CN106995829A - A kind of method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone - Google Patents
A kind of method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone Download PDFInfo
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- CN106995829A CN106995829A CN201710332977.7A CN201710332977A CN106995829A CN 106995829 A CN106995829 A CN 106995829A CN 201710332977 A CN201710332977 A CN 201710332977A CN 106995829 A CN106995829 A CN 106995829A
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Abstract
Multicomponent flavone glycoside generation epimedium aglucone in a kind of method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone, the dual-enzyme system collaboration conversion barren wort total chromocor being made up of heat-resisting α L rhamnosidases and heat-resisting β glucuroides.The present invention can be by multicomponent flavone compound in barren wort total chromocor（Icariin, towards Huo Ding A, towards Huo Ding B and towards Huo Ding C etc.）It is almost completely converted into purpose product；Optimum temperature is high so that substrate and intermediate product all have good solubility, it is not necessary to cosolvent；Enzymolysis time is short, and yield is high.The epimedium aglucone that the present invention is prepared is significantly higher than each monomer of barren wort total chromocor before conversion for the inhibited proliferation of liver cancer, lung cancer, colon cancer and breast cancer.
Description
Technical field
The invention belongs to enzyme engineering and biomedicine field, and in particular to restructuring enzyme system is in catalysis barren wort total chromocor system
Method in standby epimedium aglucone.
Background technology
Epimedium aglucone (Icaritin) is a kind of polyhydroxy flavonoids list in Berberidaceae barrenwort barrenwort
Body composition, it is extremely low in barrenwort in-vivo content, is the hydrolysate of icariin, be immunized with estrogen-like action, regulation,
Promote cardiac muscle mitochondria and differentiation, promote sclerotin protection, establishing-Yang, anti-liver injury and delay liver fibrosis, a variety of medicines such as antitumor
Reason activity.With discovery both at home and abroad to the new drug effect of epimedium aglucone, the international market demand of following epimedium aglucone is very big.
Therefore prepare epimedium aglucone method with how developing high-efficiency environment friendly is the emphasis studied at present.
Because icariin is one of content most higher flavonoid glycoside compound of barrenwort, it has with epimedium aglucone
There is identical parent nucleus, therefore, the preparation of current epimedium aglucone is mainly for icariin, using enzyme process or acid system by its parent nucleus
Glycosyl on No. 3 and No. 7 carbon potentials removes to obtain epimedium aglucone.Such as CN 103305564 is combined by enzymolysis, acidolysis,
Icariin is converted into icariine, but due to acidolysis reaction poor process control, accessory substance is more, and product yield is low, it is crucial
It is that strong acidic condition easily changes the structure of flavones parent nucleus and is particularly the methoxyl groups of 4 ' numbers positions, makes its technique that there is limitation；CN
Icariin is converted into epimedium aglucone by 101302548 by a kind of beta-glucosidase of commercialization, although the enzyme can be obtained
Epimedium aglucone, but its hydrolysis time length (24 hours), highest enzymolysis yield is also only 55% or so, according to hydrolysis result
And enzyme specificity characteristic, it is known that the beta-glucosidase used ferments from traditional microbiological, and its enzyme is not pure β-grape
Glycosidase；Icariin is converted into epimedium aglucone by CN 104561178 using naringinase.Naringinase is sent out by microorganism
The complex enzyme with alpha-L-Rhamnosidase and activity of beta-glucosidase that ferment is prepared, single-minded characteristic is mainly specificity
Hydrolyze aurantiin.In this method alpha-L-Rhamnosidase and beta-glucosidase ratio it is uncontrollable, effect sequencing can not
Control, and reaction time length (30 hours), efficiency are low (patent only illustrates that epimedium aglucone can be generated).
Comprehensive existing result of study is can be found that：(1) current research technique can only prepare excessive using icariin as substrate
Sheep leaves of pulse plants aglycon, can not also be same by the Main Components of barren wort total chromocor (icariin, towards Huo Ding A, towards Huo Ding B and towards Huo Ding C)
When be converted into epimedium aglucone；(2) yield and efficiency for preparing epimedium aglucone at present need to be improved.(3) in prior art,
In preparation process, first have to obtain Shorthorned Epimedium P.E, then isolated icariin, then passes through enzyme process or microorganism
Method is converted, and not only considerably increases cost, and without fully efficiently using towards Huo Ding A, towards Huo Ding B, towards Huo Ding C.The opposing party
Face so that a large amount of icariin resource problems that obtain are protruded very much, it is impossible to ensure prepare with scale epimedium aglucone.And it is of the invention
A kind of method for preparing epimedium aglucone there is provided enzymatic conversion method barren wort total chromocor, just can be high only by two kinds of glycosidases
By barren wort total chromocor, almost conversion prepares epimedium aglucone completely on effect ground.
