CN106995829B - Method for preparing icariin by converting total flavonoids of epimedium herb through enzyme method - Google Patents

Method for preparing icariin by converting total flavonoids of epimedium herb through enzyme method Download PDF

Info

Publication number
CN106995829B
CN106995829B CN201710332977.7A CN201710332977A CN106995829B CN 106995829 B CN106995829 B CN 106995829B CN 201710332977 A CN201710332977 A CN 201710332977A CN 106995829 B CN106995829 B CN 106995829B
Authority
CN
China
Prior art keywords
icariin
rhamnosidase
beta
alpha
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201710332977.7A
Other languages
Chinese (zh)
Other versions
CN106995829A (en
Inventor
赵林果
张珊珊
解静聪
王靖秋
吴涛
葛林
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Forestry University
Original Assignee
Nanjing Forestry University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Forestry University filed Critical Nanjing Forestry University
Priority to CN201710332977.7A priority Critical patent/CN106995829B/en
Publication of CN106995829A publication Critical patent/CN106995829A/en
Application granted granted Critical
Publication of CN106995829B publication Critical patent/CN106995829B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0104—Alpha-L-rhamnosidase (3.2.1.40)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

A method for preparing icariin by converting total flavonoids of herba Epimedii with enzyme method comprises synergistically converting multi-component flavonoid glycoside in total flavonoids of herba Epimedii with a two-enzyme system composed of heat-resistant alpha-L-rhamnosidase and heat-resistant beta-glucosidase to generate icariin. The invention can almost completely convert multi-component flavonoid compounds (icariin, epimedin A, epimedin B, epimedin C and the like) in the epimedium total flavone into target products; the optimal action temperature is high, so that the substrate and the intermediate product have good solubility, and a cosolvent is not needed; short enzymolysis time and high yield. The proliferation inhibition effect of the icariin prepared by the invention on liver cancer, lung cancer, colon cancer and breast cancer is obviously higher than that of each monomer of epimedium total flavonoids before conversion.

