CN101380551A - Preparation method of nylon affinity membrane and use thereof - Google Patents
Preparation method of nylon affinity membrane and use thereof Download PDFInfo
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- CN101380551A CN101380551A CNA2008100349835A CN200810034983A CN101380551A CN 101380551 A CN101380551 A CN 101380551A CN A2008100349835 A CNA2008100349835 A CN A2008100349835A CN 200810034983 A CN200810034983 A CN 200810034983A CN 101380551 A CN101380551 A CN 101380551A
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Abstract
The invention discloses a method for preparing nylon affinity membranes, in which Reactive Red 120 is taken as ligand, and the application thereof. The preparation method comprises the following steps: after the modification by chitosan, the nylon membrane after being activated is immerged into Reactive Red 120 dye, the nylon affinity membrane that takes Reactive Red 120 as the ligand is obtained; the prepared nylon affinity membranes are applied to purifying pawpaw protease, the purification method is not only fast, simple and convenient, but also large in the quantity of separation, good activity of the extracted enzyme high in purity, and compared with the column affinity chromatogram method, the preparation method is more applicable to scaled purification and separation.
Description
Technical field
The invention belongs to the nylon affinity membrane field, particularly relating to a kind of is the preparation method and the application in the separation and purification papain thereof of the nylon affinity membrane of aglucon with Reactive Red 120.
Background technology
Papain Papain (EC3.4.22.2) is a class thiol protease, extensively is present in root, stem, leaf and the fruit of papaya (Caricapapaya), and wherein content is the abundantest 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 of meat.The purifying major part of papain is to adopt the precipitation method, but this method still is mixed with other protease, can not reach the demand of pharmaceuticals industry.
At present, the method of separation and purification papain comprises that crude separation separates with refining, wherein crude separation comprises salting out method, isoelectric point precipitation, organic solvent classification partition method etc., and refining separation comprises gel filtration, ion-exchange chromatography, adsorption chromatography, hydrophobic chromatography and covalent chromatography etc.Although these methods all have certain advantage, all there are shortcomings such as complex operation, the sample activity recovery is low, purification effect is undesirable, when especially from the weak solution of large volume, extracting a spot of bioactivator.
The affinity film chromatography The Application of Technology can make the protein purification multiple improve; and have select that specificity is good, pressure drop is little, weak point consuming time, large biological molecule sex change probability in separation process are little, allow faster charging rate, characteristics such as can reuse, effectively reduce cost; compare easier realization scale purifies and separates with the post affinity chromatography.
Summary of the invention
One of technical problem to be solved by this invention provides a kind of preparation method of nylon affinity membrane;
Two of technical problem to be solved by this invention provides the application of a kind of nylon affinity membrane in the separation and purification papain, has overcome loaded down with trivial details, the unfavorable shortcoming of purification effect of method of operating in the prior art, applicable to the scale purifies and separates.
The preparation method of a kind of nylon affinity membrane of the present invention comprises the following steps:
(1) nylon membrane after the activation immerses in the chitosan solution, and behind the room temperature reaction, 80-90 ℃ of oven dry dashed with 1vol.% acetic acid and deionized water, carries out the modification of nylon membrane;
(2) nylon membrane after chitin modified immerses in Reactive Red 120 dye solutions, and behind the insulation 30-50min, adding mass fraction is 15%～20%NaCl solution, 55 ℃～60 ℃ constant temperature oscillating reactions 30-40min, and the bath temperature that raises then adds Na
2CO
3Solution, 65 ℃～80 ℃ are continued constant temperature oscillating reactions 4-5h, use the deionized water of heat respectively, methanol solution, 2mol/L NaCl solution, urea liquid and deionized water fully wash, and are able to Reactive Red 120 and are the nylon affinity membrane of aglucon.
Described nylon membrane activation is meant that nylon membrane after the hydrolysis, activates with formaldehyde in 25-30 ℃ of 1mol/LHCl;
Described chitosan solution is meant that mass fraction is 2% chitosan solution (with the 1vol.% acetate dissolution);
Described Reactive Red 120 dyes concentrations are 5mg/mL～15mg/mL, preferred 8mg/mL～12mg/mL;
Described Na
2CO
3Solution is meant that mass fraction is 15%～20%Na
2CO
3Solution;
Described methanol solution is meant that volume fraction is 10% methanol solution;
Described urea liquid is meant the 6mol/L urea liquid.
The application of a kind of nylon affinity membrane of the present invention in the separation and purification papain, this method comprises:
Papaya powder in Tris-HCl buffer solution, being splined on Reactive Red 120 is the nylon affinity membrane of aglucon, after Tris-HCl buffer solution flushing, uses the NaSCN eluant solution, obtains papain.