The content of the invention
The technical problem of solution：The invention provides the side that a kind of enzymatic conversion method barren wort total chromocor prepares epimedium aglucone
Method, successfully screening obtains one kind and can effectively degraded icariin parent nucleus knot from microbe-derived glycoside hydrolase gene pool
Glucose residue and xylose residues in the alpha-L-Rhamnosidase of sandlwood saccharide residue and a kind of efficient degradation its mother nucleus structure on structure
The difunctional beta-glucosidase of high temperature resistant, just can efficiently will be a variety of in barren wort total chromocor only by two kinds of glycosidases
Almost conversion prepares epimedium aglucone to icariin completely.Effectively reduce preparation cost and improve resources effective utilization
Rate.
Technical scheme：A kind of method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone, by heat-resisting α-L- sandlwoods
The dual-enzyme system of glycosidase and heat-resisting beta-glucosidase composition can convert the multicomponent flavone glycoside in barren wort total chromocor
Epimedium aglucone is generated, the multicomponent flavone glycoside is icariin, determines C towards Huo Ding A, towards Huo Ding B and Chao Huo.
Above-mentioned heat-resisting alpha-L-Rhamnosidase derives from Thermotoga petrophila DSM 13995 α-L- sandlwoods
The Escherichia coli recombinant strain of glycosidase genes.
Above-mentioned heat-resisting beta-glucosidase derives from Dictyoglomus thermophilum DSM 3960GH3 β-grape
The Escherichia coli recombinant strain of glycosidase genes.
The method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone, prepares pH 4.5,50mmol/L citric acids-phosphorus
Sour disodium hydrogen buffer solution, it is 5g/L to add barren wort total chromocor to concentration, while adding Thermotoga petrophila DSM
13995 heat-resisting alpha-L-Rhamnosidase is to 30U/mL, and Dictyoglomus thermophilum DSM 3960GH3 families
The heat-resisting beta-glucosidase of race reacts 1h to 25U/mL at 85 DEG C.
The dual-enzyme system being made up of heat-resisting alpha-L-Rhamnosidase and heat-resisting beta-glucosidase is always yellow in conversion barrenwort
Multicomponent flavone glycoside in ketone is the application in epimedium aglucone.
The enzymatic compositions that the multicomponent flavone glycoside in barren wort total chromocor is epimedium aglucone are converted, by heat-resisting α-L- mouse
Lee's glycosidase and heat-resisting beta-glucosidase composition.
Epimedium aglucone made from the above method.
Above-mentioned the answering in treatment breast cancer, lung cancer, colon cancer, liver-cancer medicine is prepared rich in epimedium aglucone product
With.
Alpha-L-Rhamnosidase used in this method have from Aspergillus terreus CCF 3059,
Aspergillus niger NL-1、Bacteroides thetaiotaomicron VPI-5482、Thermotoga
Petrophila DSM 13995, used beta-glucosidase has from Thermotoga thermarum DSM
5069GH1 families, Thermotoga thermarum DSM 5069GH3 families, Thermotoga petrophila DSM
13995GH1 families, Thermotoga petrophila DSM 13995GH3 families, Dictyoglomus thermophilum
DSM3960GH3 families.
Optimal double enzymes barren wort total chromocor of degrading prepares the acquisition process of epimedium aglucone technique and is：Compare 1) simultaneously
Calais comes from Thermotoga petrophila DSM 13995 alpha-L-Rhamnosidase and from Dictyoglomus
Thermophilum DSM3960GH3 families beta-glucosidase/β-xyloside bifunctional enzyme；2) first Calais comes from
Calais comes from after beta-glucosidase/β-xyloside bifunctional enzyme of Dictyoglomus thermophilum DSM3960 families
Thermotoga petrophila DSM 13995 alpha-L-Rhamnosidase；3) first Calais is come from from Thermotoga
Calais comes from Dictyoglomus thermophilum after petrophila DSM13995 alpha-L-Rhamnosidase
It is prepared by the different enzyme addition conversion barren wort total chromocors of 3 kinds of beta-glucosidase/β-xyloside bifunctional enzyme of DSM3960 families
The molar yield of epimedium aglucone, it is determined that first plus after alpha-L-Rhamnosidase add beta-glucosidase/β-xyloside difunctional
Its conversion ratio highest of the mode of enzyme.
