CN101139579B - Method for preparing cross-linking bacillus subtilis proteinase crystal - Google Patents
Method for preparing cross-linking bacillus subtilis proteinase crystal Download PDFInfo
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- CN101139579B CN101139579B CN2007100257824A CN200710025782A CN101139579B CN 101139579 B CN101139579 B CN 101139579B CN 2007100257824 A CN2007100257824 A CN 2007100257824A CN 200710025782 A CN200710025782 A CN 200710025782A CN 101139579 B CN101139579 B CN 101139579B
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Abstract
The invention discloses a preparing method for a cross-linked subtilisin crystal that is of simple process and steady performance, which comprises the following procedures: (I) mixing subtilisin solution 2-10% (mass concentration) and salt solution 10-30% (mass concentration) in proportion 1:1 swung dash 1:5, agitating till crystal separates from the solution; (II) filtering the separated crystaland scrubbing by salt solution at least 4 times; (III) adding the crystal into salt solution to get suspension fluid of subtilisin crystal; (IV) adding slowly double-functional cross-linking agent into the suspension fluid of subtilisin crystal and agitating 3-16 hours, the added amount is 1-5 times by weight of subtilisin crystal; (V) filtering or centrifuging the reaction solution to get coarsecross-linked subtilisin crystal, scrubbing 2-8 times by de-ionized water the coarse crystal, then scrubbing by a buffering liquid, till getting the desired cross-linked subtilisin crystal.
Description
Technical field
The present invention relates to a kind of preparation method of cross-linking bacillus subtilis proteinase crystal.
Background technology
Enzyme (Enzyme) is the protein with height specificity and catalytic efficiency that produces in the viable cell, is called biological catalyst again.Organism is in metabolic processes, and nearly all chemical reaction all is to carry out under the catalysis of enzyme.Human of long standing and well established to the understanding of enzyme.The summer Yu epoch of China before more than 4,000 year, just wine brewing was in vogue, had begun the Zhou Dynasty to make vinegar, sauce, and had treated maldigestion with song.The systematic study of enzyme originates in the middle of the 19th century to the essential research of fermenting.1858, French chemistry and biologist Louis pasteur (Louis Pasteur) were the phenomenons relevant with life with the fermentation of a series of article proof.But 1897, German famous chemist Edward Bi Xina (Eduard Buchner) successfully realized fermentation with not celliferous yeast juice, illustrates that the material with fermentative action is present in the cell, does not rely on viable cell.This material is exactly an enzyme.Nineteen twenty-six American Sumner (James Batcheller Sumner) extracts urase first, and carries out crystallization, and points out that the essence of enzyme is protein.
These scientific researches have effectively promoted human understanding and use to enzyme.Now, existing two over thousands of kinds of enzymes are identified out, wherein have 200 surplus kind obtain crystallization.Particularly in the last thirty years, along with the progress of protein stripping technique, the molecular structure of enzyme, the research of enzyme mechanism are developed, and the structure of some enzyme and the mechanism of action are illustrated.
As biological catalyst (biological catalyst), enzyme has the characteristic of two aspects.Its existing and general identical catalytic property of catalyzer has the feature of the unexistent biomacromolecule of general catalyzer again.For example, enzyme is the same with general catalyzer, and the chemical reaction that can only catalysis thermodynamics allows shortens and reaches the time of chemical equilibrium, and does not change trim point.In addition, enzyme does not have the change of quality and quantity as catalyzer in the front and back of chemical reaction, and the enzyme of trace just can be brought into play bigger katalysis.The mechanism of action of enzyme and general catalyzer all is to reduce the activation energy (activation energy) of reaction.
Simultaneously, enzyme has the catalytic efficiency of height, the specificity of height, the controllability of enzymic activity, the characteristics such as unstable of enzymic activity again as the protein catalyzed reaction.Wherein, the preceding several characteristics of enzyme allows people see the bright prospect that zymochemistry is used, and still, the unstable of enzyme has but seriously restricted further developing of enzyme.As protein, enzymic catalytic reaction requires gentle condition such as certain pH value, temperature, and any chemical factors of protein denaturation that makes such as strong acid, highly basic, organic solvent, heavy metallic salt, high temperature, ultraviolet ray, concuss all may make enzyme denaturation and lose its catalytic activity.
Subtilisin is a kind of very important industrial enzymes.Widespread use washing composition, makeup and asymmetry catalysis are synthetic.But subtilisin is very unstable.Understand self hydrolysis and inactivation.The crosslinked enzyme crystal technology is a kind of very effective enzyme stabilization technology.But still do not find the preparation method of relevant cross-linking bacillus subtilis proteinase at present.
Summary of the invention
Technical problem to be solved by this invention is: will provide that a kind of technology is simple, the preparation method of the cross-linking bacillus subtilis proteinase crystal of stable performance.