The method of nylon affinity membrane separation and purification papain, concrete steps comprise:
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 with Tris-HCl buffer solution balance 10-20min, flow velocity is 0.1-1.0mL/min, send into papaya powder solution by the film bridge with peristaltic pump subsequently, then with protein that Tris-HCl buffer solution flush away does not adsorb; Eluent with NaSCN carries out wash-out at last, obtains the target protein papain.
Described Tris-HCl buffer solution is the Tris-HCl buffer solution of 0.05mol/L～1mol/L pH8.0～8.5, Tris-HCl buffer solution of preferred 0.05mol/L pH8.5;
The papaya powder solution that described papaya powder solution is 10.0～15.0mg/mL;
Described NaSCN solution is meant the NaSCN solution of 1mol/L pH6.0.
The papain that obtains by said method can tested enzyme activity and protein content, calculates the purifying multiple.
Beneficial effect of the present invention:
(1) isolation and purification method of the present invention is simple to operate, and is consuming time less, and purifying multiple and enzymatic activity are higher;
(2) sample source is convenient, compares with post affinity chromatography method and is convenient to realize the scale purifies and separates.
Description of drawings
Fig. 1 is protein content and the enzyme curve alive that utilizes nylon affinity membrane absorb-elute of separation and purification papain from papaya powder.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
The activation of nylon membrane and bonding shitosan carry out modification, and concrete steps are as follows:
(1) the hydrolysis 24h in 25 ℃ of 1mol/L HCl of nylon membrane elder generation activates with formaldehyde then.The nylon membrane of 10 hydrolysis immerses 20mL formalin (〉 36.5wt.%), add 0.2mL phosphoric acid (85wt.%), in 60 ℃ of reaction 7h, 40-50 ℃ of hot water washing is repeatedly.
(2) nylon membrane that above-mentioned formaldehyde is activated, immersion 10mL mass fraction is 2% chitosan solution (with the 1vol.% acetate dissolution), and room temperature reaction 1h changes 80 ℃ of baking ovens then over to, take out behind the 1h, use 1vol.% acetic acid and the unreacted shitosan of deionized water flush away respectively.Shitosan content on the nylon membrane can be tested by the method for ninhydrin.With Reactive Red 120 is the preparation of the nylon affinity membrane of aglucon, and concrete steps are as follows:
It is in Reactive Red 120 dye solutions of 10mg/mL that above-mentioned nylon membrane after chitin modified immerses concentration, behind 60 ℃ of insulation 40min, adding mass fraction is 20% NaCl solution, constant temperature oscillating reactions 30min, raise then bath temperature to 80 ℃, adding mass fraction is 25% Na
2CO
3Solution, continue constant temperature oscillating reactions 4h, use the deionized water of heat respectively, volume fraction is 10% methanol solution, 2mol/L NaCl solution, 6mol/L urea liquid and deionized water fully wash, up to cleaning solution be colourless till, obtaining with Reactive Red 120 is the nylon affinity membrane of aglucon, is stored in the distilled water.
Utilize nylon affinity membrane separation and purification papain from papaya powder solution, concrete steps are as follows:
Build up membrane stack on 15 nylon affinity membranes, put into the film bridge, send into buffer solution and eluent with peristaltic pump, at first be Tris-HCl buffer solution balance 15min of 8.5 with 0.05mol/L pH, flow velocity is 1.0mL/min, the papaya powder solution of 10mg/mL of sending into 35mL with peristaltic pump is by the film bridge subsequently, then with the protein that Tris-HCl buffer solution flush away does not adsorb of 0.05mol/L pH8.5; At last the eluent (pH6.0) with the NaSCN of 1mol/L carries out wash-out, obtains the target protein papain.
With the casein is the ultraviolet spectrophotometry enzyme sign alive of substrate, and concrete steps are as follows:
Get the enzyme liquid of 0.1mL, add 0.7mL 0.05mol/L Tris-HCl buffer solution (pH8.0), (above-mentioned buffer solution includes the L-cysteine of 0.5mol/L and the EDTA of 0.02mol/L to add the papain activator of 0.2mL again, pH8.0) 35 ℃ of insulations are constant, 1% (W/V) casein solution (the above-mentioned buffer solution preparation) 1mL that adds same preheating, 35 ℃ of reaction 15mim add 3mL5% trichloroacetic acid (TCA) solution cessation reaction (the control group experiment adds substrate after adding TCA earlier).Leave standstill, 8000 rev/mins of centrifugal 20min get filtrate colorimetric under the 280nm wavelength.
Claims (13)
1. the preparation method of a nylon affinity membrane comprises the following steps:
(1) nylon membrane after the activation immerses in the chitosan solution, and behind the room temperature reaction, 80-90 ℃ of oven dry dashed with 1vol.% acetic acid and deionized water, carries out the modification of nylon membrane;
(2) nylon membrane after chitin modified immerses in Reactive Red 120 dye solutions, and behind the insulation 30-50min, adding mass fraction is 15%～20%NaCl solution, 55 ℃～60 ℃ constant temperature oscillating reactions 30-50min, and the bath temperature that raises then adds Na
2CO
3Solution, 65 ℃～80 ℃ are continued constant temperature oscillating reactions 4-5h, and deionized water, methanol solution, NaCl solution, urea liquid and the deionized water with heat fully washs respectively, is able to Reactive Red 120 and is the nylon affinity membrane of aglucon.