Description

Method for preparing icariin by converting total flavonoids of epimedium herb through enzyme method
Technical Field
The invention belongs to the field of enzyme engineering and biological medicine, and particularly relates to a method for preparing epimedium aglycone by catalyzing epimedium total flavonoids with a recombinant enzyme system.
Background
Icaritin (Icaritin) is a polyhydroxy flavonoid monomer component in epimedium herb of Epimedium of berberidaceae, has extremely low content in epimedium herb, is a hydrolysate of icariin, and has estrogen-like effect, and has effects of regulating immunity, promoting regeneration and differentiation of cardiac muscle cells, promoting bone protection, tonifying yang, resisting liver injury, delaying hepatic fibrosis, resisting tumor, etc. With the discovery of new drug effects of the icariin at home and abroad, the international market demand of the icariin in the future is large. Therefore, how to develop a method for efficiently and environmentally preparing icariin is the focus of the current research.
Because icariin is one of flavonoid glycoside compounds with the highest content of epimedium herb and has the same mother nucleus as icariin, the icariin is prepared mainly aiming at icariin at present, and glycosyl groups on carbon positions of No. 3 and No. 7 of the mother nucleus of the icariin are removed by adopting an enzyme method or an acid method so as to obtain the icariin. For example, CN 103305564 converts icariin into icaritin by combining enzymolysis and acidolysis, but because the acidolysis reaction process has poor controllability, a large number of byproducts and low product yield, the key is that the strong acid condition easily changes the structure of the flavone mother nucleus, particularly the methoxy group at the 4' position, so that the process has limitation; CN 101302548 converts icariin into icariin through a commercial beta-glucosidase, although the icariin can be obtained by the enzyme, the hydrolysis time is long (24 hours), the highest enzymolysis yield is only about 55%, according to the enzymolysis effect and the specificity of the enzyme, the used beta-glucosidase comes from the traditional microorganism fermentation, and the enzyme is not pure beta-glucosidase; CN 104561178 adopts naringinase to convert icariin into icariin aglycone. Naringinase is a complex enzyme with activities of alpha-L-rhamnosidase and beta-glucosidase, which is prepared by microbial fermentation, and has the specific characteristic of specifically hydrolyzing naringin. In the method, the proportion of alpha-L-rhamnosidase and beta-glucosidase is not controllable, the sequence of action is not controllable, the reaction time is long (30 hours), and the efficiency is low (the patent only shows that the icariin can be generated).
The results of the existing research can be combined to find that: (1) the existing research process can only prepare the icariin by taking the icariin as a substrate, and can not simultaneously convert the main components (icariin, epimedin A, epimedin B and epimedin C) of the total flavonoids of the epimedium into the icariin; (2) the yield and efficiency of the current preparation of icariin are still to be improved. (3) In the prior art, during the preparation process, epimedium extract is firstly obtained, then icariin is obtained by separation, and then the icariin is converted by an enzymatic method or a microbiological method, so that the cost is greatly increased, and the epimedin A, the epimedin B and the epimedin C are not fully and efficiently utilized. On the other hand, the problem of obtaining a large amount of icariin resources is very prominent, and the large-scale preparation of icariin cannot be guaranteed. The invention provides a method for preparing icariin by converting epimedium total flavonoids by an enzyme method, which can efficiently convert the epimedium total flavonoids almost completely to prepare the icariin only by two glycosidases.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides a method for preparing icariin by converting epimedium total flavonoids by an enzyme method, which obtains alpha-L-rhamnosidase capable of effectively degrading rhamnose residues on an icariin mother nucleus structure and high-temperature resistant bifunctional beta-glucosidase capable of efficiently degrading glucose residues and xylose residues in the mother nucleus structure by successfully screening from a glycoside hydrolase gene library derived from microorganisms, and can efficiently almost completely convert a plurality of types of icariin in the epimedium total flavonoids to prepare the icariin only by two types of glycosidases. Effectively reduces the preparation cost and improves the high-efficiency utilization rate of resources.
The technical scheme is as follows: a method for preparing icariin by converting total flavonoids of epimedium through an enzyme method comprises the step of converting multi-component flavonoid glycosides in the total flavonoids of epimedium into the icariin by a double-enzyme system consisting of heat-resistant alpha-L-rhamnosidase and heat-resistant beta-glucosidase, wherein the multi-component flavonoid glycosides are icariin, epimedin A, epimedin B and epimedin C.
The heat-resistant alpha-L-rhamnosidase is derived from Escherichia coli recombinant bacteria of alpha-L-rhamnosidase gene of Thermotoga petrophila DSM 13995.
The heat-resistant beta-glucosidase is derived from Escherichia coli recombinant strain of Dictyoglycous thermophilum DSM3960GH3 beta-glucosidase gene.
A method for preparing icariin by converting total flavonoids of epimedium through an enzyme method comprises the steps of preparing a citric acid-disodium hydrogen phosphate buffer solution with the pH value of 4.5 and the concentration of 50mmol/L, adding total flavonoids of epimedium to the concentration of 5g/L, simultaneously adding heat-resistant alpha-L-rhamnosidase of Thermotoga petrophila DSM13995 to the concentration of 30U/mL and heat-resistant beta-glucosidase of Dictyoglyces thermophilum DSM3960GH3 family to the concentration of 25U/mL, and reacting for 1h at the temperature of 85 ℃.
The application of a double-enzyme system consisting of heat-resistant alpha-L-rhamnosidase and heat-resistant beta-glucosidase in converting multicomponent flavone glycoside in epimedium total flavone into icariin aglycone.
An enzyme composition for converting multicomponent flavonoid glycoside in total flavonoids of epimedium into anhydroicaritin comprises heat-resistant alpha-L-rhamnosidase and heat-resistant beta-glucosidase.
The icariin is prepared by the method.
The icariin-rich product can be used for preparing medicines for treating breast cancer, lung cancer, colon cancer and liver cancer.
