CN101586100A - Immobilized cholinesterase and preparation and application thereof - Google Patents
Immobilized cholinesterase and preparation and application thereof Download PDFInfo
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- CN101586100A CN101586100A CNA2009100986749A CN200910098674A CN101586100A CN 101586100 A CN101586100 A CN 101586100A CN A2009100986749 A CNA2009100986749 A CN A2009100986749A CN 200910098674 A CN200910098674 A CN 200910098674A CN 101586100 A CN101586100 A CN 101586100A
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- water
- pseudocholinesterase
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- soluble material
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- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 108090000322 Cholinesterases Proteins 0.000 title abstract description 5
- 229940048961 cholinesterase Drugs 0.000 title abstract description 5
- 102000003914 Cholinesterases Human genes 0.000 title abstract 4
- 239000000243 solution Substances 0.000 claims abstract description 30
- 102000004190 Enzymes Human genes 0.000 claims abstract description 29
- 108090000790 Enzymes Proteins 0.000 claims abstract description 29
- 229940088598 enzyme Drugs 0.000 claims abstract description 29
- 239000002195 soluble material Substances 0.000 claims abstract description 24
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- 239000002502 liposome Substances 0.000 claims abstract description 8
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 7
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 7
- 241000978776 Senegalia senegal Species 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 238000001035 drying Methods 0.000 claims abstract description 6
- 239000011343 solid material Substances 0.000 claims abstract description 6
- 239000000544 cholinesterase inhibitor Substances 0.000 claims abstract description 5
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims abstract description 5
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims abstract description 5
- 238000012216 screening Methods 0.000 claims abstract description 5
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims abstract description 4
- 235000019422 polyvinyl alcohol Nutrition 0.000 claims abstract description 3
- 102100032404 Cholinesterase Human genes 0.000 claims description 46
- 108010053652 Butyrylcholinesterase Proteins 0.000 claims description 40
- 239000002953 phosphate buffered saline Substances 0.000 claims description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 8
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 230000001681 protective effect Effects 0.000 claims description 6
- 102100033639 Acetylcholinesterase Human genes 0.000 claims description 5
- 108010022752 Acetylcholinesterase Proteins 0.000 claims description 5
- 101710083761 Cholinesterase Proteins 0.000 claims description 5
- 229940022698 acetylcholinesterase Drugs 0.000 claims description 5
- 239000000284 extract Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 102000009027 Albumins Human genes 0.000 claims description 4
- 108010088751 Albumins Proteins 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 4
- 239000008187 granular material Substances 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 3
- 239000003905 agrochemical Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- 229920003023 plastic Polymers 0.000 claims description 3
- 239000004033 plastic Substances 0.000 claims description 3
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 3
- 235000006491 Acacia senegal Nutrition 0.000 claims description 2
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 210000001124 body fluid Anatomy 0.000 claims description 2
- 239000010839 body fluid Substances 0.000 claims description 2
- 239000011159 matrix material Substances 0.000 claims description 2
- 210000005036 nerve Anatomy 0.000 claims description 2
- 231100000614 poison Toxicity 0.000 claims description 2
- 239000002574 poison Substances 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 9
- 239000000447 pesticide residue Substances 0.000 abstract description 6
- 230000003197 catalytic effect Effects 0.000 abstract description 5
- 229920000084 Gum arabic Polymers 0.000 abstract description 4
- 235000010489 acacia gum Nutrition 0.000 abstract description 4
- 239000000205 acacia gum Substances 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 239000002245 particle Substances 0.000 abstract description 2