2. the preparation method of a kind of nylon affinity membrane according to claim 1 is characterized in that, described nylon membrane activation is meant that nylon membrane after the hydrolysis, activates with formaldehyde in 25-30 ℃ of 1mol/L HCl.
3. the preparation method of a kind of nylon affinity membrane according to claim 1 is characterized in that, described chitosan solution is that the mass fraction with the 1vol.% acetate dissolution is 2% chitosan solution.
4. the preparation method of a kind of nylon affinity membrane according to claim 1 is characterized in that, described Reactive Red120 dyes concentration is 5mg/mL～15mg/mL.
5. the preparation method of a kind of nylon affinity membrane according to claim 4 is characterized in that, described Reactive Red120 dyes concentration is 8mg/mL～12mg/mL.
6. the preparation method of a kind of nylon affinity membrane according to claim 1 is characterized in that, described Na
2CO
3Solution is meant that mass fraction is 15%～20%Na
2CO
3Solution.
7. the preparation method of a kind of nylon affinity membrane according to claim 1, it is characterized in that described washing is to be that 10% methanol solution, 2mol/L NaCl solution, 6mol/L urea liquid and deionized water fully wash with hot deionized water, volume fraction respectively.
8, a kind of application of nylon affinity membrane is characterized in that, nylon affinity membrane is used for the separation and purification papain.
9. the application of a kind of nylon affinity membrane according to claim 8, it is characterized in that, the method of nylon affinity membrane separation and purification papain comprises: the papaya powder in Tris-HCl buffer solution, being splined on Reactive Red 120 is the nylon affinity membrane of aglucon, after Tris-HCl buffer solution flushing, use the NaSCN eluant solution, obtain papain;
10. the application of a kind of nylon affinity membrane according to claim 8 is characterized in that, described Tris-HCl buffer solution is the Tris-HCl buffer solution of 0.05M～1M pH8.0～8.5.
11. the application of a kind of nylon affinity membrane according to claim 9 is characterized in that, described Tris-HCl buffer solution is Tris-HCl buffer solution of 0.05M pH8.5.
12. the application of a kind of nylon affinity membrane according to claim 8 is characterized in that, the papaya powder solution that described papaya powder solution is 10.0～15.0mg/mL.
13. the application of a kind of nylon affinity membrane according to claim 8 is characterized in that, described NaSCN solution is meant the NaSCN solution of 1mol/L pH6.0.
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| CN2008100349835A CN101380551B (en) | 2008-03-21 | 2008-03-21 | Preparation method of nylon affinity membrane and use thereof |
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|---|---|---|---|
| CN2008100349835A CN101380551B (en) | 2008-03-21 | 2008-03-21 | Preparation method of nylon affinity membrane and use thereof |
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| CN101380551A true CN101380551A (en) | 2009-03-11 |
| CN101380551B CN101380551B (en) | 2012-07-18 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199876A (en) * | 2011-04-19 | 2011-09-28 | 五邑大学 | Chitosan modified nylon fabric and manufacturing method thereof |
| CN102512982A (en) * | 2012-01-11 | 2012-06-27 | 浙江理工大学 | Antibacterial oxidation-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 |
| CN115069099A (en) * | 2022-07-04 | 2022-09-20 | 东华大学 | Preparation method and application of polymer affinity membrane with efficient nucleic acid separation and purification function |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100446844C (en) * | 2006-09-15 | 2008-12-31 | 东华大学 | Nylon affinity membrane preparing method and use |
-
2008
- 2008-03-21 CN CN2008100349835A patent/CN101380551B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199876A (en) * | 2011-04-19 | 2011-09-28 | 五邑大学 | Chitosan modified nylon fabric and manufacturing method thereof |
| CN102199876B (en) * | 2011-04-19 | 2013-01-16 | 五邑大学 | Chitosan modified nylon fabric and manufacturing method thereof |
| CN102512982A (en) * | 2012-01-11 | 2012-06-27 | 浙江理工大学 | Antibacterial oxidation-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 |
| CN115069099A (en) * | 2022-07-04 | 2022-09-20 | 东华大学 | Preparation method and application of polymer affinity membrane with efficient nucleic acid separation and purification function |
| CN115069099B (en) * | 2022-07-04 | 2023-09-26 | 东华大学 | Preparation method and application of polymer affinity membrane with efficient nucleic acid separation and purification functions |
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| Publication number | Publication date |
|---|---|
| CN101380551B (en) | 2012-07-18 |
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