CN1935339A - Nylon affinity membrane preparing method and use - Google Patents
Nylon affinity membrane preparing method and use Download PDFInfo
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- CN1935339A CN1935339A CN 200610116076 CN200610116076A CN1935339A CN 1935339 A CN1935339 A CN 1935339A CN 200610116076 CN200610116076 CN 200610116076 CN 200610116076 A CN200610116076 A CN 200610116076A CN 1935339 A CN1935339 A CN 1935339A
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Abstract
The present invention discloses a preparation method of nylon affinity membrane. Said method includes the following steps:(1) on the nylon membrane chemically-bonding chitosan so as to obtain the chitosan modified nylon membrane; and (2) bonding the chitosan modified nylon membrane and affinity dye Cibacron Blue F3GA so as to obtain the described nylon affinity membrane which can be used for separating and purifying papain.
Description
Technical field
The present invention relates to a kind of preparation method and purposes of nylon affinity membrane, particularly relate to a kind of be matrix with nylon, with dyestuff Cibacron Blue F3GA be affine base affinity membrane the preparation method and in the application aspect the separation and purification papain.
Background technology
Papain (EC3.4.22.2) is a class thiol protease, extensively is present in root, stem, leaf and the fruit of papaya (Caricapapaya), wherein content the abundantest [1-2] in immature milk.Be widely used in food service industry, as the clarification of beer and tenderization and leather, weaving, daily use chemicals and the pharmaceuticals industry [3-5] of meat.The purifying major part of papain is to adopt the precipitation method, but this method still is mixed with other protease [6], can not reach the demand of pharmaceuticals industry.
Along with people propose further requirement to purity and security, chromatographic technique is applied to the purifying of papain, as ion-exchange chromatography, affinity chromatography [7-9], such filler specificity height, purifying multiple height, but separating rate is limited by intergranular diffusion and low axial rate, pressure drop is big, disengaging time is long, and treating capacity is little, can not use high charging rate.And the film separation is a kind of technology of separating according to bulk of molecule, characteristics are that surface area is big, the evolving path short, operation is forced down, treating capacity is big, but the conventional film isolation technics has only and separates relative molecular mass and differ system more than 10 times, can not satisfy the biochemical needs that separate.
List of references
[1]M.Azarkan，A.E.Moussaoui，D.W.Wuytswinkel，G.Dehon，Y.Looze.J.Chromatogr.B，2003，790：229-238.
[2] Deng Jing (Deng Jing), Wu Huachang (Wu Huachang), Zhou Jian (Zhou Jian) .G.X.Light Ind. (Guangxi light industry), 2003,3:5-7.
[3]Q.H.Chen，GQ.He，Y.C.Jiao，H.Ni.Food?Chem.，2006，98：624-629..
[4]S.S.Khaparde，R.S.Singhal.Bioresource?Technol.，2001，78：1-4.
[5]F.S.Ian，M.Paul，S.Geisela.Bums，1999，25：636-639.
[6]S.Nitsawang，H.K.Rajni，P.Kanasawud，Enzyme?Microb.Tech.，2006，in?press.
[7]D.Tombaccini，A.Mocali，E.Weber，F.Paoletti.Anal.Biochem.，2001，289：231-238.
[8]E.Govrin，A.Levine.Protein?Expres.Purif.，1999，15：247-350.
[9]W.J.Syu，S.H.Wu，K.T.Wang.J.Chromatogr.，1983，262：346-351.
[10]L.Yang，W.W.Hsiao，P.Chen，J.Membr.Sci.197(2002)185.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method and application of nylon affinity membrane, to remedy the deficiencies in the prior art or defective, meets the demands of production and living.
In order to solve the problems of the technologies described above, one of the technical solution adopted in the present invention is: a kind of preparation method of nylon affinity membrane comprises the steps:
(1) nylon membrane is separated 10-72h at 0.1-10M hydrochloric acid heavy water earlier, then with the glutaraldehyde activation processing of 10%-25%, nylon membrane after the activation places the chitosan solution of 0.5%-5% to carry out chemical bonding, dries in 70-120 ℃ baking oven then, promptly gets chitin modified nylon membrane;
(2) the nylon membrane 2-10g after the chitin modified processing being placed concentration is the 50-350mlCibacron Blue F3GA dye solution of 5.0-30mg/ml, 60 ℃ of reaction 0.5-3h, adding 20-50ml concentration then is 20%NaCl solution, continue reaction 1.0-3.0h, add 5.0-20ml concentration then and be 20% NaCO3 solution and carry out the fixation reaction, behind the reaction 2-10h, take out, successively with hot water, methyl alcohol, 2M NaCl, 6M urea, deionized water washs, obtain described nylon affinity membrane, 4 ℃ of preservations, standby.