The alpha-L-rhamnosidase used in the method is derived from Aspergillus terreus CCF 3059, Aspergillus niger NL-1, Bacillus theotaomicron VPI-5482, Thermotoga petrophila DSM13995, and the beta-glucosidase is derived from Thermotoga thermus DSM 5069GH1 family, Thermotoga thermalis DSM 5069GH3 family, Thermotoga petrophila DSM13995 GH1 family, Thermotoga petrophila DSM13995 GH3 family, Dictyoglomyus thermophilus DSM3960GH3 family.
The optimal process for preparing the icariin by degrading the total flavonoids of the epimedium with double enzymes comprises the following steps: comparing 1) the simultaneous addition of alpha-L-rhamnosidase from Thermotoga petrophila DSM13995 and beta-glucoside/beta-xyloside bifunctional enzyme from Dictyoglomonus thermophilum DSM3960GH3 family; 2) adding beta-glucoside/beta-xyloside bifunctional enzyme derived from Dictyoglycous thermophilum DSM3960 family and then adding alpha-L-rhamnosidase derived from Thermotoga petrophila DSM 13995; 3) the molar conversion rate of epimedium total flavone prepared by converting epimedium total flavone in a mode of adding alpha-L-rhamnosidase from Thermotoga petrophila DSM13995 firstly and then adding 3 different enzymes of beta-glucoside/beta-xyloside bifunctional enzyme from Dictyoglomonus thermophilum DSM3960 family to prepare the icaritin is determined, and the conversion rate is the highest in a mode of adding the alpha-L-rhamnosidase firstly and then adding the beta-glucoside/beta-xyloside bifunctional enzyme.
Has the advantages that: 1. the invention has wide range of conversion substrates, and the conversion target is expanded from icariin to icariin, epimedin A, epimedin B, epimedin C and the like in epimedium total flavonoids. Improves the yield of the icariin and the utilization rate of the epimedium. 2. The invention uses two recombinant enzymes with definite components to catalyze the epimedium total flavonoids to prepare the icariin, and the molar conversion rate of enzymatic conversion is more than 97 percent. 3. The invention screens alpha-L-rhamnosidase from different sources and provides the alpha-L-rhamnosidase which is derived from Thermotoga petrophila DSM13995 and can efficiently degrade epimedium flavonoid glycoside rhamnose residues, the optimal reaction temperature of the alpha-L-rhamnosidase is 90 ℃, and the temperature stability is good. 4. The invention screens beta-glucosidase from different sources to obtain the bifunctional GH3 family beta-glucosidase capable of efficiently degrading icariin glucose residues and xylose residues, and the optimal reaction temperature of the beta-glucosidase is 85 ℃, and the catalytic efficiency is high. 5. The invention provides a method for efficiently and environmentally preparing icariin, and the prepared icariin has stronger biological activity. Multi-component flavonoid compounds in the epimedium total flavonoids can be almost completely converted into target products; the optimal action temperature is high, so that the substrate and the intermediate product have good solubility, and a cosolvent is not needed; short enzymolysis time and high yield. 6. The product with epimedium aglycone as main flavone component is obtained after double-enzyme conversion of epimedium total flavone, and has activity in inhibiting proliferation of human liver cancer cell HepG2, lung cancer cell A549 and mouse colon cancer cell CT26, which is obviously stronger than that of untransformed epimedium total flavone.
Drawings
FIG. 1 is a schematic diagram of preparing icariin from multicomponent flavone glycoside in total flavonoids of epimedium by enzymatic conversion, wherein the multicomponent of total flavonoids of epimedium comprises epimedin A, epimedin B, epimedin C, icariin and the like. Their specific structural features are shown in the table on the right side of fig. 1.
FIG. 2 is an HPLC chromatogram of a standard substance of the main component in epimedium total flavonoids, wherein 1, epimedin A; 2. fix towards houding B; 3. joading C; 4. icariin, a rare component 5 (baohuoside I) of the icariin and a target product 6 (icariin), and shows that the components can be effectively analyzed by the conventional HPLC method, and the method is efficient and stable.
FIG. 3 is a graph showing the conversion efficiency of various sources of alpha-L-rhamnosidase and various sources of beta-glucoside to epimedium flavone glucoside, respectively. Wherein A is a conversion efficiency chart of converting icariin into icariside I by using alpha-L-rhamnosidase from different sources in 4, and the conversion efficiency is calculated by the content of the icariside I; b is a conversion efficiency chart of 5 different sources of different families of beta-glucoside for respectively converting epimedin A, epimedin B and icariin to produce the baohuoside I, and the conversion rate is calculated by the content of the baohuoside I. Note: in A TPERha represents alpha-L-rhamnosidase derived from Thermotoga petrophila DSM13995, AtRhha represents alpha-L-rhamnosidase derived from Aspergillus terreus CCF 3059, AnRhha represents alpha-L-rhamnosidase derived from Aspergillus niger NL-1, BtRha represents alpha-L-rhamnosidase derived from Bacteroides thetaiotaomicron VPI-5482; TtBGL1 in B is derived from Thermotoga thermomarum DSM 5069GH1 family; TpBGL1 represents a β -glucosidase from the Thermotoga petrophila DSM13995 GH1 family; TtBGL3 represents a protein derived from Thermotoga thermomarum DSM 5069GH3 family; TpBGL3 represents a β -glucosidase from the Thermotoga petrophila DSM13995 GH3 family; DthBGL3 denotes the beta-glucosidase enzyme from Dictyoglomonus thermophilum DSM 3960.
FIG. 4 is a graph comparing the conversion rates of three conversion methods, wherein the conversion rates are calculated by the content of icaritin. The method 1 comprises adding the glucoside/xyloside bifunctional enzyme and then adding the rhamnosidase, the method 2 comprises adding the glucosidase and the rhamnosidase at the same time, and the method 3 comprises adding the rhamnosidase and then adding the glucoside/xyloside bifunctional enzyme.
FIG. 5 is an HPLC chart before and after the conversion of the synergistic degradation of icariin by alpha-L-rhamnosidase and beta-glucosidase. Wherein A is herba Epimedii total flavone control, B is liquid chromatogram obtained by adding glucosidase/xylosidase bifunctional enzyme and rhamnosidase for reaction in method 1, C is liquid chromatogram obtained by adding glucosidase and rhamnosidase simultaneously in method 2, and D is liquid chromatogram obtained by adding rhamnosidase and glucoside/xylosidase for reaction in method 3. (the name of the compound corresponding to each peak in the figure: 1, epimedin A; 2, epimedin B; 3, epimedin C; 4, icariin; 5, icariin)
FIG. 6 is a comparison of antitumor activities of individual flavonoids in total flavonoids of Epimedium herb. Wherein HepG2 is human liver cancer cell, A549 is human lung cancer cell, CT26 is mouse colon cancer cell, 4T1 is mouse breast cancer cell.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting.
