CN113125482A - Eutectic solvent for sample preparation of cryoelectron microscope and preparation method thereof - Google Patents
Eutectic solvent for sample preparation of cryoelectron microscope and preparation method thereof Download PDFInfo
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- CN113125482A CN113125482A CN202110425865.2A CN202110425865A CN113125482A CN 113125482 A CN113125482 A CN 113125482A CN 202110425865 A CN202110425865 A CN 202110425865A CN 113125482 A CN113125482 A CN 113125482A
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- 239000002904 solvent Substances 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 32
- 230000005496 eutectics Effects 0.000 title claims abstract description 31
- 239000001257 hydrogen Substances 0.000 claims abstract description 31
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 31
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 24
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims abstract description 11
- 235000019743 Choline chloride Nutrition 0.000 claims abstract description 11
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims abstract description 11
- 229960003178 choline chloride Drugs 0.000 claims abstract description 11
- 239000000203 mixture Substances 0.000 claims abstract description 11
- -1 amine compound Chemical class 0.000 claims abstract description 7
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 5
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims abstract description 5
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims abstract description 5
- 229960003237 betaine Drugs 0.000 claims abstract description 5
- 229930182821 L-proline Natural products 0.000 claims abstract description 4
- 229960002429 proline Drugs 0.000 claims abstract description 4
- 235000000346 sugar Nutrition 0.000 claims abstract description 4
- 150000007524 organic acids Chemical class 0.000 claims abstract description 3
- 239000007788 liquid Substances 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 17
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 7
- 238000010438 heat treatment Methods 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- 235000013772 propylene glycol Nutrition 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 239000004202 carbamide Substances 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 claims description 2
- 239000011976 maleic acid Substances 0.000 claims description 2
- 150000008163 sugars Chemical class 0.000 claims description 2
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims 1
- 235000001727 glucose Nutrition 0.000 claims 1
- 235000005985 organic acids Nutrition 0.000 claims 1
- 229920005862 polyol Polymers 0.000 claims 1
- 150000003077 polyols Chemical class 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 12
- 108090000790 Enzymes Proteins 0.000 abstract description 12
- 239000000758 substrate Substances 0.000 abstract description 10
- 102000005744 Glycoside Hydrolases Human genes 0.000 abstract description 9
- 108010031186 Glycoside Hydrolases Proteins 0.000 abstract description 9
- 239000003795 chemical substances by application Substances 0.000 abstract description 7
- 230000003197 catalytic effect Effects 0.000 abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 5
- 238000003860 storage Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 150000005846 sugar alcohols Polymers 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 28
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 18
- 239000000243 solution Substances 0.000 description 18
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical compound C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 16
- 239000007853 buffer solution Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 12
- 102000004357 Transferases Human genes 0.000 description 11
- 108090000992 Transferases Proteins 0.000 description 11
- 238000002156 mixing Methods 0.000 description 10
- 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 8
- 235000010290 biphenyl Nutrition 0.000 description 8
- 239000004305 biphenyl Substances 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 229930003944 flavone Natural products 0.000 description 8
- 235000011949 flavones Nutrition 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- 150000002212 flavone derivatives Chemical class 0.000 description 7
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 230000002210 biocatalytic effect Effects 0.000 description 6
- 229910052802 copper Inorganic materials 0.000 description 6
- 239000010949 copper Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000000411 inducer Substances 0.000 description 6
- 229930027917 kanamycin Natural products 0.000 description 6
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 6
- 229960000318 kanamycin Drugs 0.000 description 6
- 229930182823 kanamycin A Natural products 0.000 description 6
- 229930014626 natural product Natural products 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 5
- 229930182478 glucoside Natural products 0.000 description 5
- 229960004063 propylene glycol Drugs 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 239000006184 cosolvent Substances 0.000 description 3
- 210000004748 cultured cell Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 239000012520 frozen sample Substances 0.000 description 3
- 150000008195 galaktosides Chemical class 0.000 description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 102000051366 Glycosyltransferases Human genes 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 1
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229940098895 maleic acid Drugs 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940045136 urea Drugs 0.000 description 1
- 238000002424 x-ray crystallography Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/2202—Preparing specimens therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a eutectic solvent for preparing a sample by a cryoelectron microscope and a preparation method thereof, and particularly relates to the field of preparing a sample by a cryoelectron microscope. The hydrogen bond donor agent is selected from one of polyalcohol, sugar, organic acid and organic amine compound, or their mixture. The hydrogen bond receptor reagent is at least one selected from the group consisting of choline chloride, betaine and L-proline. The molar ratio of the hydrogen bond donor reagent to the hydrogen bond acceptor reagent is 1-2: 2-4. By using the eutectic solvent, the problem of unclear result background caused by poor water solubility of a substrate during sample preparation of a cryoelectron microscope is solved, and the catalytic activity, the thermal stability, the storage time and other enzyme activity properties of the glycosidase are improved.