In order to solve the problems of the technologies described above, two of the technical solution adopted in the present invention is: described nylon affinity membrane be used for the separation and purification papain, comprise the steps:
10-20 opens and builds up membrane stack on the nylon affinity membrane, puts into the film bridge, sends into buffer solution and eluent with peristaltic pump, at first is 7.0 phosphate buffer balance 10-20min with 0.05M, pH, and flow velocity is 0.1-1.0ml/min; Send into the papain solution of 1.0-5.0mg/ml of 15-50ml by film bridge, the protein that does not adsorb with 0.05M, pH7.0 phosphate buffer flush away then with peristaltic pump subsequently; Be that the 1.0M NaSCN that 8.0 phosphate buffers are prepared carries out wash-out with 0.05M, pH at last, obtain the target protein papain.
The invention has the beneficial effects as follows: separate with traditional film, affinity chromatography compares; the nylon affinity membrane of the present invention's preparation not only has purifying multiple height, pressure drop is little; analysis time is short, large biological molecule sex change probability in separation process is little; allow characteristics such as charging rate faster, and than the easier realization scale of post affinity chromatography purifies and separates.
The specific embodiment
Below in conjunction with specific embodiment the present invention is further elaborated.
Embodiment 1
Nylon membrane is earlier separated 72h at 0.1M hydrochloric acid heavy water, and then with 10% glutaraldehyde activation processing, the nylon membrane of activation places 0.5% chitosan solution to carry out chemical bonding then, dries in 70 ℃ baking oven then, promptly gets chitin modified nylon membrane.The shitosan content of bonding is tested by document [10] disclosed method on the nylon membrane, and the content of test result shitosan is the 89.6mg/g film.
It is the 50mlCibacronBlue F3GA dye solution of 5.0mg/ml that nylon membrane 2g after the chitin modified processing is placed concentration, 60 ℃ of reaction 0.5h, and adding 20ml concentration then is 20%NaCl solution, continue reaction 1.0h, add 5.0ml concentration then and be 20% NaCO3 solution and carry out the fixation reaction, behind the reaction 2h, take out, successively with hot water, methyl alcohol, 2M NaCl, 6M urea, deionized water washs, 4 ℃ of preservations, standby.
Affinity membrane composition membrane stack carries out separation and purification to papain and comprises the steps:
Build up membrane stack on the affinity membrane of 10 method for preparing, put into the film bridge, send into buffer solution and eluent etc. with peristaltic pump.At first with 0.05M phosphate buffer (pH7.0) balance 10min, flow velocity: 0.1ml/min; Follow sample presentation, the papain solution of the 1.0mg/ml of 15ml, the protein that does not adsorb with 0.05M phosphate buffer (pH7.0) flush away then.1.0MNaSCN. with 0.05M phosphate buffer (pH8.0) preparation carries out wash-out at last, obtains the target protein papain.Obtain the activity preservation rate of papain and eluting rate all 95%.
Embodiment 2
Nylon membrane is earlier separated 10h at 10M hydrochloric acid heavy water, and then with 25% glutaraldehyde activation processing, the nylon membrane of activation places 5% chitosan solution to carry out chemical bonding then, dries in 120 ℃ baking oven then, promptly gets chitin modified nylon membrane.The shitosan content of bonding is tested by document [10] disclosed method on the nylon membrane, and the content of test result shitosan is the 90.6mg/g film.
It is the 350mlCibacronBlue F3GA dye solution of 30mg/ml that nylon membrane 10g after the chitin modified processing is placed concentration, 60 ℃ of reaction 3h, and adding 50ml concentration then is 20%NaCl solution, continue reaction 3.0h, add 20ml concentration then and be 20% NaCO3 solution and carry out the fixation reaction, behind the reaction 10h, take out, successively with hot water, methyl alcohol, 2M NaCl, 6M urea, deionized water washs, 4 ℃ of preservations, standby.
Affinity membrane composition membrane stack carries out separation and purification to papain and comprises the steps:
Build up membrane stack on the affinity membrane of 20 method for preparing, put into the film bridge, send into buffer solution and eluent etc. with peristaltic pump.At first with 0.05M phosphate buffer (pH7.0) balance 20min, flow velocity: 0.1-1.0ml/min; Follow sample presentation, the papain solution of the 5.0mg/ml of 50ml, the protein that does not adsorb with 0.05M phosphate buffer (pH7.0) flush away then.1.0M NaSCN. with 0.05M phosphate buffer (pH8.0) preparation carries out wash-out at last, obtains the target protein papain.Obtain the activity preservation rate of papain and eluting rate all 93%.