Example 1: the invention relates to the acquisition of the alpha-rhamnosidase gene and the construction of the recombinant plasmid pET-TPERha
1.1 culture of Thermotoga petrophila DSM13995
Thermotoga petrophila DSM13995 purchased from DSMZ culture Collection (www.dsmz.de) with the number 13995 and the formula of the culture medium is: 10g/L starch, 5g/L tryptone, 3g/L yeast extract, 5g/L meat extract, 10 g/L2-equilin ethanesulfonic acid, 10mg/L iron sulfate heptahydrate, and 1mg/L resazurin, and the pH was adjusted to 7.2. Inoculating with 0.5% inoculating amount by syringe, static culturing at 85 deg.C for 24 hr, and collecting cells.
1.2 extraction of genomic DNA
(1) The Thermotoga petrophila DSM13995 was cultured by standing for about 24 hours, and 30mL of the lysate was centrifuged at 4,000g for 10min to collect cells.
(2) The cells were resuspended in 9.5mL of TE buffer, 0.5mL of 10% Sodium Dodecyl Sulfate (SDS) and 50. mu.L of proteinase K (20mg/mL) were added, mixed well, and incubated at 37 ℃ for 1 h.
(3) 1.8mL of 5mol/L NaCl, 1.5mL of cetyltriethylammonium bromide (CTAB)/NaCl were added, mixed well and incubated at 65 ℃ for 20 min.
(4) Adding equal volume of chloroform/isoamyl alcohol, mixing, and centrifuging at 6,000g for 10 min.
(5) To prevent genomic DNA fragmentation due to shear forces, the supernatant was transferred to another centrifuge tube using a wide-mouthed pipette, mixed with an equal volume of phenol/chloroform/isoamyl alcohol and centrifuged at 6,000g for 10 min.
(6) In another centrifuge tube, 0.6 volume of isopropanol was added and gently shaken until white filamentous DNA precipitate was clearly visible.
(7) The DNA was wound with a pipette and washed in 70% alcohol.
(8) The DNA was scraped from the pipette with a sterile toothpick and transferred to a 1.5mL centrifuge tube.
(9) Air-dried at room temperature, and dissolved in 500. mu.L of TE buffer.
(10) 50. mu.L of the DNA was assayed by a nucleic acid protein detector.
1.3 construction of recombinant plasmid pET-BGL
A primer synthesized by Shanghai bioengineering, Inc. was designed according to the known Thermotoga petrophila DSM13995 high sugar-resistant α -rhamnosidase gene (accession No.: WP-011944094.1). Performing PCR amplification by using the extracted genome DNA of Thermotoga petrophila DSM13995 as a template and synthesized primers at 95 deg.C for 5 min; timing, adding Pyrobest polymerase, adding 40 mu L of paraffin oil and sealing; 28 cycles (94 ℃, 30 s; 58 ℃, 30 s; 72 ℃, 2.9 min); 72 ℃ for 10 min; the reaction was stopped and the temperature was maintained at 4 ℃. And purifying the PCR amplification product by using a gel recovery kit. Obtaining the DNA molecule of the alpha-rhamnosidase TPERha.
Carrying out double enzyme digestion on the obtained DNA molecule of the alpha-rhamnosidase TPERha and pET-20b by Nde I and NotI respectively, tapping and recovering the gel respectively, concentrating the gel, connecting the gel at 16 ℃ overnight, transforming a connecting product into escherichia coli JM109 competent cells, screening positive clones, and carrying out sequence analysis; selecting the clone with correct sequence to extract plasmid, and obtaining the recombinant plasmid pET-TPERha containing novel alpha-rhamnosidase DNA molecules.
Example 2: preparation of the heat-resistant alpha-rhamnosidase of the invention
The recombinant plasmid pET-TPERha was transformed into Escherichia coli JM109(DE3) host bacteria (purchased from Novagen Co.), cultured overnight at 37 ℃ on an LB plate (LB medium: tryptone 10g/L, yeast extract 5g/L, NaCl5g/L, agar 15g/L) containing kanamycin (50. mu.g/mL), the transformants were picked up in 200mL of LB medium (50. mu.g/mL kanamycin) at 37 ℃ and cultured with shaking at 200rpm until OD600 became 0.6, an inducer of isopropyl beta-D-thiogalactopyranoside (IPTG) was added to the culture solution to give a final concentration of 0.5mM, cultured at 30 ℃ for 6 hours, and the culture solution was centrifuged at 13,000rpm for 15 minutes at 4 ℃ by a high-speed refrigerated centrifuge to collect the cells.
Since the recombinant plasmid pET-TPERha contains a His-tag, Purification was carried out using His Bind Purification Kit (available from Novagen) to obtain a purified recombinant enzyme. The specific operation process comprises the following steps:
A. sample processing
(1) The washed cells were resuspended in 1X Binding Buffer (8 mL) and disrupted by sonication.
(2) After breaking the cell wall, centrifuging for 30min at 13,000g, and taking the supernatant as a sample.
B. Treating columns
(1) 1mL of the packing was packed into a column.
(2) The column was washed with 3mL of sterile water.
(3) The column was washed with 5mL of 1 XCharge Buffer.
(4) The column was washed with 3mL of 1 XBinding Buffer.
C. Sample loading
(1) The sample was added to the column and the flow rate was controlled to be about 6 drops per minute.
(2) The column was washed with 3mL of 1 XBinding Buffer to remove unbound protein.
(3) The column was washed with 4mL of an eluate containing 20mM imidazole to remove the contaminating proteins.
(4) The column was washed with 80mmol/L imidazole eluent to elute the desired protein.
(5) The column was washed with 4mL of 1 × Strip Buffer.
Purified alpha-rhamnosidase is obtained by this process.
Example 3: the invention relates to the acquisition of beta-glucoside/beta-xyloside bifunctional enzyme gene and the construction of recombinant plasmid pET-DthBGL3
3.1 cultivation of Dictyoglycomus thermophilum DSM3960
Dictyoglomonus thermophilum DSM3960 is purchased from DSMZ strain collection (www.dsmz.de) with the number of 3960, and the culture medium formula is as follows: 1.5g/L potassium dihydrogen phosphate, 4.2g/L disodium hydrogen phosphate dodecahydrate, 0.5g/L ammonium chloride, 0.38g/L magnesium chloride hexahydrate, 0.06g/L calcium chloride dihydrate, 0.04g/L ferric ammonium sulfate hexahydrate, 2.9mg/L cobalt chloride hexahydrate, 2.4mg/L sodium molybdate dihydrate, 1.7mg/L sodium selenate pentahydrate, 2mg/L manganese chloride tetrahydrate, 2.8mg/L zinc sulfate, 5g/L soluble starch, 2g/L peptone, 2g/L yeast extract, 1g/L sodium carbonate, 1g/L cysteine hydrochloride, 1g/L sodium celosite, deoxygenated under nitrogen atmosphere, and pH is adjusted to 7.2. Inoculating with 0.5% inoculating amount by syringe, static culturing at 85 deg.C for 24 hr, and collecting cells.
3.2 extraction of genomic DNA
(1) Performing static culture on Dictyoglycomus thermophilum DSM3960 for about 24 hours, and centrifuging 30mL of bacterial liquid for 10min at 4,000g to collect cells.
(2) The cells were resuspended in 9.5mL of TE buffer, 0.5mL of 10% Sodium Dodecyl Sulfate (SDS) and 50. mu.L of proteinase K (20mg/mL) were added, mixed well, and incubated at 37 ℃ for 1 h.