Description
Technical Field
The invention relates to the field of sample preparation of a cryoelectron microscope, in particular to a eutectic solvent for sample preparation of a cryoelectron microscope and a preparation method thereof.
Background
Cryoelectron microscopy is an important technique in the field of structural biology and is commonly used for the resolution of large molecular weight, difficult to crystallize biomolecular structures. With the development of the cryoelectron microscopy technology, the resolution at the atomic level can be resolved, and the cryoelectron microscopy technology gradually becomes a biomacromolecule structure resolution technology which is complementary with the X-ray crystallography technology. Is especially suitable for resolving the interaction mechanism between macromolecules or with small molecules.
Glycosyltransferases are enzymes that link catalytically activated sugars, such as UDP-glucose, to different acceptor molecules in vivo, and the glycosylated products have many biological functions. The nonpolar compound flavone, biphenyl and other compounds in the natural medicinal active matters can change the polarity thereof through glycosylation to realize polarity inversion, thereby promoting the absorption and transportation of the compounds in organisms and improving the biological activity of the compounds.
In the process of researching the catalytic mechanism of the glucoside transferase, a cryoelectron microscope is used for researching the structure of the glucoside transferase, but the sample preparation of the cryoelectron microscope is generally carried out in a buffer solution environment with water as a main body, so that the problem of precipitation due to poor solubility of a nonpolar substrate is easy to occur, and the enzymatic reaction is further hindered.
Disclosure of Invention
In view of the above, in the process of preparing a sample by a cryoelectron microscope, the eutectic solvent is used to solve the problem of unclear result background caused by poor water solubility of a substrate in the process of preparing the sample by the cryoelectron microscope, and improve the enzyme activity properties of the glycosidase, such as catalytic activity, thermal stability, storage time and the like. The DES-buffer solvent disclosed by the invention is simple to prepare, mild in reaction conditions, non-toxic and degradable, conforms to the development of green chemistry, and has a wide industrial production prospect.
In order to achieve the purpose, the invention provides the following technical scheme:
a eutectic solvent for preparing a sample by a cryoelectron microscope comprises a hydrogen bond donor reagent and a hydrogen bond acceptor reagent.
The hydrogen bond donor agent is selected from one of polyalcohol, sugar, organic acid and organic amine compound, or their mixture.
The hydrogen bond receptor reagent is at least one selected from the group consisting of choline chloride, betaine and L-proline.
The molar ratio of the hydrogen bond donor reagent to the hydrogen bond acceptor reagent is 1-2: 2-4.
Preferably, the hydrogen bond donor agent is selected from one of propylene glycol, 1, 2-propylene glycol, glucose, maleic acid and urea, or a mixture thereof.
Preferably, the hydrogen bond receptor agent is at least one of choline chloride, betaine and L-proline.
Preferably, the hydrogen bond donor agent is selected from 1, 2-propanediol.
Preferably, the molar ratio of hydrogen bond donor agent to hydrogen bond acceptor agent is 1: 2.