Embodiment 3
Nylon membrane is earlier separated 50h at 5M hydrochloric acid heavy water, and then with 15% glutaraldehyde activation processing, the nylon membrane of activation places 3% chitosan solution to carry out chemical bonding then, dries in 100 ℃ baking oven then, promptly gets chitin modified nylon membrane.The shitosan content of bonding is tested by document [10] disclosed method on the nylon membrane, and the content of test result shitosan is the 89.2mg/g film.
It is the 200mlCibacronBlue F3GA dye solution of 20mg/ml that nylon membrane 5g after the chitin modified processing is placed concentration, 60 ℃ of reaction 2h, and adding 30ml concentration then is 20%NaCl solution, continue reaction 2h, add 10ml concentration then and be 20% NaCO3 solution and carry out the fixation reaction, behind the reaction 5h, take out, successively with hot water, methyl alcohol, 2M NaCl, 6M urea, deionized water washs, 4 ℃ of preservations, standby.
Affinity membrane composition membrane stack carries out separation and purification to papain and comprises the steps:
Build up membrane stack on the affinity membrane of 15 method for preparing, put into the film bridge, send into buffer solution and eluent etc. with peristaltic pump.At first with 0.05M phosphate buffer (pH7.0) balance 15min, flow velocity: 0.5ml/min; Follow sample presentation, the papain solution of the 3.0mg/ml of 30ml, the protein that does not adsorb with 0.05M phosphate buffer (pH7.0) flush away then.1.0MNaSCN. with 0.05M phosphate buffer (pH8.0) preparation carries out wash-out at last, obtains the target protein papain.Obtain the activity preservation rate of papain and eluting rate all 90%.
Embodiment 4
Nylon affinity membrane of the present invention comprises the steps: the absorption property test of papain
Accurately take by weighing a certain amount of nylon affinity membrane of preparation as stated above, place 0.05M, in the papain solution (2.0mg/ml) of the buffer solution preparation of different pH values, behind the room temperature concussion different time, measure the absorbance of absorption front and back protein solution under 280nm, be calculated as follows adsorbance:
Q＝[(c
i-c
t)Vs]/m
In the formula: the Q-adsorbance; c
iConcentration before the-absorption;
c
t-absorption back concentration; The Vs-liquor capacity; The m-film quality
Test result is: 8.0,25 ℃ of pH values, and adsorption time 3h, the papain original concentration is 4.0mg/ml, adsorbance can reach 235.3mg/g.
Claims (3)
1, a kind of preparation method of nylon affinity membrane is characterized in that, comprises the steps:
(1) nylon membrane is separated 10-72h at 0.1-10M hydrochloric acid heavy water earlier, then with the glutaraldehyde activation processing of 10%-25%, nylon membrane after the activation places the chitosan solution of 0.5%-5% to carry out chemical bonding, dries in 70-120 ℃ baking oven then, promptly gets chitin modified nylon membrane;
(2) the nylon membrane 2-10g after the chitin modified processing being placed concentration is the 50-350mlCibacron Blue F3GA dye solution of 5.0-30mg/ml, 60 ℃ of reaction 0.5-3h, adding 20-50ml concentration then is 20%NaCl solution, continue reaction 1.0-3.0h, add 5.0-20ml concentration then and be 20% NaCO3 solution and carry out the fixation reaction, behind the reaction 2-10h, take out, successively with hot water, methyl alcohol, 2M NaCl, 6M urea, deionized water washs, obtain described nylon affinity membrane, 4 ℃ of preservations, standby.
2, a kind of application of nylon affinity membrane as claimed in claim 1 is characterized in that, is used for the separation and purification papain.
3, the application of nylon affinity membrane according to claim 2 is characterized in that, nylon affinity membrane separation and purification papain comprises the steps:
10-20 opens and builds up membrane stack on the nylon affinity membrane, put into the film bridge, send into buffer solution and eluent with peristaltic pump, at first be 7.0 phosphate buffer balance 10-20min with 0.05M, pH, flow velocity is 0.1-1.0ml/min, send into the papain solution of 1.0-5.0mg/ml of 15-50ml by film bridge, the protein that does not adsorb with 0.05M, pH7.0 phosphate buffer flush away then with peristaltic pump subsequently; Be that the 1.0M NaSCN that 8.0 phosphate buffers are prepared carries out wash-out with 0.05M, pH at last, obtain the target protein papain.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100531872C (en) * | 2007-08-03 | 2009-08-26 | 厦门大学 | Method for preparing polypyrole-polycarbonate composite membrane of fixed load cibacron blue |
| CN101259405B (en) * | 2007-12-13 | 2010-06-02 | 东华大学 | Preparation and application of nylon affinity membrane with reactive blue 4 as ligand |
| CN101249990B (en) * | 2008-03-11 | 2010-06-09 | 东华大学 | Method for rapidly removing metal copper ion from waste water |
| US7943597B2 (en) | 2008-04-08 | 2011-05-17 | Cypress Pharmaceutical, Inc. | Phosphate-binding chitosan and uses thereof |
| CN101380551B (en) * | 2008-03-21 | 2012-07-18 | 东华大学 | Preparation method of nylon affinity membrane and use thereof |
| CN102773028A (en) * | 2012-05-09 | 2012-11-14 | 江苏维赛科技生物发展有限公司 | Method for preparing hybond-N plus surface cross-linking chitosan modified membrane material |
| CN101565739B (en) * | 2009-06-09 | 2013-06-05 | 东华大学 | Method for measuring purity of papain by fast protein liquid chromatogram |
| CN103691328A (en) * | 2014-01-09 | 2014-04-02 | 常州斯威克光伏新材料有限公司 | Method for preparing contamination-resistant composite reverse osmosis membrane |
| CN104084060A (en) * | 2014-07-10 | 2014-10-08 | 杭州天创环境科技股份有限公司 | Antipollution modifying method of compound reverse osmosis membrane |
| CN108160056A (en) * | 2017-12-28 | 2018-06-15 | 大连海洋大学 | A kind of nylon membrane preparation method for adsorbing heavy metal |
| CN108993147A (en) * | 2018-08-27 | 2018-12-14 | 杭州易膜环保科技有限公司 | A kind of resistance to chlorine type reverse osmosis composite membrane and preparation method thereof |
-
2006
- 2006-09-15 CN CNB2006101160766A patent/CN100446844C/en not_active Expired - Fee Related
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100531872C (en) * | 2007-08-03 | 2009-08-26 | 厦门大学 | Method for preparing polypyrole-polycarbonate composite membrane of fixed load cibacron blue |
| CN101259405B (en) * | 2007-12-13 | 2010-06-02 | 东华大学 | Preparation and application of nylon affinity membrane with reactive blue 4 as ligand |
| CN101249990B (en) * | 2008-03-11 | 2010-06-09 | 东华大学 | Method for rapidly removing metal copper ion from waste water |
| CN101380551B (en) * | 2008-03-21 | 2012-07-18 | 东华大学 | Preparation method of nylon affinity membrane and use thereof |
| US7943597B2 (en) | 2008-04-08 | 2011-05-17 | Cypress Pharmaceutical, Inc. | Phosphate-binding chitosan and uses thereof |
| CN101565739B (en) * | 2009-06-09 | 2013-06-05 | 东华大学 | Method for measuring purity of papain by fast protein liquid chromatogram |
| CN102773028A (en) * | 2012-05-09 | 2012-11-14 | 江苏维赛科技生物发展有限公司 | Method for preparing hybond-N plus surface cross-linking chitosan modified membrane material |
| CN103691328A (en) * | 2014-01-09 | 2014-04-02 | 常州斯威克光伏新材料有限公司 | Method for preparing contamination-resistant composite reverse osmosis membrane |
| CN104084060A (en) * | 2014-07-10 | 2014-10-08 | 杭州天创环境科技股份有限公司 | Antipollution modifying method of compound reverse osmosis membrane |
| CN104084060B (en) * | 2014-07-10 | 2016-03-30 | 杭州天创环境科技股份有限公司 | A kind of antipollution method of modifying of complex reverse osmosis membrane |
| CN108160056A (en) * | 2017-12-28 | 2018-06-15 | 大连海洋大学 | A kind of nylon membrane preparation method for adsorbing heavy metal |
| CN108160056B (en) * | 2017-12-28 | 2020-12-18 | 大连海洋大学 | Preparation method of nylon membrane for adsorbing heavy metals |
| CN108993147A (en) * | 2018-08-27 | 2018-12-14 | 杭州易膜环保科技有限公司 | A kind of resistance to chlorine type reverse osmosis composite membrane and preparation method thereof |
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| CN100446844C (en) | 2008-12-31 |
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