(3) 1.8mL of 5mol/L NaCl, 1.5mL of cetyltriethylammonium bromide (CTAB)/NaCl were added, mixed well and incubated at 65 ℃ for 20 min.
(4) Adding equal volume of chloroform/isoamyl alcohol, mixing, and centrifuging at 6,000g for 10 min.
(5) To prevent genomic DNA fragmentation due to shear forces, the supernatant was transferred to another centrifuge tube using a wide-mouthed pipette, mixed with an equal volume of phenol/chloroform/isoamyl alcohol and centrifuged at 6,000g for 10 min.
(6) In another centrifuge tube, 0.6 volume of isopropanol was added and gently shaken until white filamentous DNA precipitate was clearly visible.
(7) The DNA was wound with a pipette and washed in 70% alcohol.
(8) The DNA was scraped from the pipette with a sterile toothpick and transferred to a 1.5mL centrifuge tube.
(9) Air-dried at room temperature, and dissolved in 500. mu.L of TE buffer.
(10) 50. mu.L of the DNA was assayed by a nucleic acid protein detector.
3.3 construction of recombinant plasmid pET-DthBGL3
A primer synthesized by Shanghai bioengineering, Inc. was designed according to the known novel beta-glucosidase gene of Dictyoglomyus thermophilum DSM3960 (accession number: WP-041723615.1). Performing PCR amplification by using the extracted genomic DNA of Dictyoglossus thermophilum DSM3960 as a template and synthetic primers at 94 deg.C for 3 min; 30 cycles (94 ℃, 10 s; 58 ℃, 30 s; 72 ℃, 2.7 min); 72 ℃ for 5 min; the reaction was stopped and the temperature was maintained at 4 ℃. And purifying the PCR amplification product by using a gel recovery kit. Obtaining the DNA molecule of beta-glucosidase DthBgl 3.
Carrying out double enzyme digestion on the DNA molecule of the obtained beta-glucosidase DthBgl3 and pET-28a by using Nco I and XhoI respectively, carrying out tapping recovery respectively, concentrating, connecting overnight at 16 ℃, transforming a connecting product into escherichia coli JM109 competent cells, screening positive clones, and carrying out sequence analysis; selecting the clone with correct sequence to extract plasmid, and obtaining the recombinant plasmid pET-DthBGL3 containing novel beta-glucosidase DNA molecule.
Example 4: preparation of the beta-glucosidase of the invention
The recombinant plasmid pET-DthBGL3 was transformed into Escherichia coli JM109(DE3) host bacteria (purchased from Novagen) and cultured overnight at 37 ℃ on an LB plate (LB medium: tryptone 10g/L, yeast extract 5g/L, NaCl5g/L, agar 15g/L) containing kanamycin (50. mu.g/mL), the transformants were picked up in 200mL of LB medium (50. mu.g/mL kanamycin) at 37 ℃ and cultured with shaking at 200rpm until OD600 became 0.6, an isopropyl beta-D-thiogalactopyranoside (IPTG) inducer was added to the culture medium to a final concentration of 0.005-0.01mM, cultured at 30 ℃ for 6 hours, and the culture was centrifuged at 13,000rpm for 15min at 4 ℃ by a high-speed refrigerated centrifuge to collect the cells.
The recombinant plasmid pET-DthBGL3 contains a His-tag, and is purified by His Bind Purification Kit (available from Novagen), thereby obtaining a purified recombinant enzyme. The specific operation process comprises the following steps:
A. sample processing
(1) The washed cells were resuspended in 1X Binding Buffer (8 mL) and disrupted by sonication.
(2) After breaking the cell wall, centrifuging for 30min at 13,000g, and taking the supernatant as a sample.
B. Treating columns
(1) 1mL of the packing was packed into a column.
(2) The column was washed with 3mL of sterile water.
(3) The column was washed with 5mL of 1 XCharge Buffer.
(4) The column was washed with 3mL of 1 XBinding Buffer.
C. Sample loading
(1) The sample was added to the column and the flow rate was controlled to be about 6 drops per minute.
(2) The column was washed with 3mL of 1 XBinding Buffer to remove unbound protein.
(3) The column was washed with 8mL of an eluate containing 20mM imidazole to remove the contaminating proteins.
(4) The column was washed with 200mmol/L imidazole eluent to elute the desired protein.
(5) The column was washed with 4mL of 1 × Strip Buffer.
The purified β -glucosidase was obtained by this procedure, and the purity of β -glucosidase was identified by staining after SDS-PAGE electrophoresis, and the results are shown in fig. 2.
The expression level of the beta-glucosidase gene in a host bacterium JM109(DE3) is high, after the target protein is purified by a HisTag label, the purity of the beta-glucosidase Dth3 in eluent is high, a single band exists at 80kDa, and the electrophoresis purity level is achieved.
Example 5: determination of enzyme activity of alpha-L-rhamnosidase and beta-glucosidase
P-nitrophenol-alpha-rhamnoside (pNP-R) is used as a substrate, the p-nitrophenol obtained by hydrolysis and sodium carbonate are subjected to a color reaction, and the absorbance of the product is measured at the wavelength of 405 nm. The 100 μ L reaction system comprises 75 μ L of 100mM buffer solution with optimum pH, 20 μ L of 5mM substrate, mixing, preheating, adding 5 μ L diluted enzyme solution, reacting at optimum temperature for 10min, and adding 0.3mL of 1M NaCO3The reaction is stopped, and the mixture is evenly mixed and then is measured by an enzyme-labeling instrument under the condition of 405 nm. A control with enzyme solution and no substrate and a control with substrate and no enzyme solution were performed simultaneously.
P-nitrophenol-beta-glucoside (pNP-G) is used as a substrate, the p-nitrophenol obtained by hydrolysis and sodium carbonate are subjected to a color reaction, and the absorbance of the product is measured at the wavelength of 405 nm.The 100 μ L reaction system comprises 90 μ L100 mM optimum pH buffer solution, 5 μ L20 mM substrate, mixing well, preheating, adding 5 μ L diluted enzyme solution, reacting at optimum temperature for 10min, and adding 0.3mL 1M NaCO3The reaction is stopped, and the mixture is evenly mixed and then is measured by an enzyme-labeling instrument under the condition of 405 nm. A control with enzyme solution and no substrate and a control with substrate and no enzyme solution were performed simultaneously.
P-nitrophenol-beta-xyloside (pNP-X) is used as a substrate, the p-nitrophenol obtained by hydrolysis and sodium carbonate are subjected to a color reaction, and the absorbance of the product is measured at the wavelength of 405 nm. The 100 μ L reaction system comprises 90 μ L100 mM optimum pH buffer solution, 5 μ L20 mM substrate, mixing well, preheating, adding 5 μ L diluted enzyme solution, reacting at optimum temperature for 10min, and adding 0.3mL 1M NaCO3The reaction is stopped, and the mixture is evenly mixed and then is measured by an enzyme-labeling instrument under the condition of 405 nm. A control with enzyme solution and no substrate and a control with substrate and no enzyme solution were performed simultaneously.
One enzyme activity unit (U) is defined as: under the most suitable conditions, the hydrolysis releases 1. mu. mol of the enzyme required for the p-nitrophenol.
