimization,the activity of?-L-rhamnosidase produced by Aspergillus Niger NCU-317 was 675.8 U/m L,1.4 times of the initial PDA medium.By using ammonium sulfate graded precipitation and gel chromatographic chromatography,the separation and purification of?-L-rhamnosidase from Aspergillus Niger NCU-317 was carried out.When the purification multiple was 1.27and 3.51 respectively,the recovery rate was 80.00%and 57.86%respectively.The molecular weight of the enzyme protein determined by SDS-PAGE gel electrophoresis was 58.1 kDa.The enzymatic properties of?-L-rhamnosidase were investigated.The enzyme was stable to high temperature and acidity.At 70?for 2 h,the enzyme activity was 262 U/mL,about 1/3 of the enzyme activity at the optimum temperature.The enzyme activity was measured at pH 2⁷ for 2 h,and the enzyme activity was almost unaffected.The optimum reaction temperature was 50?,the optimum pH was 5,Mg²⁺and Mn²⁺could increase the enzyme activity,Fe²⁺,Cu²⁺,and Hg²⁺.Inhibit enzyme activity.As a result,the Michaelis constant K_m was 14.03mmol/?L·min?,and the maximum reaction rate V_max was 22.47 mmol/?L·min?.The ?-L-rhamnosidase produced by Aspergillus Niger NCU-317 was used to hydrolyze rutin to prepare isoquercitrin.The conversion rate of rutin was up to 95%,and the product was separated and purified by silica gel column chromatography.The purity of isoquercetin was as high as 99.1%by HPLC.The structure of the product was characterized by NMR and MS.