- 239000002131 composite material Substances 0.000 abstract 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 abstract 1
- 229940098773 bovine serum albumin Drugs 0.000 abstract 1
- 239000010408 film Substances 0.000 abstract 1
- 239000008363 phosphate buffer Substances 0.000 abstract 1
- 239000003223 protective agent Substances 0.000 abstract 1
- 238000002791 soaking Methods 0.000 abstract 1
- 239000010409 thin film Substances 0.000 abstract 1
- 239000012528 membrane Substances 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000000152 carbamate pesticide Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 235000013311 vegetables Nutrition 0.000 description 5
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 4
- 238000013016 damping Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000003987 organophosphate pesticide Substances 0.000 description 3
- 231100000167 toxic agent Toxicity 0.000 description 3
- 239000003440 toxic substance Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000361919 Metaphire sieboldi Species 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 2
- YRIBGSCJIMXMPJ-UHFFFAOYSA-N butyrylcholine Chemical compound CCCC(=O)OCC[N+](C)(C)C YRIBGSCJIMXMPJ-UHFFFAOYSA-N 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000002985 plastic film Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 1
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- CCBICDLNWJRFPO-UHFFFAOYSA-N 2,6-dichloroindophenol Chemical compound C1=CC(O)=CC=C1N=C1C=C(Cl)C(=O)C(Cl)=C1 CCBICDLNWJRFPO-UHFFFAOYSA-N 0.000 description 1
- NTBLZMAMTZXLBP-UHFFFAOYSA-M 2-acetylsulfanylethyl(trimethyl)azanium;iodide Chemical class [I-].CC(=O)SCC[N+](C)(C)C NTBLZMAMTZXLBP-UHFFFAOYSA-M 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 150000001840 cholesterol esters Chemical class 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- AUZONCFQVSMFAP-UHFFFAOYSA-N disulfiram Chemical compound CCN(CC)C(=S)SSC(=S)N(CC)CC AUZONCFQVSMFAP-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000009461 vacuum packaging Methods 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
一种固定的胆碱酯酶及其制备和应用，其固定所用的载体是能够干燥成膜并在水溶液中迅速溶解的水溶性材料，如聚乙烯醇、聚乙烯吡咯烷酮或阿拉伯树胶；也包括上述水溶性材料与脂质体形成的复合材料。也包括上述水溶性材料与脂质体形成的复合材料。选用1～10mg/mL的牛血白蛋白磷酸盐缓冲液做保护剂；采用水溶性材料固定胆碱酯酶，用滴加或浸泡的方法直接将胆碱酯酶水溶性材料溶液附着到固体材料上，干燥成薄膜，或滴加到模具中干燥成酶颗粒，使用时投入溶液中立即溶解释放其中的酶分子，在均相中行使催化功能，具有更好的催化效率和抑制灵敏度，可以应用在农药残留快速检测或者胆碱酯酶抑制剂的筛选。A kind of immobilized cholinesterase and its preparation and application, the carrier used in the immobilization is a water-soluble material capable of drying into a film and rapidly dissolving in an aqueous solution, such as polyvinyl alcohol, polyvinylpyrrolidone or gum arabic; also includes the above-mentioned Composite material formed by water-soluble material and liposome. Composite materials formed by the above-mentioned water-soluble materials and liposomes are also included. Choose 1-10mg/mL bovine serum albumin phosphate buffer as the protective agent; use water-soluble materials to immobilize cholinesterase, and directly attach the cholinesterase water-soluble material solution to the solid material by dripping or soaking and dry it into a thin film, or drop it into a mold and dry it into enzyme particles. When it is put into the solution, it will dissolve and release the enzyme molecules in it immediately, and perform the catalytic function in a homogeneous phase. It has better catalytic efficiency and inhibition sensitivity, and can be applied In the rapid detection of pesticide residues or the screening of cholinesterase inhibitors.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of enzyme product and processing thereof and application, be specially a kind of with water-soluble material fixed Pseudocholinesterase and preparation thereof, and in the application of the screening of Fast Determination of Pesticide Residue or anticholinesterase.
Background technology
Pseudocholinesterase is the intravital important enzyme of humans and animals, and it comprises acetylcholinesterase and butyrylcholine esterase.Organophosphorus, carbamate chemicals for agriculture or toxic agent can suppress the activity of Pseudocholinesterase.Many biotoxins or active ingredient also suppress cholinesterase activity, can be applied to the treatment of senile dementia and many nervous system diseases.Utilize degree of inhibition of AchE can detect pesticide residue in food or the environment, also can be used for the drug screening of nervous system disease.
But Pseudocholinesterase is preserved external being not easy, and needs freezing preservation usually, needs to dissolve packing before the use, uses very inconvenient.Various physics or chemical process are used to the fixing of Pseudocholinesterase, but these methods often cause the loss of enzymic activity.Fixing inside or the surface that always enzyme is fixed on solid material of common enzyme, the reaction of enzyme is an inhomogeneous reaction, catalytic efficiency is low, nor is convenient to detect.In addition, conventional enzyme technique for fixing is wished by fixedly making enzyme repeatedly to use, fixedly Pseudocholinesterase is used for Pesticides Testing and but is not suitable for repeatedly using, because the activity of agricultural chemicals meeting inhibitory enzyme to be measured, make immobilized enzyme disposablely to use, even can adopt some drugs to make Pseudocholinesterase partly reply activity, but complex operation, cost height, circulation ratio are also bad.
Summary of the invention
The present invention is directed to the Pseudocholinesterase poor stability, fixing back enzymic activity reduces, and can not carry out problems such as homogeneous reaction during use, and a kind of fixed Pseudocholinesterase is provided, with and preparation method thereof.
A kind of fixed Pseudocholinesterase of the present invention, its fixedly the used carrier of Pseudocholinesterase be can drying and forming-film and in the aqueous solution dissolved water-soluble material rapidly; Described water-soluble material is polyvinyl alcohol, polyvinylpyrrolidone or Sudan Gum-arabic; Also comprise the matrix material that above-mentioned water-soluble material and liposome form.
The preparation of a kind of fixed Pseudocholinesterase of the present invention may further comprise the steps:
(1) from the in vitro tissue of animal or body fluid, extracts Pseudocholinesterase, or directly to adopt commercialization acetylcholinesterase or butyrylcholine esterase be raw material, with the protective material dilution is the Pseudocholinesterase diluent of desired concn, and used protective material is selected the ox blood albumin phosphate buffered saline buffer of 1～10mg/mL for use;
(2) water-soluble material described in the claim 1 is water-soluble according to 1%～50% percent weight in volume, obtain water-soluble material solution;
(3) Pseudocholinesterase diluent and the water-soluble material solution volume ratio by 1: 4～4: 1 is mixed, obtain Pseudocholinesterase water-soluble material solution;
(4) directly Pseudocholinesterase water-soluble material solution is attached on the solid material with the method that drips or soak, is dried to film, or be added drop-wise to and be dried to enzyme granulate in the mould; Described solid material is plastics.
The Thiomersalate that contains percent weight in volume 0.1%～1% in the described protective material of step (1).
Described liposome is one or more the mixture in phosphatide or phosphatide and the following material: cholesterol, stearylamine, phosphatidic acid and oleic acid; Described phosphatide is Yelkin TTS, phosphatidylcholine, phosphatidylethanolamine, fabaceous lecithin two palmityl phosphatide, sphingophospholipid or distearoyl phosphatidylcholine.
Described in the aqueous solution rapidly the dissolved aqueous solution be meant water, or the organic solvent or the two or more blended aqueous solution of these organic solvents of water and ethanol, acetone, octane-iso or hexanaphthene formation.
The application characteristic of a kind of fixed Pseudocholinesterase of the present invention is: during use described a kind of fixed Pseudocholinesterase is dropped in the solution to be measured, be directly used in the detection of agricultural chemicals and nerve poison or be used for the screening of anticholinesterase.
Adopt fixedly Pseudocholinesterase of water-soluble material, fixing Pseudocholinesterase after drying film forming, drop into the neutral instant release enzyme molecule wherein of separating of solution during use, in homogeneous phase, exercise catalysis, have better catalytic efficiency and suppress sensitivity, be applied in the screening of Fast Determination of Pesticide Residue or anticholinesterase.
The advantage of a kind of fixed Pseudocholinesterase of the present invention is:
The water soluble film or the block that will contain enzyme during use are put in the solution that may contain organophosphorus or carbamate pesticide, add enzyme substrate solution, and visual inspection or employing instrument are quantitative.Because water-soluble material is dissolving fast in water, discharges Pseudocholinesterase wherein, make inhibition and catalytic process all in homogeneous phase, carry out, enzymic activity increases, and suppresses sensitivity and improves.