Beneficial effect：1. the scope of conversion of substrate of the present invention is wide, the target of conversion is expanded to barrenwort by icariin
Icariin in general flavone, towards Huo Ding A, towards Huo Ding B, towards Huo Ding C etc..The yield of epimedium aglucone is improved, is also improved
The utilization rate of barrenwort.2. the present invention prepares icariin using two kinds of restructuring enzymatic barren wort total chromocors of definite ingredients
Member, the molar yield of enzymatic conversion method is more than 97%.3. the present invention has screened the alpha-L-Rhamnosidase of separate sources and offer
What efficient degradation epimedium flavone glycosides sandlwood saccharide residue was capable of in a kind of acquisition derives from Thermotoga petrophila DSM
13995 alpha-L-Rhamnosidase, the optimal reactive temperature of the enzyme is 90 DEG C, and temperature stability is preferable.4. the present invention is screened
The beta-glucosidase of separate sources obtains the difunctional GH3 family of energy efficient degradation icariin glucose residue and xylose residues
The beta-glucosidase of race, the enzyme optimal reactive temperature is 85 DEG C, high catalytic efficiency.5. the invention provides a kind of efficient, ring
The method for preparing epimedium aglucone with protecting, the epimedium aglucone of preparation has stronger bioactivity.Can be by barren wort total chromocor
Middle multicomponent flavone compound is almost completely converted into purpose product；Optimum temperature is high so that substrate and intermediate product
All there is good solubility, it is not necessary to cosolvent；Enzymolysis time is short, and yield is high.6. barren wort total chromocor is obtained after double enzymatic conversions
To the product using epimedium aglucone as main flavone component, suppressing human liver cancer cell HepG2, lung cell A549 and mouse knot
Activity in terms of colon-cancer cell CT26 propagation, is significantly stronger than unconverted barren wort total chromocor.
Brief description of the drawings
Fig. 1 prepares the schematic diagram of epimedium aglucone for multicomponent flavone glycoside in enzymatic conversion method barren wort total chromocor, wherein
The multicomponent of barren wort total chromocor is included towards Huo Ding A, towards Huo Ding B, towards Huo Ding C and icariin etc..Their concrete structure is special
Levy and see form on the right side of Fig. 1.
Fig. 2 is the HPLC spectrograms of the standard items of the key component in barren wort total chromocor, wherein 1. towards Huo Ding A；2. towards suddenly
Determine B；3. towards Huo Ding C；4. icariin and its rare component 5 (precious glycosides I suddenly) and target product 6 (epimedium aglucone), show mesh
Preceding HPLC methods can be analyzed effectively this several component, and method efficient stable.
Fig. 3 is that a variety of separate sources alpha-L-Rhamnosidases and a variety of separate sources beta-glucosidases are yellow to barrenwort respectively
The transformation efficiency figure of ketoside.Wherein A is separate sources alpha-L-Rhamnosidase conversion icariin generation icariside in 4
I transformation efficiency figure, its transformation efficiency is with the content meter of icariside I；B is the different family β-glucose of 5 kinds of separate sources
Glycosides is converted towards Huo Ding A, towards Huo Ding B and the transformation efficiency figure of the precious glycosides I suddenly of icariin production respectively, and its conversion ratio is with precious glycosides I suddenly
Content meter.Note：TPERha represents the alpha-L-rhamnoside from Thermotoga petrophila DSM 13995 in A
Enzyme, AtRha represent to represent source from Aspergillus terreus CCF 3059 alpha-L-Rhamnosidase, AnRha
Alpha-L-Rhamnosidase, BtRha in Aspergillus niger NL-1 represent to derive from Bacteroides
Thetaiotaomicron VPI-5482 alpha-L-Rhamnosidase；TtBGL1 represents to derive from Thermotoga in B
Thermarum DSM 5069GH1 families；TpBGL1 represents to derive from Thermotoga petrophila DSM 13995GH1
The beta-glucosidase of family；TtBGL3 represents to derive from Thermotoga thermarum DSM 5069GH3 families；
TpBGL3 represents the beta-glucosidase from Thermotoga petrophila DSM 13995GH3 families；DthBGL3
Represent Dictyoglomus thermophilum DSM3960 beta-glucosidase.
Fig. 4 compares figure for the conversion ratio of three kinds of method for transformation, and wherein conversion ratio is with the content meter of epimedium aglucone.It is wherein square
Method 1 is first plus after glucoside/xyloside bifunctional enzyme adds rhamnosidase, and method 2 is while adds glucuroide and mouse
Lee's glycosidase, method 3 adds glucoside/xyloside bifunctional enzyme first to add after rhamnosidase.
Fig. 5 is the HPLC figures before and after alpha-L-Rhamnosidase and the conversion of beta-glucosidase Synergistic degradation icariin.Its
Middle A compares for barren wort total chromocor, and B is method 1 first plus after glucoside/xyloside bifunctional enzyme plus after rhamnoside enzyme reaction
Liquid phase spectrogram, C is that method 2 adds reaction product liquid chromatography(LC figure after glucuroide and rhamnosidase simultaneously, and D is that method 3 is first
Plus add product liquid phase spectrogram after glucoside/xyloside bifunctional enzyme reaction after rhamnosidase.(the corresponding chemical combination in each peak in figure
Name claims：1, Epimedin A；2, Epimedin B；3, epimedin C；4, icariin；5, epimedium aglucone)
Fig. 6 is that the antitumor activity of each flavones monomer in barren wort total chromocor compares figure.Wherein HepG2 is that human liver cancer is thin
Born of the same parents, A549 is human lung carcinoma cell, and CT26 is mouse colonic cell, and 4T1 is mouse mastopathy cell.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is described in further detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention,
It is not used to limit invention.