A preparation method of a eutectic solvent comprises the following steps:
and S01, mixing the hydrogen bond acceptor reagent and the hydrogen bond donor reagent according to a preset molar ratio, and adding the mixture into the flask.
S02, adding a stirring bar into the flask, stirring the mixture by using a magnetic stirrer at the temperature of 70-80 ℃, heating for 2-10 h until colorless transparent liquid is formed, cooling to room temperature, and storing the colorless transparent liquid in a silica gel drier for 14 d.
Preferably, in step S02, the reaction temperature is 75 ℃.
Preferably, in step S02, the heating time is 6 h.
Compared with the prior art, the invention has the beneficial effects that:
compared with the traditional buffer solvent, the DES-buffer solvent is prepared firstly, and then the traditional buffer solvent is replaced by the DES-buffer solvent in the process of glycosidase transferase, the eutectic solvent is simple to prepare, mild in reaction condition, non-toxic and degradable, accords with the development of green chemistry, and has wide industrial production prospect.
Meanwhile, the eutectic solvent provided by the invention solves the problem of influence of substrate solubility on preparation of a sample by a cryoelectron microscope, promotes the biocatalytic conversion of small molecules of natural products such as flavone and biphenyl, and optimizes the reaction environment by using the eutectic solvent.
Detailed Description
The following examples may help one skilled in the art to better understand the present invention, but are not intended to limit the present invention in any way.
Example 1
The invention provides a eutectic solvent for preparing a sample by a cryoelectron microscope and a preparation method thereof, solves the problem of influence of substrate solubility on the sample preparation by the cryoelectron microscope, promotes the biocatalytic conversion of small molecules of natural products such as flavone and biphenyl, and optimizes the reaction environment by using the eutectic solvent. The eutectic solvent is prepared by mixing choline chloride and glycerol with the purity of more than or equal to 99% according to the proportion of 1: 2.
The method for preparing the low cosolvent comprises the following steps: mixing choline chloride and glycerol with purity of more than or equal to 99% in a flask according to the proportion of 1:2, adding a stirrer into the flask, stirring the mixture by using a magnetic stirrer at the temperature of 70-80 ℃, and heating for more than 2 hours until colorless transparent liquid is formed. After cooling to room temperature, these clear liquids were stored in a silica gel desiccator and dried for two weeks. And mixing the dried eutectic solvent with the original buffer solvent in a ratio of 1:99 to obtain the DES-buffer solution.
The invention provides a preparation method of a DES-buffer solution applied to a glycosidase cryoelectron microscope detection sample, which comprises the following steps:
inoculating 10 μ L of bacterial liquid into 5mL of culture solution in a test tube, adding 5 μ L of kanamycin, placing in a shaking table at the temperature of 37 ℃ and the rotation speed of 180-. The bacterial solution in the tube was poured into 200mL of medium, and 200. mu.L of kanamycin was added. Placing into a shaking table, culturing at 37 deg.C and 200r/min for 2-4 h. 200 μ L inducer, galactoside Inducer (IPTG), was added to the shaker. The temperature is 25 ℃, the rotating speed is 150r/min, and the induction lasts 18-20 h. After induction, the bacterial liquid is centrifuged, and the supernatant is discarded to obtain the thallus. Resuspending the thallus with imidazole, disrupting the cultured cells in a high-pressure homogenizing and disrupting machine, centrifuging, collecting the supernatant, and packaging to obtain crude enzyme solution. The crude enzyme solution is filtered by a filter membrane of 0.45 mu m and then the protein is separated and purified by adopting a Ni-NTA affinity chromatography column. And concentrating the obtained enzyme solution to remove imidazole components, and replacing the original buffer solution with DES-buffer solution in the concentration process to obtain the glycosidase transferase sample.
And dripping the sample in the solution state on a copper net to form a thin sample liquid layer, and then putting the copper net into liquid ethane for quick freezing. Such frozen samples were kept at a low temperature and observed under a transmission electron microscope, thereby obtaining the structure of the biomacromolecule.