Enzyme activity was calculated against the standard curve:
enzyme activity (U/mL) ═ c × V1/(t × V2) × N
c: the p-nitrophenol content (mu mol/mL) after the enzyme reaction is calculated by a p-nitrophenol standard equation;
v1: total reaction system volume (mL);
t: enzyme and substrate reaction time (min);
v2: volume of enzyme solution (mL) at the time of enzyme reaction;
n: and (5) diluting the enzyme solution by multiple times.
Example 6: comparison of multicomponent catalytic ability of different sources of recombinase to hydrolyze epimedium flavone
6.1 comparing the catalytic ability of alpha-L-rhamnosidase from different sources to icaritin
The concentration of the icariin is 1g/L, three different sources of alpha-L-rhamnosidase are added with 60U/mL enzyme under the respective optimal temperature and the optimal pH as the conversion condition, the reaction is carried out for 1h, and the detection is carried out by HPLC. The results are shown in FIG. 3A: the alpha-L-rhamnosidase from Thermotoga petrophila DSM13995 and Aspergillus terreus CCF 3059 can effectively convert icariin into icariside I, and the molar conversion rates are 45% and 12% respectively; whereas alpha-L-rhamnosidase originating from Aspergillus niger NL-1 and Bacteroides thetaiotaomicron VPI-5482 hardly converted icariin. Thus, alpha-L-rhamnosidase from Thermotoga petrophila DSM13995 was chosen for the conversion of Epimedium brevicornum total flavone extract.
6.2 comparison of the catalytic Capacity of different sources of beta-glucosides on epimedin A, epimedin B and icariin
Hydrolyzing 1g/L of epimedin A, epimedin B and icariin respectively by using five beta-glucosidases from different sources under the reaction conditions of respective optimal temperature and optimal pH, adding pure enzymes in the amounts of 25U/mL for 1h, and detecting by HPLC. As a result, as shown in FIG. 3B, 5 kinds of beta-glucosidase from different sources and families can efficiently convert epimedium A and icariin into the target product of baohuoside I, while only the beta-glucosidase DthBGL3 derived from Dictyoglyces thermophilum DSM3960 has the activity of beta-glucosidase and beta-xylosidase, so only DthBGL3 can efficiently convert epimedium B into the target product of baohuoside I, and the conversion rate reaches 99.2%. Accordingly, beta-glucoside/beta-xyloside bifunctional enzyme DthBGL3 derived from Dictyoglycous thermophilum DSM3960 is selected to convert herba Epimedii total flavone extract.
Example 7: process research for preparing icaritin by catalytic conversion of icariin by enzyme method
The selected alpha-L-rhamnosidase from Thermotoga petrophila DSM13995 and beta-glucoside/beta-xyloside bifunctional enzyme DthBGL3 from Dictyoglomonus thermophilum DSM3960 are transformed into herba Epimedii extract by different methods respectively. The herba Epimedii total flavone extract is provided by Jiangsu Kangyuan pharmaceutical industry GmbH, and its main components comprise 1.44% of epimedin A, 2.76% of epimedin B, 5.96% of epimedin C, 9.05% of icariin, and 0.95% of baohuoside I.
7.1 method 1-Add beta-glucoside/beta-xyloside bifunctional enzyme first and then add alpha-L-rhamnosidase
Herba Epimedii extract concentration is 1g/L, pH is 4.5100 mmol/L citric acid-disodium hydrogen phosphate buffer solution, 30U/mL Dictyoglycous thermophilum DSM3960GH3 family beta-glucoside/beta-xyloside bifunctional enzyme is added, reaction is continued for 1h at 90 deg.C, 75U/mL Thermotoga petrophila DSM13995 alpha-L-rhamnosidase is added, reaction is performed for 1h at 90 deg.C, and detection is performed by HPLC. The results are shown in FIG. 4: the molar conversion rate of the icariin is as follows: 87.3 percent.
7.2 method 2-Simultaneous addition of alpha-L-rhamnosidase and beta-glucoside/beta-xyloside bifunctional enzyme
Epimedium extract at a concentration of 1g/L, pH 4.5100 mmol/L citrate-disodium hydrogen phosphate buffer, was reacted with 75U/mL α -L-rhamnosidase of Thermotoga petrophila DSM13995 and 30U/mL β -glucosidase of Dictyoglyces thermophilum DSM3960GH3 at 90 ℃ for 2h and assayed by HPLC. The results are shown in FIG. 4: the molar conversion rate of the icariin is as follows: 90.9 percent.
7.3 method 3-first add alpha-L-rhamnosidase and then beta-glucoside/beta-xyloside bifunctional enzyme
The concentration of herba Epimedii extract is 1g/L, pH is 4.5100 mmol/L citric acid-disodium hydrogen phosphate buffer solution, 75U/mL alpha-L-rhamnosidase of Thermotoga petrophila DSM13995 is added for reaction at 90 deg.C for 1h, then 30U/mL beta-glucosidase of Dictyoglycous thermophilum DSM3960GH3 family is added for further reaction at 90 deg.C for 1h, and detection is performed by HPLC. The results are shown in FIG. 4: the molar conversion rate of the icariin is as follows: 95.1 percent.
Therefore, the best process for preparing icariin by transforming epimedium extract is determined as method 3-firstly adding alpha-L-rhamnosidase from Thermotoga petrophila DSM13995 and then adding beta-glucoside/beta-xyloside bifunctional enzyme DthBGL3 from Dictyoglomonus thermophilum DSM 3960.
Example 8: the epimedium total flavone has antitumor activity after being converted by DthBGL3 and TPERha
Inoculating 3000 cells into each well of a 96-well plate, culturing at 37 ℃, adding epimedium flavone with different concentrations into each well for culturing for 72 hours after the cells are attached to the wall for 6 hours, adding 20 mu L MTT (4mg/mL) into each well 4 hours before the end point of the experiment, taking out the 96-well plate at the end point of the experiment, centrifuging at 1000rcf, then sucking out the supernatant, adding 200 mu L DMSO, and measuring the light absorption value at 570 nm. Calculating the inhibition rate of the sample to be tested on the in vitro proliferation of the tumor cells according to the following formula:
inhibition rate [ 100- (OD570 (experimental well) -OD 570 (blank))/(OD (non-dosed control empty) -OD 570 (blank) × 100) ]
According to HPLC (high performance liquid chromatography) maps before and after transformation of the epimedium total flavonoids by DthBGL3 and TPERha in example 7, the epimedium total flavonoids are mainly composed of epimedin A, B, C and icariin, wherein the content of the icariin is the highest, and the transformation products are mainly icariin. As shown in figure 6, the in vitro anti-tumor activity research shows that the proliferation inhibition rates of icariin to human liver cancer HepG2, lung cancer A549, mouse colon cancer CT26 and breast cancer 4T1 are respectively up to 88%, 46%, 61% and 89% at the concentration of 100 mu M, which are obviously higher than other monomer components in epimedium total flavone. Therefore, after the epimedium total flavonoids are converted into the icaritin through DthBGL3 and TPERha, the antitumor activity is improved.