For addressing the above problem, the technical solution used in the present invention is: the preparation method of described cross-linking bacillus subtilis proteinase crystal may further comprise the steps:
(1) under 4～65 ℃, with mass concentration is that 2%～10% subtilisin solution and mass concentration are 10%～30% 1: 1 by volume～1: 5 mixed of metabisulfite solution, under the stirring velocity of 50～1000rpm/min, stirred 6～7 days, and had bacillus subtilis proteinase crystal to separate out in the solution;
(2) under 4～30 ℃, the crystal of separating out is filtered, again through metabisulfite solution washing at least 4 times;
(3) washed crystal is added in the metabisulfite solution, stirred 1～3 hour, get the bacillus subtilis proteinase crystal suspension liquid;
(4) at 4～45 ℃, under the stirring velocity of 300～1000rpm/min, with bi-functional cross-linking agent-mass concentration is that 25% glutaraldehyde slowly adds in the bacillus subtilis proteinase crystal suspension liquid, and add-on is 1～5 times of bacillus subtilis proteinase crystal weight, stirs 5 hours;
(5) after filtration or centrifugal cross-linking bacillus subtilis proteinase crystal crude product with reaction solution, the cross-linking bacillus subtilis proteinase crystal crude product that obtains is used deionized water wash 2～8 times earlier, with the damping fluid washing, stop washing less than 0.5 again up to the ultraviolet absorption value of scrub stream fluid under 280nm; Obtain required cross-linking bacillus subtilis proteinase crystal.
In actual fabrication, for ease of preserving, the ratio of cross-linking bacillus subtilis proteinase crystal according to weight ratio 5%～30% can be added in the damping fluid, stirred 1～2 hour, obtain the suspension liquid of cross-linking bacillus subtilis proteinase crystal.
Temperature in the above-mentioned steps () is preferably 44～55 ℃, and stirring velocity is preferably 280～320rpm/min.
Metabisulfite solution concentration preferred 15%～20% described in above-mentioned steps (), (two) and (three).
Preferred 28～35 ℃ of temperature of reaction described in the above-mentioned steps (four), described stirring velocity is 480～520rpm/min.In this step, enzyme crystal Weight Calculation method can detect by ultraviolet spectrophotometry and obtain, promptly get the resulting enzyme crystal suspension liquid of 1 milliliter of step (three), 300 times of dilute with waters, under 280nm, measure its uv-absorbing, be calculated as follows the weight of enzyme crystal: enzyme crystal concentration=UV 280 ultraviolet absorption values * 1.009 * 300g/L; Enzyme crystal weight=enzyme crystal concentration * enzyme crystal suspension liquid volume.
Above-mentioned damping fluid is for containing 100mM NaAc and 20mM CaCl
2The aqueous solution, the pH value of solution value is 5.5～5.85.
The invention has the beneficial effects as follows: described method cost is low, technology is simple and environmental protection, be easy to realize that the cross-linked ester crystal that makes has high stability, high purity, high reactivity, but the building-up reactions of catalysis chipal compounds.
Embodiment
The invention will be further described below by specific embodiment, but the present invention is not limited in these embodiment.
Embodiment 1:
At 35 ℃, 100ml subtilisin solution (mass concentration is about 5%) and 200ml, 15% metabisulfite solution are pressed the volume ratio of 1:2 and mixed, under the magnetic agitation of 100rpm/min, there is crystal to separate out after 6 days; Under 22 ℃, the gained crystal is filtered, wash 6 times with 15% metabisulfite solution again; The gained crystal is added in 100ml, the 15% sodium sulfate buffered soln once more, stirred 2 hours, obtain bacillus subtilis proteinase crystal suspension solution (cumulative volume is 100 milliliters).
At 35 ℃, 400rpm/min stirs down, is that 25% glutaraldehyde (be enzyme crystal weight 3.36 times) slowly adds in the bacillus subtilis proteinase crystal suspension solution with mass concentration, stirs 5 hours; With reaction solution after filtration subtilisin crosslinked enzyme crystal crude product, earlier with deionized water wash 5 times, again with pH5.7 and contain 100mM NaAc and 20mM CaCl
2Damping fluid washing, stop washing up to the ultraviolet absorption value of scrub stream fluid under 280nm less than 0.5; The cross-linking bacillus subtilis proteinase crystal that obtains added again contain 100mM NaAc and 20mM CaCl
2Damping fluid in, stirred 1 hour, promptly get the cross-linking bacillus subtilis proteinase crystal suspension liquid.
Embodiment 2:
With embodiment 1 described preparation method, different is at 50 ℃, it is that 20% metabisulfite solution mixes by the volume ratio of 1:2 that mass concentration is about 5% subtilisin solution and concentration, under the magnetic agitation of 100rpm/min, has crystal to separate out after 6 days.All the other steps are identical.