Example 2
The invention provides a preparation method of a eutectic solvent for a glycoside transferase cryoelectron microscope detection sample, which solves the problem of influence of substrate solubility on cryoelectron microscope sample preparation, promotes biocatalytic conversion of natural product micromolecules such as flavone and biphenyl, and optimizes a reaction environment by using the eutectic solvent. The eutectic solvent is prepared by mixing choline chloride and glycol with the purity of more than or equal to 99% according to the proportion of 1: 2.
The preparation method of the low cosolvent comprises the following steps of mixing choline chloride and ethylene glycol with the purity of more than or equal to 98% in a flask according to the ratio of 1:2, adding a stirrer into the flask, stirring the mixture by using a magnetic stirrer at the temperature of 70-80 ℃, and heating for more than 2 hours until colorless transparent liquid is formed. After cooling to room temperature, these clear liquids were stored in a silica gel desiccator and dried for two weeks. And mixing the dried eutectic solvent with the original buffer solvent in a ratio of 1:99 to obtain the DES-buffer solution.
The invention provides a preparation method of a DES-buffer solution applied to a glycosidase cryoelectron microscope detection sample, which comprises the following steps:
inoculating 10 μ L of bacterial liquid into 5mL of culture solution in a test tube, adding 5 μ L of kanamycin, placing in a shaking table at the temperature of 37 ℃ and the rotation speed of 180-. The bacterial solution in the tube was poured into 200mL of medium, and 200. mu.L of kanamycin was added. Placing into a shaking table, culturing at 37 deg.C and 200r/min for 2-4 h. 200 μ L inducer, galactoside Inducer (IPTG), was added to the shaker. Inducing at 25 deg.C and 150r/min for 18-20 hr, centrifuging, removing supernatant, and collecting thallus. Resuspending the thallus with imidazole, disrupting the cultured cells in a high-pressure homogenizing and disrupting machine, centrifuging, collecting the supernatant, and packaging to obtain crude enzyme solution. The crude enzyme solution is filtered by a filter membrane of 0.45 mu m and then the protein is separated and purified by adopting a Ni-NTA affinity chromatography column. And concentrating the obtained enzyme solution to remove imidazole components, and replacing the original buffer solution with DES-buffer solution in the concentration process to obtain the glycosidase transferase sample.
And dripping the sample in the solution state on a copper net to form a thin sample liquid layer, and then putting the copper net into liquid ethane for quick freezing. Such frozen samples were kept at a low temperature and observed under a transmission electron microscope, thereby obtaining the structure of the biomacromolecule.
Example 3
The invention provides a preparation method of a eutectic solvent for a glycoside transferase cryoelectron microscope detection sample, which solves the problem of influence of substrate solubility on cryoelectron microscope sample preparation, promotes biocatalytic conversion of natural product micromolecules such as flavone and biphenyl, and optimizes a reaction environment by using the eutectic solvent. The eutectic solvent is prepared by mixing choline chloride and 1, 2-propylene glycol with the purity of more than or equal to 99% according to the proportion of 1: 2.
The method for preparing the low cosolvent comprises the steps of mixing choline chloride and 1, 2-propylene glycol with the purity of more than or equal to 99% in a flask according to the proportion of 1:2, adding a stirrer into the flask, stirring the mixture by using a magnetic stirrer at the temperature of 70-80 ℃, and heating for more than 2 hours until colorless transparent liquid is formed. After cooling to room temperature, these clear liquids were stored in a silica gel desiccator and dried for two weeks. And mixing the dried eutectic solvent with the original buffer solvent in a ratio of 1:99 to obtain the DES-buffer solution.
The invention provides a preparation method of a DES-buffer solution applied to a glycosidase cryoelectron microscope detection sample, which comprises the following steps:
inoculating 10 μ L of bacterial liquid into 5mL of culture solution in a test tube, adding 5 μ L of kanamycin, placing in a shaking table at the temperature of 37 ℃ and the rotation speed of 180-. The bacterial solution in the tube was poured into 200mL of medium, and 200. mu.L of kanamycin was added. Placing into a shaking table, culturing at 37 deg.C and 200r/min for 2-4 h. 200 μ L inducer, galactoside Inducer (IPTG), was added to the shaker. Inducing at 25 deg.C and 150r/min for 18-20 hr, centrifuging, removing supernatant, and collecting thallus. Resuspending the thallus with imidazole, disrupting the cultured cells in a high-pressure homogenizing and disrupting machine, centrifuging, collecting the supernatant, and packaging to obtain crude enzyme solution. The crude enzyme solution is filtered by a filter membrane of 0.45 mu m and then the protein is separated and purified by adopting a Ni-NTA affinity chromatography column. And concentrating the obtained enzyme solution to remove imidazole components, and replacing the original buffer solution with DES-buffer solution in the concentration process to obtain the glycosidase transferase sample.
And dripping the sample in the solution state on a copper net to form a thin sample liquid layer, and then putting the copper net into liquid ethane for quick freezing. Such frozen samples were kept at a low temperature and observed under a transmission electron microscope, thereby obtaining the structure of the biomacromolecule.
The results of the invention are:
through detection, the method solves the problem of influence of substrate solubility on sample preparation by a cryoelectron microscope, promotes the biocatalytic conversion of natural product micromolecules such as flavone, biphenyl and the like, and optimizes the reaction environment by using the eutectic solvent. And the catalytic activity, the thermal stability and the storage time of the glucoside transferase are greatly improved. The application of the invention is that the nonpolar compounds such as flavone and biphenyl in the natural medicinal active substances can change the polarity thereof through glycosylation to realize polarity inversion, thereby promoting the absorption and transportation in organisms and improving the biological activity thereof. In the process of researching the catalytic mechanism of the glucoside transferase, a cryoelectron microscope is used for researching the structure of the glucoside transferase, but the sample preparation of the cryoelectron microscope is usually carried out in a pure water environment, so that the substrate is separated out due to poor solubility, and the enzymatic reaction is hindered. In order to solve the problem that a nonpolar substrate is difficult to dissolve in a water environment reaction system and promote the biocatalytic conversion of small molecules of natural products such as flavone, biphenyl and the like, the invention optimizes the reaction environment by using a eutectic solvent.
Claims (8)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110425865.2A CN113125482A (en) | 2021-04-20 | 2021-04-20 | Eutectic solvent for sample preparation of cryoelectron microscope and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110425865.2A CN113125482A (en) | 2021-04-20 | 2021-04-20 | Eutectic solvent for sample preparation of cryoelectron microscope and preparation method thereof |
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Citations (3)
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|---|---|---|---|---|
| US20140295404A1 (en) * | 2013-03-01 | 2014-10-02 | Andrew Simon Goldsborough | Sample fixation and stabilisation |
| CN107136559A (en) * | 2017-07-06 | 2017-09-08 | 中国烟草总公司郑州烟草研究院 | Natural eutectic solvent humectant for smoke of glucose-type and its preparation method and application |
| CN110824030A (en) * | 2019-08-27 | 2020-02-21 | 杭州师范大学 | A kind of extraction method of pesticide in turmeric |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140295404A1 (en) * | 2013-03-01 | 2014-10-02 | Andrew Simon Goldsborough | Sample fixation and stabilisation |
| CN107136559A (en) * | 2017-07-06 | 2017-09-08 | 中国烟草总公司郑州烟草研究院 | Natural eutectic solvent humectant for smoke of glucose-type and its preparation method and application |
| CN110824030A (en) * | 2019-08-27 | 2020-02-21 | 杭州师范大学 | A kind of extraction method of pesticide in turmeric |
Non-Patent Citations (1)
| Title |
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| JOHNATHAN GORKE 等: "Toward Advanced Ionic Liquids. Polar, Enzyme-friendly Solvents for Biocatalysis", BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, vol. 15, 18 March 2010 (2010-03-18), pages 40 - 53, XP055285343, DOI: 10.1007/s12257-009-3079-z * |
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