Embodiment 1：The acquisition of α of the present invention-rhamnoside enzyme gene and recombinant plasmid pET-TPERha structure
1.1Thermotoga petrophila DSM 13995 culture
Thermotoga petrophila DSM 13995 are purchased from DSMZ DSMZs (www.dsmz.de) numbering
For 13995, its culture medium prescription is：10g/L starch, 5g/L tryptones, 3g/L yeast extracts, 5g/L meat extracts, 10g/L
2- horse quinolines ethyl sulfonic acid, 10mg/L iron sulfate heptahydrates, 1mg/L resazurins, adjustment pH are 7.2.With syringe according to 0.5% inoculation
Amount inoculation, 85 DEG C of static gas wave refrigerator 24h collect cell.
The extraction of 1.2 genomic DNAs
(1) quiescent culture Thermotoga petrophila DSM 13995 about 24 hours, take 30mL bacterium solutions 4,000g from
Heart 10min collects cell.
(2) thalline is resuspended with 9.5mL TE buffer solutions, adds the lauryl sodium sulfate of 0.5mL 10% (SDS) and 50 μ L eggs
White enzyme K (20mg/mL), is well mixed, 37 DEG C of insulation 1h.
(3) 1.8mL 5mol/L NaCl, 1.5mL cetyltriethylammonium bromides (CTAB)/NaCl is added, is mixed, 65
DEG C incubate 20min.
(4) isometric chloroform/isoamyl alcohol is added, is mixed, 6,000g centrifugation 10min.
(5) to prevent shearing force from causing genomic DNA to be broken, supernatant is transferred in another centrifuge tube with thick mouth suction pipe, plus
Enter isometric phenol/chloroform/isoamyl alcohol to mix, 6,000g centrifugation 10min.
(6) in another centrifuge tube, 0.6 times of volume isopropanol is added, gently rocks to white thread DNA precipitations and clearly may be used
See.
(7) it is with suction pipe that DNA is wound on it, cleaned in 70% alcohol.
(8) DNA is scraped from suction pipe with sterile toothpick, be transferred in 1.5mL centrifuge tubes.
(9) air-dry at room temperature, plus 500 μ L TE buffer solutions.
(10) 50 μ L are taken to detect DNA concentration with nucleic acid-protein detector.
1.3 recombinant plasmid pET-BGL structure
(logged according to the resistance to high sugar α-rhamnoside enzyme genes of known Thermotoga petrophila DSM 13995
Number：WP_011944094.1 primer) is designed, primer is synthesized by Shanghai bioengineering Co., Ltd.With the Thermotoga of extraction
Petrophila DSM 13995 genomic DNA is template, and entering performing PCR with the primer of synthesis expands, and the condition of amplification is 95
DEG C, 5min；Time out, plus Pyrobest polymerases, plus 40 μ L paraffin oil seals；28 circulations (94 DEG C, 30s；58 DEG C,
30s；72 DEG C, 2.9min)；72 DEG C, 10min；Reaction stops, 4 DEG C of insulations.By gel reclaims kit to pcr amplification product
Purified.Obtain α-rhamnosidase TPERha DNA molecular.
Obtained α-rhamnosidase TPERha DNA molecular and pET-20b are subjected to double enzymes with Nde I and NotI respectively
Cut, and rubber tapping is reclaimed respectively, 16 DEG C of connections are stayed overnight after concentration, and connection product is converted into escherichia coli jm109 competent cell, sieve
Positive colony is selected, sequence analysis is carried out；Select sequence and correctly clone extraction plasmid, obtain and contain novel alpha-rhamnosidase
The recombinant plasmid pET-TPERha of DNA molecular.
Embodiment 2：The preparation of heat-resisting α-rhamnosidase of the present invention
Recombinant plasmid pET-TPERha is converted into e. coli jm109 (DE3) Host Strains (being purchased from Novagen companies),
LB flat boards (LB culture mediums containing kanamycins (50 μ g/mL)：Tryptone 10g/L, yeast extract 5g/L, NaCl5g/L,
Agar 15g/L) on by 37 DEG C of overnight incubations, choose transformant (50 μ g/mL kanamycins) 37 into 200mL LB culture mediums
DEG C, when 200rpm shaken cultivations to OD600 are 0.6, add final concentration of 0.5mM isopropyl ss-D- Thiogalactopyranosides
(IPTG) derivant, 30 DEG C culture 6h, with high speed freezing centrifuge by nutrient solution at 4 DEG C, with 13,000rpm centrifuge 15min,
Collect thalline.
Due to containing His-tag labels in recombinant plasmid pET-TPERha, pass through HisBind Purification
Kit (being purchased from Novagen companies) is purified, the recombinase purified.Specific operation process：
A. the processing of sample
(1) by washed thalline, it is resuspended with 1 × Binding Buffer 8mL, supersonic wave wall breaking.
(2) after broken wall, 13,000g centrifugation 30min take supernatant as sample.
B. pillar is handled
(1) 1mL fillers are taken to fill post.
(2) with 3mL sterile washing pillar.
(3) pillar is washed with 5mL 1 × Charge Buffer.
(4) pillar is washed with 3mL 1 × Binding Buffer.
C. loading
(1) sample is added into pillar, coutroi velocity 6 drop about per minute.
(2) pillar is washed with 1 × Binding of 3mL Buffer, uncombined protein is removed.
(3) eluent for containing 20mM imidazoles with 4mL washes pillar, except foreigh protein removing.
(4) pillar is washed with the eluent of 80mmol/L imidazoles, destination protein is eluted.
(5) pillar is washed with 1 × Strip of 4mL Buffer.
α-the rhamnosidase purified by this process.
Embodiment 3：The acquisition of beta-glucosidase/β of the present invention-xyloside bifunctional enzyme gene and recombinant plasmid pET-
DthBGL3 structure
3.1Dictyoglomus thermophilum DSM 3960 culture
Dictyoglomus thermophilum DSM 3960 are purchased from DSMZ DSMZs (www.dsmz.de) volume
Number it is 3960, its culture medium prescription is：Potassium dihydrogen phosphate 1.5g/L, disodium hydrogen phosphate dodecahydrate 4.2g/L, ammonium chloride 0.5g/
L, Magnesium dichloride hexahydrate 0.38g/L, two water and calcium chloride 0.06g/L, six ferric sulfate hydrate ammonium 0.04g/L, CoCL2 6H2O
2.9mg/L, two molybdic acid hydrate sodium 2.4mg/L, five are hydrated sodium selenate 1.7mg/L, four water and manganese chloride 2mg/L, zinc sulfate
2.8mg/L, soluble starch 5g/L, peptone 2g/L, yeast extract 2g/L, sodium carbonate 1g/L, cysteine hydrochloride 1g/L,
Resazurin sodium 1g/L, deoxidation in a nitrogen environment, adjustment pH is 7.2.It is inoculated with syringe according to 0.5% inoculum concentration, 85 DEG C quiet
24h is only cultivated, cell is collected.
The extraction of 3.2 genomic DNAs
(1) quiescent culture Dictyoglomus thermophilum DSM 3960 about 24 hours, take 30mL bacterium solutions 4,
000g centrifugations 10min collects cell.
(2) thalline is resuspended with 9.5mL TE buffer solutions, adds the lauryl sodium sulfate of 0.5mL 10% (SDS) and 50 μ L eggs
White enzyme K (20mg/mL), is well mixed, 37 DEG C of insulation 1h.
(3) 1.8mL 5mol/L NaCl, 1.5mL cetyltriethylammonium bromides (CTAB)/NaCl is added, is mixed, 65
DEG C incubate 20min.
(4) isometric chloroform/isoamyl alcohol is added, is mixed, 6,000g centrifugation 10min.
(5) to prevent shearing force from causing genomic DNA to be broken, supernatant is transferred in another centrifuge tube with thick mouth suction pipe, plus
Enter isometric phenol/chloroform/isoamyl alcohol to mix, 6,000g centrifugation 10min.
(6) in another centrifuge tube, 0.6 times of volume isopropanol is added, gently rocks to white thread DNA precipitations and clearly may be used
See.
(7) it is with suction pipe that DNA is wound on it, cleaned in 70% alcohol.
(8) DNA is scraped from suction pipe with sterile toothpick, be transferred in 1.5mL centrifuge tubes.
(9) air-dry at room temperature, plus 500 μ L TE buffer solutions.
(10) 50 μ L are taken to detect DNA concentration with nucleic acid-protein detector.
3.3 recombinant plasmid pET-DthBGL3 structure
(logged according to the new beta-glucosidase genes of known Dictyoglomus thermophilum DSM 3960
Number：WP_041723615.1 primer) is designed, primer is synthesized by Shanghai bioengineering Co., Ltd.With extraction
Dictyoglomus thermophilum DSM 3960 genomic DNA is template, and entering performing PCR with the primer of synthesis expands,
The condition of amplification is 94 DEG C, 3min；30 circulations (94 DEG C, 10s；58 DEG C, 30s；72 DEG C, 2.7min)；72 DEG C, 5min；Reaction
Stop, 4 DEG C of insulations.Pcr amplification product is purified by gel reclaims kit.Obtain beta-glucosidase DthBgl3
DNA molecular.
Obtained beta-glucosidase DthBgl3 DNA molecular and pET-28a are carried out with Nco I and XhoI respectively double
Digestion, and rubber tapping is reclaimed respectively, 16 DEG C of connections are stayed overnight after concentration, and connection product is converted into escherichia coli jm109 competent cell,
Screening positive clone, carries out sequence analysis；Select sequence and correctly clone extraction plasmid, obtain and contain new beta-glucosidase
The recombinant plasmid pET-DthBGL3 of DNA molecular.
Embodiment 4：The preparation of beta-glucosidase of the present invention
Recombinant plasmid pET-DthBGL3 is converted into e. coli jm109 (DE3) Host Strains (being purchased from Novagen companies),
LB flat boards (LB culture mediums containing kanamycins (50 μ g/mL)：Tryptone 10g/L, yeast extract 5g/L, NaCl 5g/
L, agar 15g/L) on by 37 DEG C of overnight incubations, choose transformant (50 μ g/mL kanamycins) 37 into 200mL LB culture mediums
DEG C, when 200rpm shaken cultivations to OD600 are 0.6, add final concentration of 0.005-0.01mM isopropyl ss-D- thio-pyryliums half
Lactoside (IPTG) derivant, 30 DEG C culture 6h, with high speed freezing centrifuge by nutrient solution at 4 DEG C, with 13,000rpm centrifuge
15min, collects thalline.
Due to containing His-tag labels in recombinant plasmid pET-DthBGL3, pass through HisBind Purification
Kit (being purchased from Novagen companies) is purified, the recombinase purified.Specific operation process：
A. the processing of sample
(1) by washed thalline, it is resuspended with 1 × Binding Buffer 8mL, supersonic wave wall breaking.
(2) after broken wall, 13,000g centrifugation 30min take supernatant as sample.
B. pillar is handled
(1) 1mL fillers are taken to fill post.
(2) with 3mL sterile washing pillar.
(3) pillar is washed with 5mL 1 × Charge Buffer.
(4) pillar is washed with 3mL 1 × Binding Buffer.
C. loading
(1) sample is added into pillar, coutroi velocity 6 drop about per minute.
(2) pillar is washed with 1 × Binding of 3mL Buffer, uncombined protein is removed.
(3) eluent for containing 20mM imidazoles with 8mL washes pillar, except foreigh protein removing.
(4) pillar is washed with the eluent of 200mmol/L imidazoles, destination protein is eluted.
(5) pillar is washed with 1 × Strip of 4mL Buffer.
The beta-glucosidase purified by this process, beta-glucosidase is identified by SDS-PAGE electrophoresis poststaining
The purity of enzyme, as a result as shown in Figure 2.
Beta-glucosidase gene expression quantity in Host Strains JM109 (DE3) is higher, and destination protein passes through HisTag labels
After purification, beta-glucosidase Dth3 purity is higher in its eluent, there is single band at 80kDa, reaches the pure level of electrophoresis
Not.
Embodiment 5：Alpha-L-Rhamnosidase and beta-glucosidase enzyme activity determination
With p-nitrophenol-α-rhamnoside (pNP-R) for substrate, hydrolyze obtained p-nitrophenol and occur with sodium carbonate
Chromogenic reaction, determines the absorbance of product under 405nm wavelength.100 μ L reaction systems include 75 μ L 100mM optimal pHs
Buffer solution, 20 μ L 5mM substrates mix to add after preheating under 5 μ L dilution enzyme liquids, optimum temperature and react 10min, then add
0.3mL 1M NaCO3Terminating reaction, ELIASA is determined under the conditions of 405nm after mixing.Do control of the enzyme liquid without substrate simultaneously
And have control of the substrate without enzyme liquid.
With p-nitrophenol-beta-glucosidase (pNP-G) for substrate, hydrolyze obtained p-nitrophenol and occur with sodium carbonate
Chromogenic reaction, determines the absorbance of product under 405nm wavelength.100 μ L reaction systems are slow including 90 μ L 100mM optimal pHs
Fliud flushing, 5 μ L 20mM substrates mix to add after preheating under 5 μ L dilution enzyme liquids, optimum temperature and react 10min, then add 0.3mL
1M NaCO3Terminating reaction, ELIASA is determined under the conditions of 405nm after mixing.Control of the enzyme liquid without substrate is done simultaneously and has bottom
Control of the thing without enzyme liquid.
With p-nitrophenol-β-xyloside (pNP-X) for substrate, hydrolyze obtained p-nitrophenol and occur to show with sodium carbonate
Colour response, determines the absorbance of product under 405nm wavelength.100 μ L reaction systems are buffered including 90 μ L 100mM optimal pHs
Liquid, 5 μ L 20mM substrates mix to add after preheating under 5 μ L dilution enzyme liquids, optimum temperature and react 10min, then add 0.3mL
1M NaCO3Terminating reaction, ELIASA is determined under the conditions of 405nm after mixing.Control of the enzyme liquid without substrate is done simultaneously and has bottom
Control of the thing without enzyme liquid.
One enzyme-activity unit (U) is defined as：Under conditions of being best suitable for, the enzyme needed for hydrolysis 1 μm of ol paranitrophenol of release
Amount.
Reference standard curve, calculates enzyme activity：
Enzyme activity (U/mL)=c × V 1/ (t × V2) × N
c：Content of p-nitrophenol (μm ol/mL) after the enzyme reaction calculated by p-nitrophenol normal equation；
V1：Reaction system cumulative volume (mL)；
t：The enzyme-to-substrate reaction time (min)；
V2：The volume (mL) of enzyme liquid during enzyme reaction；
N：Enzyme liquid extension rate.
Embodiment 6：The comparison of the restructuring enzyme hydrolysis epimedium flavone multicomponent catalytic capability of separate sources
6.1 compare catalytic capability of the separate sources alpha-L-Rhamnosidase to epimedium aglucone
The concentration of icariin is 1g/L, with the alpha-L-Rhamnosidase of three kinds of separate sources in respective optimum temperature, most
Suitable pH is conversion condition, adds 60U/mL enzyme, reacts 1h, is detected by HPLC.As a result as shown in Figure 3A：Derive from
Thermotoga petrophila DSM 13995 and Aspergillus terreus CCF 3059 alpha-L-Rhamnosidase
Icariin can effectively be converted for icariside I, its molar yield is respectively 45% and 12%；And derive from
Aspergillus niger NL-1 and Bacteroides thetaiotaomicron VPI-5482 alpha-L-Rhamnosidase
Icariin can not almost be converted.Therefore, source Thermotoga petrophila DSM 13995 α-L- rhamnoses are chosen
Glycosides enzyme is used for the conversion of barren wort total chromocor extract.
6.2 compare separate sources beta-glucosidase to towards Huo Ding A, the catalytic capability towards Huo Ding B and icariin
1g/L is hydrolyzed towards Huo Ding A, towards Huo Ding B and icariin with the beta-glucosidase of five kinds of separate sources respectively, instead
It is respective optimum temperature and optimal pH to answer condition, and enzyme addition is 25U/mL pure enzyme, and reaction time 1h is carried out by HPLC
Detection.As a result the beta-glucosidase of the different families of 5 kinds of separate sources can be efficiently by Chao Dinghuo A and excessive sheep as shown in Figure 3 B
Leaves of pulse plants glycosides is converted into the precious glycosides I suddenly of target product, however only from Dictyoglomus thermophilum DSM3960 β-
Glucuroide DthBGL3 has beta-glucosidase and xylobiase enzyme activity, therefore only DthBGL3 can be by towards suddenly
Determine B and be efficiently converted into the precious glycosides I suddenly of target product, its conversion ratio reaches 99.2%.Accordingly, from from Dictyoglomus
Thermophilum DSM3960 beta-glucosidase/β-xyloside bifunctional enzyme DthBGL3 conversion barren wort total chromocors are extracted
Thing.
Embodiment 7：Enzyme law catalysis conversion icariin prepares the technical study of epimedium aglucone
By selection is from Thermotoga petrophila DSM 13995 alpha-L-Rhamnosidase and derives from
Dictyoglomus thermophilum DSM3960 beta-glucosidase/β-xyloside bifunctional enzyme DthBGL3 is not respectively with
Shorthorned Epimedium P.E is converted with method.Wherein barren wort total chromocor extract is provided by Jiangsu Kang Yuan medicine companies limited company,
The content of its main component is as follows, Epimedin A:1.44%th, Epimedin B:2.76%th, epimedin C:5.96%th, icariin:
9.05%th, precious glycosides I suddenly:0.95%.
7.1 method 1- first add after beta-glucosidase/β-xyloside bifunctional enzyme plus alpha-L-Rhamnosidase
The concentration of Shorthorned Epimedium P.E is 1g/L, and the 100mmol/L citrate-phosphate disodium hydrogen buffer solutions of pH 4.5 first add
Beta-glucosidase/β-the xyloside for entering 30U/mL Dictyoglomus thermophilum DSM3960GH3 families is difunctional
Enzyme, then continues reaction 1h at 90 DEG C, adds 75U/mL Thermotoga petrophila DSM 13995 α-L- mouse again
Lee's glycosidase reacts 1h at 90 DEG C and detected by HPLC.As a result it is as shown in Figure 4：The molar yield of epimedium aglucone
For：87.3%.
7.2 method 2- add alpha-L-Rhamnosidase and beta-glucosidase/β-xyloside bifunctional enzyme simultaneously
The concentration of Shorthorned Epimedium P.E is 1g/L, and the 100mmol/L citrate-phosphate disodium hydrogen buffer solutions of pH 4.5 are added
75U/mL Thermotoga petrophila DSM 13995 alpha-L-Rhamnosidase and 30U/mL Dictyoglomus
The beta-glucosidase of thermophilum DSM3960GH3 families, reacts 2h at 90 DEG C, is detected by HPLC.Knot
Fruit is as shown in Figure 4：The molar yield of epimedium aglucone is：90.9%.
7.3 method 3- first add alpha-L-Rhamnosidase to add beta-glucosidase/β-xyloside bifunctional enzyme again
The concentration of Shorthorned Epimedium P.E is 1g/L, and the 100mmol/L citrate-phosphate disodium hydrogen buffer solutions of pH 4.5 first add
The alpha-L-Rhamnosidase for entering 75U/mL Thermotoga petrophila DSM 13995 reacts 1h, Ran Houzai at 90 DEG C
The beta-glucosidase for adding 30U/mL Dictyoglomus thermophilum DSM3960GH3 families continues anti-at 90 DEG C
1h is answered, is detected by HPLC.As a result it is as shown in Figure 4：The molar yield of epimedium aglucone is：95.1%.
Come from accordingly, it is determined that conversion Shorthorned Epimedium P.E prepares the optimised process of epimedium aglucone for the first Calais of method 3-
Calais comes from Dictyoglomus to Thermotoga petrophila DSM 13995 alpha-L-Rhamnosidase again
Thermophilum DSM3960 beta-glucosidase/β-xyloside bifunctional enzyme DthBGL3.
Embodiment 8：Barren wort total chromocor of the present invention antitumor activity after DthBGL3 and TPERha conversions
3000 cells are inoculated with per hole in 96 orifice plates, 37 DEG C of cultures are put into, after after cell attachment 6h, added per hole different
The epimedium flavone culture 72h of concentration, adds 20 μ L MTT (4mg/mL) to every hole in 4h before experimental endpoints, is taken during experimental endpoints
Go out 96 orifice plates, in 1000rcf centrifugations, then suck supernatant, add and light absorption value is surveyed at 200 μ L DMSO, 570nm.According to following public affairs
Formula calculates inhibiting rate of the testing sample to Proliferation of Tumor Cells In Vitro：
Inhibiting rate=[100- (OD570 (experimental port)-OD570 (blank control))/(OD (not dosing control is empty)-
OD570 (blank control) × 100) %
HPLC collection of illustrative plates before and after being converted according to barren wort total chromocor in embodiment 7 through DthBGL3 and TPERha is visible, excessive sheep
Leaves of pulse plants general flavone is mainly made up of Epimedin A, B, C and icariin originally, wherein, the content highest of icariin, converted product
Predominantly epimedium aglucone.Anti tumor activity in vitro research as shown in Figure 6 shows, under 100 μM of concentration, epimedium aglucone
To human liver cancer HepG2, lung cancer A549, mouse junction cancer CT26, breast cancer 4T1 proliferation inhibition rate respectively be up to 88%, 46%,
61% and 89%, it is significantly higher than the other monomers composition in barren wort total chromocor.Therefore, barren wort total chromocor through DthBGL3 and
TPERha is converted into after epimedium aglucone, and antitumor activity is improved.
Claims (8)
1. a kind of method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone, it is characterised in that by heat-resisting α-L- rhamnoses
The dual-enzyme system of glycosides enzyme and heat-resisting beta-glucosidase composition can convert the multicomponent flavone glycoside life in barren wort total chromocor
Into epimedium aglucone, the multicomponent flavone glycoside is icariin, determines C towards Huo Ding A, towards Huo Ding B and Chao Huo.
2. the method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone according to claim 1, it is characterised in that institute
Heat-resisting alpha-L-Rhamnosidase is stated to derive fromThermotoga petrophilaDSM 13995 alpha-L-Rhamnosidase base
The Escherichia coli recombinant strain of cause.
3. the method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone according to claim 1, it is characterised in that institute
Heat-resisting beta-glucosidase is stated to derive fromDictyoglomus thermophilumThe GH3 beta-glucosidase bases of DSM 3960
The Escherichia coli recombinant strain of cause.
4. the method that enzymatic conversion method barren wort total chromocor prepares epimedium aglucone according to claim 1, it is characterised in that match somebody with somebody
The mmol/L citrate-phosphate disodium hydrogen buffer solutions of pH 4.5,50 processed, it is 5 g/L to add barren wort total chromocor to concentration, simultaneously
AddThermotoga petrophila DSM 13995 heat-resisting alpha-L-Rhamnosidase to 30 U/mL, andDictyoglomus thermophilumThe heat-resisting beta-glucosidase of the GH3 families of DSM 3960 is to 25 U/mL, at 85 DEG C
React 1 h.
5. the dual-enzyme system being made up of heat-resisting alpha-L-Rhamnosidase and heat-resisting beta-glucosidase is in conversion barren wort total chromocor
In multicomponent flavone glycoside be epimedium aglucone in application.
6. convert the enzymatic compositions that the multicomponent flavone glycoside in barren wort total chromocor is epimedium aglucone, it is characterised in that by resistance to
Hot alpha-L-Rhamnosidase and heat-resisting beta-glucosidase composition.
7. the obtained product for being rich in epimedium aglucone of claim 1 methods described.
8. the product rich in epimedium aglucone is preparing treatment breast cancer, lung cancer, colon cancer, liver cancer according to claim 7
Application in medicine.
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