Embodiment 3:
With embodiment 1 described preparation method, different is at 10 ℃, and the metabisulfite solution that mass concentration is about 5% subtilisin solution and 10% mixes by the volume ratio of 1:2, under the magnetic agitation of 100rpm/min, has crystal to separate out after 6 days.All the other steps are identical.
Claims (6)
1. the preparation method of a cross-linking bacillus subtilis proteinase crystal is characterized in that: may further comprise the steps:
(1) under 4～65 ℃, with mass concentration is that 2%～10% subtilisin solution and mass concentration are 10%～30% 1: 1 by volume～1: 5 mixed of metabisulfite solution, under the stirring velocity of 50～1000rpm/min, stirred 6～7 days, and had bacillus subtilis proteinase crystal to separate out in the solution;
(2) under 4～30 ℃, the crystal of separating out is filtered, again through metabisulfite solution washing at least 4 times;
(3) washed crystal is added in the metabisulfite solution, stirred 1～3 hour, get the bacillus subtilis proteinase crystal suspension liquid;
(4) at 4～45 ℃, under the stirring velocity of 300～1000rpm/min, with bi-functional cross-linking agent--mass concentration is that 25% glutaraldehyde slowly adds in the bacillus subtilis proteinase crystal suspension liquid, and add-on is 1～5 times of bacillus subtilis proteinase crystal weight, stirs 5 hours;
(5) after filtration or centrifugal cross-linking bacillus subtilis proteinase crystal crude product with reaction solution, the cross-linking bacillus subtilis proteinase crystal crude product that obtains is used deionized water wash 2～8 times earlier, with the damping fluid washing, stop washing less than 0.5 again up to the ultraviolet absorption value of scrub stream fluid under 280nm; Obtain required cross-linking bacillus subtilis proteinase crystal.
2. preparation method according to claim 1, it is characterized in that: for ease of preserving, the ratio of cross-linking bacillus subtilis proteinase crystal according to weight ratio 5%～30% added in the damping fluid, stirred 1～2 hour, obtain the suspension liquid of cross-linking bacillus subtilis proteinase crystal.
3. preparation method as claimed in claim 1 or 2 is characterized in that: the temperature in the step () is preferably 44～55 ℃, and stirring velocity is preferably 280～320rpm/min.
4. preparation method as claimed in claim 1 or 2 is characterized in that: the metabisulfite solution concentration described in step (), (two) and (three) is 15%～20%.
5. preparation method as claimed in claim 1 or 2 is characterized in that: the temperature of reaction described in the step (four) is 28～35 ℃, and described stirring velocity is 480～520rpm/min.
6. preparation method as claimed in claim 1 or 2 is characterized in that: described damping fluid is for containing 100mM NaAc and 20mM CaCl
2The aqueous solution, the pH value of solution value is 5.5～5.85.
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| CN2007100257824A CN101139579B (en) | 2007-08-02 | 2007-08-02 | Method for preparing cross-linking bacillus subtilis proteinase crystal |
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| CN2007100257824A CN101139579B (en) | 2007-08-02 | 2007-08-02 | Method for preparing cross-linking bacillus subtilis proteinase crystal |
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| CN101139579B true CN101139579B (en) | 2010-12-08 |
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| CN101892203A (en) * | 2010-07-21 | 2010-11-24 | 中国人民解放军第三军医大学 | Crystal of thymidylate synthetase (ThyX) of helicobacter pylori and preparation method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5976529A (en) * | 1990-08-03 | 1999-11-02 | Vertex Pharmaceuticals, Inc. | Methods of enzyme therapy by orally administering crosslinked enzyme crystals |
| CN1408855A (en) * | 2001-09-19 | 2003-04-09 | 上海化工研究院 | method for improving lipase activity stability |
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2007
- 2007-08-02 CN CN2007100257824A patent/CN101139579B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5976529A (en) * | 1990-08-03 | 1999-11-02 | Vertex Pharmaceuticals, Inc. | Methods of enzyme therapy by orally administering crosslinked enzyme crystals |
| CN1408855A (en) * | 2001-09-19 | 2003-04-09 | 上海化工研究院 | method for improving lipase activity stability |
Non-Patent Citations (4)
| Title |
|---|
| Paul A. Fitzpatrick et al..X-ray crystal structure of cross-linked subtilisin carlsberg inwater vs. acetonitrile.BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATION198 2.1994,198(2),675-681. |
| Paul A. Fitzpatrick et al..X-ray crystal structure of cross-linked subtilisin carlsberg inwater vs. acetonitrile.BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATION198 2.1994,198(2),675-681. * |
| 潘力等.碱性蛋白酶交联酶晶体的制备及稳定性.华南理工大学学报（自然科学版）26 9.1998,26(9),22-26. |
| 潘力等.碱性蛋白酶交联酶晶体的制备及稳定性.华南理工大学学报(自然科学版)26 9.1998,26(9),22-26. * |
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