Because enzyme is disperseed to be fixed in the enzyme membrane, does not need steps such as weighing, dissolving, packing during use.Directly enzyme membrane is put into and detected in the liquid, very easy to use.In addition, enzyme membrane can adopt vacuum packaging, directly room temperature preservation, perhaps 4 ℃ of refrigerator cold-storages.
Embodiment
Embodiment 1:
(1) acetylcholinesterase is fixed
(1) configuration 10mM pH 8 phosphate buffered saline buffers (PBS).
(2) the pure acetylcholinesterase of the commercialization of buying is diluted with the ox blood albumin phosphate buffered saline buffer that contains 1～10mg/mL, concentration is 1U/mL.
(3) the weighing polyvinylpyrrolidone is water-soluble, by weight the polyvinylpyrrolidonesolution solution of volume percent configuration 1～10%.
(4) according to 1: 1 volume ratio mixed enzyme solution and polyvinylpyrrolidonesolution solution, abundant mixing.
(5) draw 60 μ l and be added drop-wise to drying and forming-film on the plastic sheet.
(2) the water-soluble enzyme membrane of acetylcholinesterase-polyvinylpyrrolidone is used for the detection of organophosphorus toxicants
Take by weighing vegetables sample to be measured, extract remains of pesticide 50 times with 10mM pH 8 damping fluid joltings according to the 2ml/g ratio.Put into enzyme membrane in centrifuge tube, Dropwise 50 μ l extracting solution suppressed 10 minutes, added 50 μ l2mg/ml, 2,6 dichloroindophenol acetate solution (solvent is the PBS solution that contains 5% acetone) reaction 5 minutes, can judge the degree of inhibition by observing change in color.Blueness is dark more, illustrates that the inhibition degree is low, and organophosphorus or carbamate pesticide are few; Blueness is shallow more, and inhibition degree height is described, the vegetable pesticide residue amount is big.
Embodiment 2:
(1) preparation of blank liposome
(1) configuration 10mM pH 8 phosphate buffered saline buffers (PBS).
(2) take by weighing 0.9g phosphatide and 0.3g cholesterol in the 50ml small beaker, add dehydrated alcohol 1～2ml, place 65～70 ℃ of water-baths, stir and make dissolving, rotate ethanol liquid film forming on wall of cup that this small beaker makes phosphatide, dry up with nitrogen.
(3) get PBS 30ml in addition in small beaker,, be incubated with placing 65～70 ℃ of water-baths, stand-by.
(4) get the phosphate buffered saline buffer 30ml of preheating, add in the small beaker that contains phosphatide and cholesterol ester plasma membrane 65～70 ℃ of stirred in water bath aquation 10min.Subsequently small beaker is placed on the magnetic stirring apparatus, room temperature stirs 30～60min, if liquor capacity reduces, can mend and add water to 30ml, and mixing, promptly.
(2) butyrylcholine esterase-liposome membrane is fixing
(1) the pure butyrylcholine esterase of the commercialization of buying is diluted with the ox blood albumin phosphate buffered saline buffer that contains 1～10mg/mL, concentration is 1U/mL, adds 0.01%～1% Thiomersalate again.
(2) with 50 times of dilutions of gained liposome solutions, and and butyrylcholine esterase equal-volume mixing.
(3) the weighing polyvinyl alcohol is water-soluble, disposes every liter of solution of 20% that contains polyvinyl alcohol 200 gram.
(4) according to 1: 4 volume ratio mixed enzyme solution and polyvinyl alcohol solution, fully mixing is drawn 80 μ l and is added drop-wise to drying and forming-film on the plastic sheet.
(3) the water-soluble enzyme membrane of butyrylcholine esterase-polyvinyl alcohol is used for the detection of organophosphorus toxicants
Take by weighing vegetables sample to be measured, extract remains of pesticide 50 times with 10mM pH 8 damping fluid joltings according to the 2ml/g ratio.Put into enzyme membrane in centrifuge tube, Dropwise 50 μ l extracting solution suppressed 10 minutes, added 50 μ l 2mg/ml indophenols acetate solution (solvent is the PBS solution that contains volume ratio 5% acetone) reaction 5 minutes, can judge the degree of inhibition by observing change in color.Blueness is dark more, illustrates that the inhibition degree is low, and organophosphorus or carbamate pesticide are few; Blueness is shallow more, and inhibition degree height is described, the vegetable pesticide residue amount is big.
