CN110272929A - A kind of enzymic catalytic reaction system producing isoquercitrin using microlayer model catalysis rutin - Google Patents
A kind of enzymic catalytic reaction system producing isoquercitrin using microlayer model catalysis rutin Download PDFInfo
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- CN110272929A CN110272929A CN201910495452.4A CN201910495452A CN110272929A CN 110272929 A CN110272929 A CN 110272929A CN 201910495452 A CN201910495452 A CN 201910495452A CN 110272929 A CN110272929 A CN 110272929A
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- microlayer model
- rutin
- isoquercitrin
- catalytic reaction
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- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 39
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 title claims abstract description 38
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 title claims abstract description 38
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 title claims abstract description 38
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 235000005493 rutin Nutrition 0.000 title claims abstract description 38
- 229960004555 rutoside Drugs 0.000 title claims abstract description 38
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 title claims abstract description 37
- OVSQVDMCBVZWGM-QSOFNFLRSA-N quercetin 3-O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-QSOFNFLRSA-N 0.000 title claims abstract description 34
- OVSQVDMCBVZWGM-IDRAQACASA-N Hirsutrin Natural products O([C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1)C1=C(c2cc(O)c(O)cc2)Oc2c(c(O)cc(O)c2)C1=O OVSQVDMCBVZWGM-IDRAQACASA-N 0.000 title claims abstract description 33
- FVQOMEDMFUMIMO-UHFFFAOYSA-N Hyperosid Natural products OC1C(O)C(O)C(CO)OC1OC1C(=O)C2=C(O)C=C(O)C=C2OC1C1=CC=C(O)C(O)=C1 FVQOMEDMFUMIMO-UHFFFAOYSA-N 0.000 title claims abstract description 33
- GXMWXESSGGEWEM-UHFFFAOYSA-N isoquercitrin Natural products OCC(O)C1OC(OC2C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)C(O)C1O GXMWXESSGGEWEM-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 108090000790 Enzymes Proteins 0.000 claims abstract description 22
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- 238000006911 enzymatic reaction Methods 0.000 claims abstract 2
- 238000006243 chemical reaction Methods 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 13
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- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000007789 sealing Methods 0.000 claims description 5
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- 230000001939 inductive effect Effects 0.000 claims description 4
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- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
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- 150000001875 compounds Chemical class 0.000 claims description 2
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- 108010044879 alpha-L-rhamnosidase Proteins 0.000 abstract description 10
- 239000000872 buffer Substances 0.000 abstract description 6
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- 229940079593 drug Drugs 0.000 abstract description 3
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- 238000011105 stabilization Methods 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000000034 method Methods 0.000 description 8
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- 238000005516 engineering process Methods 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
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- 241000406668 Loxodonta cyclotis Species 0.000 description 1
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 description 1
- 241001480055 Quercus mongolica Species 0.000 description 1
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
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- 150000002338 glycosides Chemical class 0.000 description 1
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- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 1
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- 229960001285 quercetin Drugs 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
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- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- 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)
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/0104—Alpha-L-rhamnosidase (3.2.1.40)
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Abstract
A kind of enzymic catalytic reaction system producing isoquercitrin using microlayer model catalysis rutin.Using flow focusing cake core, the crude enzyme liquid and the buffer containing substrate obtained with recombinant bacterial strain E.coli BL21 (DE3)-pET21a-rhaB1 fermentation is mixed into dispersed phase by comparing 1:1 (v/v), silicone oil is continuous phase, the pressure limit for regulating and controlling dispersed phase and continuous phase is 25~200mbar, prepares microlayer model.The crude enzyme liquid for preparing rutin solution and alpha-L-Rhamnosidase, prepares activated product isoquercitrin using microlayer model enzyme.The conduit of the system of enzyme reaction containing microlayer model is placed in 25~60 DEG C, is taken out after 0.1~5h, it is spare in collecting duct.The present invention can prepare specific drop using micro-fluidic chip, as independent microreactor, have small in size, large specific surface area, environment closing and the advantages such as stabilization, the few, no cross contamination of drug dosage, and can improve enzyme' s catalysis efficiency.
Description
Technical field
The invention belongs to biological chemical fields, and in particular to it is a kind of using microlayer model catalysis rutin produce isoquercitrin enzyme urge
Change reaction system.
Background technique
Microlayer model refers to two kinds of mutual immiscible formed diameters of liquid in micron or nanoscale drop.It utilizes fluid
Interaction between shearing force and surface tension by continuous liquid be separated into miniature droplets (micro-nano electronic technology, 2016,
53:108-113.).Microlayer model technology has many advantages that microlayer model can be used as the reaction system of a small size, by reactant
Material is encapsulated in droplet, can dramatically speed up the reaction between the rate of mixing and substance；Reaction time is controllable, and drop size can also
Accurate control, at the same microlayer model chip it is reusable (Chemical Engineering Science, 2016,169:273-
283).In addition, the homogeneous effect that drop reactor provides is greatly improved reaction efficiency, as microlayer model can will be in conventional reactor
Only 60% hydrolysis efficiency is improved to 100% (ChemPlusChem, 2016,81:629-636), it is shown that microlayer model system
System is applied to the huge advantage of enzymic catalytic reaction.
Rutin (Rutin) can be catalytically conveted to isoquercitrin and L- rhamnose (modern food for alpha-L-Rhamnosidase
Science and technology, 2015 (1): 107-114.).Isoquercitrin category flavone compound, have anti-oxidant, antitumor, antidepression, decompression,
Numerous pharmacological actions such as lipid-loweringing (Strait Pharmaceutical Journal, 2015:223-225), it is international clinically for treating myocardial ischemia, anoxic etc.
On be processed into food additives and ancillary drug, can be used as effective ingredient and be directly used in drug production, compare precursor
Object rutin (content 5~20%) has stronger inoxidizability and anticancer activity (Biotech, 2016,6:3), and even more synthesis is new
Intermediate (the Neurochemistry of type food color EMIQ (Enzymatically Modified Isoquercitrin)
International, 2010,57:713-721), function and effect are significant, wide application.
Rhamnosidase in nature have two class of alpha-L-Rhamnosidase and beta-L-Rhamnosidase (China brewing,
2010,29:11-15).So far, since condition limits, alpha-L-Rhamnosidase not yet carries out large-scale production, causes pure
Enzyme is on the high side, and the application development of enzyme is restricted.HFM-rha78 is obtained from healthy human body excrement macro genome DNA
Novel fine bacterium source alpha-L-Rhamnosidase gene increases rutin conversion ratio with enzyme concentration at 37 DEG C and increases, rutin when acting on 10h
Conversion ratio is 41.8% (Chinese biological chemistry and molecular biosciences journal, 2018,34 (12)).
It is determining for isoquercitrin that the alpha-L-Rhamnosidase of recombinant bacterial strain expression, which is more advantageous to rutin catalysis than wild strain,
To reaction (Jiangsu University of Science and Technology, 2017).Enzyme law catalysis synthesizes isoquercitrin there are a big problem, and rutin and its hydrolysate are different
Quercitin and Quercetin all have strong anti-oxidation, by thermally labile, will lead to oxidation for a long time or other reactants generate.Cause
This, a closed system generates isoquercitrin for alpha-L-Rhamnosidase catalysis rutin, is expected to solve product, substrate shakiness
The technical bottlenecks such as determine, be oxidized easily.
Summary of the invention
The present invention is rare and expensive for the biocatalyst alpha-L-Rhamnosidase source of hydrolyzing rutin, causes
The excessively high problem of the cost of isoquercitrin is produced, it is anti-to provide a kind of enzymatic for producing isoquercitrin using microlayer model catalysis rutin
System is answered, enzymic catalytic reaction system reaction condition is mild, substrate is selectively strong, environmental-friendly for this, can handle only a small amount of liquid
Brand new technical, improve enzyme-to-substrate contact probability intracellular.
A kind of enzymic catalytic reaction system producing isoquercitrin using microlayer model catalysis rutin, the enzymic catalytic reaction system are logical
Cross following steps building:
Step 1, recombinant bacterial strain E.coli BL21 (DE3)-pET21a-rhaB1 is constructed, simultaneously inducing expression is cultivated, induces table
After reaching, it is centrifuged so that clasmatosis, takes supernatant, i.e. crude enzyme liquid is spare；
Step 2, it is the rutin solution of 0.01~1g/L with the PBS buffer solution compound concentration of pH4.0~8.0, then takes concentration
For the crude enzyme liquid of 30.00U/mg, mix in equal volume microlayer model forms required water phase i.e. dispersed phase, viscosity is the poly- diformazan of 5CS
Oil phase i.e. continuous phase needed for radical siloxane methyl-silicone oil is formed as microlayer model；
Step 3, using microlayer model chip, choosing dispersion phase pressure is 25~200mbar, continuous phase pressure is 25~
200mbar, operation microlayer model forms device and forms microlayer model, after droplets stable, uniform, be collected in sealing PTFE and carry out enzyme
Catalysis reaction, to after reaction, develop product isoquercitrin with constant flow pump, and carry out content detection.
It is that the type selecting of the microlayer model chip is flow focusing cake core as improved.
It is further improved to be, the long 22.5mm of the flow focusing cake core, wide 15.0mm, thickness 4.0mm, two intersections
Melt place, two-phase microchannel internal diameter is 250 μm, at blending after 400 μm of part internal diameter.
It is that dispersion phase pressure is 125mbar as improved, continuous phase pressure is 150mbar, and regulation prepares drop, surely
Sealing PTFE conduit is collected in after fixed.
It is that the reaction temperature of enzymic catalytic reaction is 25~60 DEG C in step 3 as improved；Reacting pH is 4.0~8.0；
Reaction time is 0.1~5h.
The utility model has the advantages that
The present invention uses microlayer model technology, with flow focusing cake core, reaction condition is mild, substrate selectivity is strong,
It is environmental-friendly, the brand new technical of only a small amount of liquid can be handled, enzyme-to-substrate contact probability intracellular is improved.Relative to traditional enzyme
The spatiotemporal efficiency of catalysis reaction is low, enzyme dosage is big and process is difficult to amplify, and causes at high cost, the low phenomenon of product rate, use is micro-
Drop technique helps to solve the above problems, to expand the application range of microlayer model enzymic catalytic reaction system.And it utilizes
When PTFE catheter collection, increases catheter length and collect drop, it is not significant on the influence of enzymic catalytic reaction result, therefore can be by microlayer model enzyme
Catalystic converter system Linear Amplifer is applied to platform and prepares activated product.Recombinant bacterial strain E.coli BL21 (DE3)-pET21a-
The alpha-L-Rhamnosidase of rhaB1 expression is more advantageous to the orientation reaction that rutin catalysis is isoquercitrin than wild strain.It utilizes
The application of the microlayer model enzymic catalytic reaction system on bioconversion rutin, reduces costs, improves efficiency of pcr product, and have
There is good industrialization prospect.
Detailed description of the invention
Fig. 1 is the HPLC chromatogram of isoquercitrin standard items；
Fig. 2 is the HPLC chromatogram of rutin after microlayer model enzymatic in system, isoquercitrin amount, wherein the different Mongolian oak of 1-
Skin glycosides, 2- rutin.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Microlayer model enzymic catalytic reaction system method is prepared in the embodiment of the present invention are as follows: formed using the microlayer model that success constructs
System, oil mutually use that stability is good, avirulent viscosity is for the dimethyl silicone polymer methyl-silicone oil of 5CS, water phase for rutin and
Alpha-L-Rhamnosidase mixed liquor forms system by operation microlayer model and adjusts continuous phase and dispersed phase pressure value, makes system institute
The drop stability of formation is good, it is appropriate to form frequency height, size dimension, with PTFE catheter collection drop, both obtains microlayer model.
It is high performance liquid chromatography that the measuring method of detection rutin and isoquercitrin qualitative, quantitative is used in the embodiment of the present invention
Method, condition are as follows: H&E Pump P3000A constant flow pump, UV-VIS detector, column model Alltima C18, length are
25mm, internal diameter are 5 μm, maintain 30 DEG C using HPLC-UV separation and measurement rutin and isoquercitrin, column temperature.Mobile phase is second
Nitrile: (0.02%) phosphoric acid (volume ratio 20:80), flow velocity 1.0mlmin-1, Detection wavelength 360nm.Sample volume is 20 μ L.Sample introduction
Before, sample passes through 0.45 μm of filter filtering.
Wherein, rutin conversion ratio and isoquercitrin yield calculation method are as follows:
Embodiment 1
Recombinant bacterial strain E.coli BL21 (DE3)-pET21a-rhaB1, obtains purpose from elephant excrement using PCR method
Gene rhaB1 (size 3081bp).
The primer used are as follows: upstream primer sequence: 5 '-CGGGTCGACAGATGTTCGGAAAGG-3 ' (underscore Sal
I restriction enzyme site), downstream primer sequence: (underscore is Hind III enzyme to 5 '-CCGAAGCTTATAGCTGAAGGTGTAATG-3 '
Enzyme site).PCR product rhaB1 and plasmid pET21a through Sal I and Hind III double digestion, then use T4DNA ligase will
PCR product is connected into pET21a, obtains recombinant plasmid pET21a-rhaB1.It is verified using double digestion and PCR.It will be verified correct
Recombinant bacterial strain transfects into E.coli DH5 α the clone for carrying out target gene.It prepares and feels with reference to competent cell reagent preparation box
By state cell E.coli BL21 (DE3), the Transfected Recombinant Plasmid for then obtaining clone is into competent cell E.coli BL21
(DE3).37 DEG C of shaking table culture E.coliBL21 to OD600=0.5~0.6, bacterium solution places 5~10min on ice in plastic tube, and 4
DEG C, 5000rpm be centrifuged 5min, bacterial sediment pre-cooling BT Buffer A be softly resuspended, on ice placement 10~15min, 4 DEG C,
5000rpm is centrifuged 5min, and the BT Buffer B of bacterial sediment pre-cooling is softly resuspended, dispenses into the centrifuge tube being pre-chilled on ice
Ultra low temperature freezer is placed to save.Competent cell is set to be melted on ice, and 10ng DNA to be transformed is added, soft to mix, and is placed on ice
Centrifuge tube is set in 42 DEG C of water-baths and is incubated for 90s by 30min, places 2~3min on ice immediately after, and 1mL is added and is preheated to 37 DEG C
Not antibiotic LB culture medium takes appropriate bacterium solution to be applied to ammonia in 37 DEG C of shaking table slow oscillation culture 60min after being mixed by inversion
In the LB plate of benzyl resistance, it is inverted overnight incubation in 37 DEG C of incubators, blue hickie is then carried out and sieves bacterium.The correct single bacterium of picking
It falls, is inoculated in (3~5mL) in the LB liquid medium containing amicillin resistance (50 μ g/mL), 37 DEG C of shaken cultivation growths
To exponential phase.Carrying out the verifying of extracting recombinant plasmid and bacterium solution in bacterium solution respectively is that template carries out PCR verifying.It is verified it is correct after
It is transferred to (500mL) in the LB liquid medium containing amicillin resistance (50 μ g/mL) according to 2% bacterium amount that connects, 22 DEG C
Constant-temperature shaking culture is to OD600When reaching 0.6~0.8,400 μM of isopropyl-β-D-thiogalactosides (IPTG) are then added,
It reduces the temperature to 17 DEG C of progress low temperature inductions and expresses 16~20h.The bacterium solution that inducing expression is terminated 8000rpm at 4 DEG C is centrifuged
5min collects thallus；Twice with pH7.2~7.4PBS buffer solution for cleaning thallus；Then thallus is resuspended with sonication buffer to carry out
Ultrasound.Sonication buffer are as follows: 1.0M Tris-HCl (pH8.0,30mL), ultrasonic power 0.6kW, ultrasonic time are
12min.12000rpm is centrifuged 30min after ultrasound, and collecting supernatant is RhaB1 crude enzyme liquid.
The rutin solution of 0.01g/L, thick enzyme obtained by use are prepared using citrate-phosphate disodium hydrogen pH of buffer 5.5
Liquid is oily phase with dimethyl silicone polymer methyl-silicone oil, microchip prepares enzymatic by being water phase compared to 1:1 (v/v) mixed liquor
React the process of drop.Using flow focusing cake core, disperse phase pressure 25mbar, continuous phase pressure 25mbar operates micro- liquid
It drips and forms device, after droplets stable, be collected in sealing PTFE conduit.
The conduit of the enzyme containing microlayer model is placed in 30 DEG C of insulating boxs, is taken out after 10min, it will be micro- in conduit using pressure pump
Drop is collected in EP pipe, is centrifuged 3min, and after treatment takes upper oil phase and lower water phase dilution in methanol solution, is carried out
HPLC detection is handled data and is learnt, when reaction environment temperature is 35 DEG C, isoquercitrin yield obtains maximum value, rutin
Conversion ratio is 50.33 ± 0.13%, and the yield of isoquercitrin is 9.80 ± 1.2%.
Embodiment 2
Except the pH 5.62 of citrate-phosphate disodium hydrogen buffer used prepares the rutin solution of 0.02g/L, dispersed phase pressure
Outside power 125mbar, continuous phase pressure 150mbar, remaining is the same as embodiment 1.
Conduit containing microlayer model is placed in 35 DEG C of insulating boxs, is taken out after 120min, it will be micro- in conduit using pressure pump
Drop is collected in EP pipe, is centrifuged 3min, and after treatment takes upper oil phase and lower water phase dilution in methanol solution, is carried out
HPLC detection is handled data and is learnt, the conversion ratio of rutin is 65.21 ± 0.06%, and the yield of isoquercitrin is 41.38 ±
0.37%, the yield of product isoquercitrin improves 4.22 times.
Embodiment 3
Except the pH 7.5 of citrate-phosphate disodium hydrogen buffer used prepares the rutin solution of 0.03g/L, disperse phase pressure
Outside 200mbar, continuous phase pressure 200mbar, remaining is the same as embodiment 1.
Conduit containing microlayer model is placed in 60 DEG C of insulating boxs, is taken out after 300min, it will be micro- in conduit using pressure pump
Drop is collected in EP pipe, is centrifuged 3min, and after treatment takes upper oil phase and lower water phase dilution in methanol solution, is carried out
HPLC detection is handled data and is learnt, the conversion ratio of rutin is 50.51 ± 0.28%, and the yield of isoquercitrin is 14.47 ±
0.04%, the yield of product isoquercitrin improves 1.48 times.
Sequence table
<120>a kind of enzymic catalytic reaction system that isoquercitrin is produced using microlayer model catalysis rutin
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cgggtcgaca gatgttcgga aagg 24
<210> 2
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccgaagctta tagctgaagg tgtaatg 27
Claims (5)
1. a kind of enzymic catalytic reaction system for producing isoquercitrin using microlayer model catalysis rutin, which is characterized in that the enzymatic
Reaction system is constructed by following steps: step 1, constructing recombinant bacterial strain E.coli BL21 (DE3)-pET21a-rhaB1, culture
And inducing expression, after inducing expression, it is centrifuged so that clasmatosis, takes supernatant, i.e. crude enzyme liquid is spare；Step 2, it uses
The PBS buffer solution compound concentration of pH4.0~8.0 is the rutin solution of 0.01~1g/L, then taking concentration is the thick enzyme of 30.00U/mg
Liquid, mix in equal volume microlayer model forms required water phase i.e. dispersed phase, viscosity is the dimethyl silicone polymer methyl-silicone oil of 5 CS
Oil phase i.e. continuous phase needed for being formed as microlayer model；Step 3, using microlayer model chip, choose dispersion phase pressure be 25~
200mbar, continuous phase pressure are 25~200mbar, and operation microlayer model forms device and forms microlayer model, to droplets stable, uniform
Afterwards, it is collected in sealing PTFE and carries out enzymic catalytic reaction, to after reaction, develop product isoquercitrin with constant flow pump,
And carry out content detection.
2. a kind of enzymic catalytic reaction system for producing isoquercitrin using microlayer model catalysis rutin according to claim 1, special
Sign is that the type selecting of the microlayer model chip is flow focusing cake core.
3. a kind of enzymic catalytic reaction system for producing isoquercitrin using microlayer model catalysis rutin according to claim 2, special
Sign is, the long 22.5mm of the flow focusing cake core, wide 15.0mm, thickness 4.0mm, at two-phase blending, two-phase microchannel
Internal diameter is 250 μm, at blending after 400 μm of part internal diameter.
4. a kind of enzymic catalytic reaction system for producing isoquercitrin using microlayer model catalysis rutin according to claim 1, special
Sign is that dispersion phase pressure is 125mbar, and continuous phase pressure is 150mbar, and regulation prepares drop, is collected in sealing after stablizing
PTFE conduit.
5. a kind of enzymic catalytic reaction system for producing isoquercitrin using microlayer model catalysis rutin according to claim 1, special
Sign is that the reaction temperature of enzymic catalytic reaction is 25~60 DEG C in step 3；Reacting pH is 4.0~8.0；Reaction time be 0.1~
5h。
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Citations (4)
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|---|---|---|---|---|
| CN101985639A (en) * | 2010-11-16 | 2011-03-16 | 江苏科技大学 | Application of alpha-L-rhamnoside enzyme in directional synthesis of isoquercitrin by biological conversion of rutin |
| CN105969826A (en) * | 2016-06-17 | 2016-09-28 | 江苏科技大学 | Method for synthesizing isoquercitrin with nano-particle immobilized enzyme special for microreactor |
| CN106119319A (en) * | 2016-08-25 | 2016-11-16 | 江苏科技大学 | Recombinant alpha L rhamnoside enzyme extract is catalyzed the method for directionally hydrolyzing flavonoid glycoside in micro passage reaction |
| CN107287223A (en) * | 2017-06-20 | 2017-10-24 | 江苏科技大学 | α L rhamnosides enzyme genes and its application |
-
2019
- 2019-06-10 CN CN201910495452.4A patent/CN110272929B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985639A (en) * | 2010-11-16 | 2011-03-16 | 江苏科技大学 | Application of alpha-L-rhamnoside enzyme in directional synthesis of isoquercitrin by biological conversion of rutin |
| CN105969826A (en) * | 2016-06-17 | 2016-09-28 | 江苏科技大学 | Method for synthesizing isoquercitrin with nano-particle immobilized enzyme special for microreactor |
| CN106119319A (en) * | 2016-08-25 | 2016-11-16 | 江苏科技大学 | Recombinant alpha L rhamnoside enzyme extract is catalyzed the method for directionally hydrolyzing flavonoid glycoside in micro passage reaction |
| CN107287223A (en) * | 2017-06-20 | 2017-10-24 | 江苏科技大学 | α L rhamnosides enzyme genes and its application |
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