Claims (3)

1. A method for preparing icariin by converting total flavonoids of epimedium herb by an enzyme method is characterized in that a double-enzyme system consisting of heat-resistant alpha-L-rhamnosidase and heat-resistant beta-glucosidase can convert multi-component flavonoid glycoside in the total flavonoids of epimedium herb to generate the icariin, wherein the heat-resistant alpha-L-rhamnosidase is derived from the heat-resistant alpha-L-rhamnosidaseThermotoga petrophilaEscherichia coli recombinant strain of alpha-L-rhamnosidase gene of DSM13995, wherein the heat-resistant beta-glucosidase is derived fromDictyoglomus thermophilumAn escherichia coli recombinant strain of a DSM3960GH3 β -glucosidase gene, wherein the multicomponent flavonoid glycosides are icariin, epimedin a, epimedin B and epimedin C; the method comprises preparing 50mmol/L citric acid-disodium hydrogen phosphate buffer solution with pH of 4.5, adding herba Epimedii total flavone to concentration of 5g/L, addingThermotoga petrophila Thermostable alpha-L-rhamnosidase of DSM13995 to 30U/mL, and addingDictyoglomus thermophilumThermostable beta-glucans of the DSM3960GH3 familyGlucosidase to 25U/mL, and reacting at 85 ℃ for 1 h.
2. A product rich in icaritin prepared by the method of claim 1.
3. The use of the icariin-rich product of claim 2 in the preparation of a medicament for treating breast cancer, lung cancer, colon cancer, and liver cancer.
CN201710332977.7A 2017-05-12 2017-05-12 Method for preparing icariin by converting total flavonoids of epimedium herb through enzyme method Active CN106995829B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710332977.7A CN106995829B (en) 2017-05-12 2017-05-12 Method for preparing icariin by converting total flavonoids of epimedium herb through enzyme method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710332977.7A CN106995829B (en) 2017-05-12 2017-05-12 Method for preparing icariin by converting total flavonoids of epimedium herb through enzyme method