Embodiment 3:
(1) extraction of Pseudocholinesterase in the earthworm
(1) configuration 10mM pH 8 phosphate buffered saline buffers (PBS).
(2) quality PBS homogenate such as fresh earthworm in vitro tissue adding is 2 minutes, and centrifugal 10 minutes of 10000 commentaries on classics/min get supernatant liquor and are used as Pseudocholinesterase.
(2) Pseudocholinesterase is fixed
(1) the weighing gum arabic is dissolved in PBS, disposes every liter of gum arabic aqueous solution that contains gum arabic 400～500 grams.
(4) according to 4: 1 volume ratio mixed enzyme solutions and gumwater, abundant mixing is drawn 200 μ l and is added drop-wise to and is dried to enzyme granulate in the mould of plastics.
(3) Pseudocholinesterase water-soluble enzyme particle is used for the detection of carbamate pesticide
Take by weighing vegetables sample to be measured, extract remains of pesticide 50 times according to the 2ml/g ratio with 10mM pH 8 damping fluid joltings, get 880 μ l extracting solutions, add enzyme granulate, suppressed 10 minutes, and added 100 μ l 2.1mM 5,5 '-dithio-two-nitrobenzoic acid (DTNB) and 100 μ l 5mM acetylthiocholine iodides successively, 37 ℃ of reactions down, quantitative by the pace of change of measuring 410nm place absorbancy to carbamate pesticide.
Claims (5)
1, a kind of fixed Pseudocholinesterase is characterized in that: its fixedly the used carrier of Pseudocholinesterase be can drying and forming-film and in the aqueous solution dissolved water-soluble material rapidly; Described water-soluble material is polyvinyl alcohol, polyvinylpyrrolidone or Sudan Gum-arabic; Also comprise the matrix material that above-mentioned water-soluble material and liposome form.
2, a kind of according to claim 1 preparation of fixed Pseudocholinesterase is characterized in that following steps:
(1) from the in vitro tissue of animal or body fluid, extracts Pseudocholinesterase, or directly to adopt commercialization acetylcholinesterase or butyrylcholine esterase be raw material, with the protective material dilution is the Pseudocholinesterase diluent of desired concn, and used protective material is selected the ox blood albumin phosphate buffered saline buffer of 1～10mg/mL for use;
(2) water-soluble material described in the claim 1 is water-soluble according to 1%～50% percent weight in volume, obtain water-soluble material solution;
(3) Pseudocholinesterase diluent and the water-soluble material solution volume ratio by 1: 4～4: 1 is mixed, obtain Pseudocholinesterase water-soluble material solution;
(4) directly Pseudocholinesterase water-soluble material solution is attached on the solid material with the method that drips or soak, is dried to film, or be added drop-wise to and be dried to enzyme granulate in the mould; Described solid material is plastics.
3, as the preparation of a kind of fixed Pseudocholinesterase as described in the claim 2, it is characterized in that: the Thiomersalate that contains percent weight in volume 0.1%～1% in the described protective material of step (1).
4, a kind of according to claim 1 fixed Pseudocholinesterase, it is characterized in that: described in the aqueous solution rapidly the dissolved aqueous solution be meant water, or the organic solvent or the two or more blended aqueous solution of these organic solvents of water and ethanol, acetone, octane-iso or hexanaphthene formation.
5, a kind of according to claim 1 application of fixed Pseudocholinesterase is characterized in that: during use described a kind of fixed Pseudocholinesterase is dropped in the solution to be measured, be directly used in the detection of agricultural chemicals and nerve poison or be used for the screening of anticholinesterase.
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| CN102134559A (en) * | 2010-11-15 | 2011-07-27 | 广州吉家庄生物科技有限公司 | Bacillus brevis, method for extracting bacillus brevis esterase and application of same |
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| CN102134559A (en) * | 2010-11-15 | 2011-07-27 | 广州吉家庄生物科技有限公司 | Bacillus brevis, method for extracting bacillus brevis esterase and application of same |
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