Publications (2)

Publication Number Publication Date
CN106995829A CN106995829A (en) 2017-08-01
CN106995829B true CN106995829B (en) 2021-03-30

Family

ID=59435226

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710332977.7A Active CN106995829B (en) 2017-05-12 2017-05-12 Method for preparing icariin by converting total flavonoids of epimedium herb through enzyme method

Country Status (1)

Country Link
CN (1) CN106995829B (en)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107641621B (en) * 2017-06-14 2021-07-23 江苏康缘药业股份有限公司 Glycosidase composition and method for preparing icariin by enzyme method
CN108384769B (en) * 2018-02-05 2021-09-10 南京林业大学 High-temperature-resistant complex enzyme and application thereof
CN108467858A (en) * 2018-02-05 2018-08-31 南京林业大学 A kind of alpha-L-Rhamnosidase and its application
CN108392487B (en) * 2018-02-24 2023-09-01 北京东方百奥医药开发有限公司 Application of icariside II or pharmaceutically acceptable carrier thereof in erectile dysfunction
CN110699263B (en) * 2019-10-29 2021-05-11 浙江工业大学 Aspergillus niger YH-6 and application thereof in improving content of icaritin in epimedium
CN111575330B (en) * 2020-05-21 2022-04-15 成都蓓乐康生物科技有限公司 Method for hydrolyzing epimedium extract by plant-derived enzyme
CN112226395B (en) * 2020-09-10 2022-07-05 广西大学 Escherichia coli engineering bacterium and method for producing icariin through whole-cell catalysis of escherichia coli engineering bacterium
CN112725396A (en) * 2020-09-22 2021-04-30 北京岳达生物科技有限公司 Method for preparing icariside II through enzyme catalysis
CN112553264B (en) * 2020-12-16 2024-07-09 金凤燮 Method for efficiently preparing icariin by enzyme conversion
CN113512542B (en) * 2021-06-22 2021-11-23 广东金骏康生物技术有限公司 Rhamnosidase mutant and preparation method and application thereof
CN113136378B (en) * 2021-06-22 2021-09-03 广东金骏康生物技术有限公司 Rhamnosidase TpeRhha mutant and preparation method and application thereof
CN113249357B (en) * 2021-06-22 2021-09-14 广东金骏康生物技术有限公司 Rhamnosidase TpeRhha-H570A mutant and preparation method and application thereof
CN114107341B (en) * 2021-11-29 2024-02-20 浙江熙正霖生物科技有限公司 Application of fungal source alpha-L-rhamnosidase in icariin production
CN114350634B (en) * 2021-12-31 2022-08-30 湖北碳元本草生物科技有限公司 Glucoside glycosyl transferase for synthesizing epimedin and coding gene and application thereof
CN117089465B (en) * 2023-08-22 2024-05-03 陕西省微生物研究所 Aspergillus wart and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101302548A (en) * 2007-05-09 2008-11-12 北京珅奥基医药科技有限公司 Preparation of icaritin
CN103305564A (en) * 2013-07-05 2013-09-18 西安纽赛生物科技有限公司 Method for converting icariin to herba epimedii
CN104762281A (en) * 2015-03-09 2015-07-08 南京林业大学 Alpha-L-rhamnosidase and preparing method and applications thereof
CN106420880A (en) * 2016-08-29 2017-02-22 江苏省中医药研究院 Herba epimedii total flavonoid enzymatic product and preparing method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101302548A (en) * 2007-05-09 2008-11-12 北京珅奥基医药科技有限公司 Preparation of icaritin
CN103305564A (en) * 2013-07-05 2013-09-18 西安纽赛生物科技有限公司 Method for converting icariin to herba epimedii
CN104762281A (en) * 2015-03-09 2015-07-08 南京林业大学 Alpha-L-rhamnosidase and preparing method and applications thereof
CN106420880A (en) * 2016-08-29 2017-02-22 江苏省中医药研究院 Herba epimedii total flavonoid enzymatic product and preparing method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
嗜热微生物来源的葡萄糖苷酶和木糖苷酶性质及功能研究;杨芯卓;《中国优秀硕士学位论文全文数据库基础信息辑》;20141115;摘要和第32-36页 *
酶解淫羊藿总黄酮制备宝藿苷Ⅰ;蒋艳荣等;《中国医院药学杂志》;20130208;第33卷(第4期);第262页右栏第3段和第263页右栏第2段 *

Also Published As

Publication number Publication date
CN106995829A (en) 2017-08-01

Similar Documents

Publication Publication Date Title
CN106995829B (en) Method for preparing icariin by converting total flavonoids of epimedium herb through enzyme method
CN107267575B (en) Method for producing N-acetyl-D-glucosamine and/or D-glucosamine salt by microbial fermentation
CN107641621B (en) Glycosidase composition and method for preparing icariin by enzyme method
CN113684164B (en) Construction method and application of microorganism for high-yield lactoyl-N-neotetraose
CN111944865B (en) Application of alpha-L-rhamnosidase from bacteria in efficient production of hesperetin-7-O-glucoside
CN113652385B (en) Construction method and application of microorganism for high-yield lactoyl-N-tetraose
CN105734092B (en) A kind of method that enzyme process prepares D-Tag
CN114874964A (en) Construction method and application of recombinant escherichia coli for high yield of 2' -fucosyllactose
CN118006590A (en) Microbacterium dextranolyticum beta-glucosidase mutant, method and application
CN116334041B (en) Rhamnosidase mutant and application thereof
CN107164436B (en) Application of beta-glucosidase in preparation of baohuoside I by converting epimedium total flavonoids
CN104480127A (en) Hyperthermophilic glycosidase mutant and application thereof in preparation of ginsenoside CK
CN113373168B (en) Bacterial-derived alpha-L-rhamnosidase gene, gene expression and application thereof
CN103820506A (en) Method for producing coenzyme Q10 by fermenting genetic recombinant bacteria
CN108265095B (en) A kind of15Preparation method of N-stable isotope labeled 5-methyl deoxycytidine
CN105039463B (en) A kind of application of glycosyl transferred beta-N- acetylaminos that regioselectivity is single-minded glycosidase
CN117965500B (en) Alpha-L rhamnosidase AfRhase and product, application and production process thereof
CN116004577B (en) alpha-L-rhamnosidase BtRha78A-F44Y mutant and preparation method and application thereof
CN111286498B (en) Efficient chitinase for producing acetamido glucose
CN114107259B (en) Application of alkaline thermophilic rhamnosidase in preparation of active bioflavonoids
CN118207186B (en) Beta-glucuronidase and recombinant expression vector, engineering bacteria, starter and method for mass production of glycyrrhetinic acid thereof
CN114958667B (en) Lactococcus for high yield of L-lactic acid and application thereof
CN116083499A (en) Application of beta-glucosidase Lf18920 in specific hydrolysis of crocin-1 to obtain crocin acid monosaccharide glycoside derivative
CN118028202A (en) Construction method and application of recombinant Escherichia coli for efficiently synthesizing lactoyl-N-disaccharide
CN118360352A (en) Method for preparing icaritin by biocatalysis of epimedium extract

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant