CN102888391B - Immobilization method of heparinase II - Google Patents
Immobilization method of heparinase II Download PDFInfo
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- CN102888391B CN102888391B CN201210423324.7A CN201210423324A CN102888391B CN 102888391 B CN102888391 B CN 102888391B CN 201210423324 A CN201210423324 A CN 201210423324A CN 102888391 B CN102888391 B CN 102888391B
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Abstract
The invention relates to an immobilization method of heparinase II, and particularly relates to a method for immobilizing heparinase II by taking chitosan microspheres as an immobilization material. The immobilized heparinase II has the advantages that the immobilized heparinase II is recyclable and reusable and enzymes can be easily separated from substrates and products and the like.
Description
Technical field
The present invention relates to a kind of process for fixation of heparinaseⅡ, particularly relating to a kind of take chitosan as the method for immobilization material to being fixed of heparinaseⅡ, realize heparinaseⅡ immobilization and can reusing.
Background technology
Heparinase refers to that a class can the enzyme of Specific lytic heparin and heparitin main chain glycosidic link, finds at first and separates, find again thereafter in some microorganisms and animal tissues, also have heparinase to exist from Flavobacterium heparinum.The nearly kind more than 10 of the heparinase having academic paper to report at present, obtain comparatively careful research only from 3 kinds of enzymes of Flavobacterium heparinum, be respectively heparinase I (EC numbering 4.2.2.7), heparinaseⅡ (without EC numbering), heparinase III (EC numbering 4.2.2.8).Heparinase I, II, III be respectively molecular weight about 43,78, the monomeric protein of 66kd, its iso-electric point is all about 9.0, and application and research is widely.
HeparinaseⅡ is a kind of can heparin and Suleparoid be the enzyme of catalyzed degradation substrate, has functions such as removing in blood remaining heparin, anti-hemostasis-coagulation, simultaneously to producing Low molecular heparin, the structure of research heparin has important effect.But free heparinaseⅡ needs when reacting to be added in substrate, after reaction, enzyme solution, substrate mix with product and are not easy to be separated, enzyme is caused to reuse, utilising efficiency is low, this is a lot of inconvenience for application brings, therefore, need to find a kind of method that can improve the utilising efficiency of heparinase.
Compared with resolvase, immobilized enzyme is while its efficiently single-minded and gentle enzymic catalytic reaction characteristic of maintenance, overcome again the weak point of resolvase, have Separation and Recovery easily, can repeatedly use, operate continuously the series of advantages such as controlled, therefore, be a kind of ideal chose improving result of use to the immobilization of heparinaseⅡ.But at present report is had no to the research of being fixed of heparinaseⅡ.
Through studying for a long period of time, the present inventor finds a kind of efficient and process for fixation of the heparinaseⅡ of low cost.Take this, The present invention gives a kind of with chitosan microball be material to the immobilized method of heparinaseⅡ, being separated of enzyme-to-substrate and product when successfully achieving use, improves application potential.
Summary of the invention
The invention provides a kind of method of chitosan microball fixing heparin enzyme II, comprise the steps:
(1) preparation of heparinaseⅡ solution: (pH7.0, containing CaCl to select Tris-HCl damping fluid
2) as the solvent of heparinaseⅡ, heparinaseⅡ is dissolved in this damping fluid, obtained heparinaseⅡ solution;
(2) heparinaseⅡ and chitosan microball is crosslinked: by chitosan microball, with Tris-HCl damping fluid, (pH 7.0, wherein containing 10mM CaCl
2) fully swelling, then get the heparinaseⅡ solution that step (1) obtains, added in this chitosan microball, be cross-linked;
(3) chitosan microball after above-mentioned being cross-linked is washed respectively to elutriant non-enzymatic activity with acetic acid-sodium acetate and Tris-HCl two kinds of damping fluids;
(4) utilize reductive agent to process the chitosan microball securing heparinaseⅡ obtained in step (3), subsequently, if needed, the reductive agent that removing is remaining, is secured to the heparinaseⅡ on chitosan microball thus.
In the above-described embodiment, be 10-25mM for the concentration of the solvent Tris-HCl dissolving heparinaseⅡ in preferred steps (1), be also the CaCl of 10-50mM containing concentration in Tris-HCl simultaneously
2, most preferably be 25mM Tris-HCl(pH 7.0, wherein containing 10mM CaCl
2);
In the above-described embodiment, in preferred steps (1), heparinaseⅡ concentration is in the solution 0.1-100IU/ml, is more preferably 1-10 IU/ml, most preferably is 1-3 IU/ml.
In the above-described embodiment, described in step (2), chitosan microball can be prepared by preparation method well known in the prior art, such as, inverse suspension method can be adopted to be prepared, the linking agent wherein used suitably can be selected by those skilled in the art, and the linking agent be such as most widely used is glutaraldehyde.Described swelling time can be controlled by those skilled in the art according to degree of swelling, such as, and swelling 20 minutes.
In the above-described embodiment, the chitosan microball used in preferred steps (2) and the volume ratio of heparinaseⅡ solution are 1/10-10/1, more preferably 1/5-5/1, most preferably 1/2-2/1.
In the above-described embodiment, preferably in step (2), crosslinking temperature is 4-10 DEG C, and crosslinking time is 8-24h, and preferred crosslinking temperature is 8-10 DEG C, and crosslinking time is 12-20h.
In the above-described embodiment, the damping fluid used in preferred steps (3) is acetic acid-sodium acetate buffer solution (pH 6.0-8.0), be more preferably 50-150mM acetic acid-sodium acetate (pH 6.5-8.0, wherein containing 50-200mM NaCl) damping fluid, most preferably be 100mM acetic acid-sodium acetate (pH 7.5, wherein containing 100mM NaCl) damping fluid.
In the above-described embodiment, the damping fluid used in preferred steps (3) is Tris-HCl damping fluid, is more preferably 50mM Tris-HCl(pH 6.5-8.0, wherein containing 10-100mM CaCl
2with 50-200mM NaCl) damping fluid, most preferably be 50mM Tris-HCl(pH 7.5, wherein containing 50mM CaCl
2with 100mM NaCl) damping fluid.
In the above-described embodiment, in the reductive agent treatment step of step (4), that the unstable C=N double bond produced after the amino on chitosan and the aldehyde radical covalent cross-linking in glutaraldehyde is reduced, any known reductive agent that may be used for reducing this double bond can be used in this reduction step, such as hydroborate, lithium aluminum hydride, Raney's nickel etc., preferred hydroborate, such as alkali metal borohydride, such as sodium borohydride, POTASSIUM BOROHYDRIDE etc.
In the above-described embodiment, the NaBH used in preferred steps (4)
4concentration is 1-10mg/ml, reaction 0.5-10h; Optimal selection concentration is the NaBH of 1-5mg/ml
4, reaction 1-3h.
In the above-described embodiment, use Tris-HCl wash buffer to remove remaining reductive agent in preferred steps (4), more preferably this damping fluid is 50mM Tris-HCl(pH 7.0, wherein containing 10mM CaCl
2).
In the above-described embodiment, after carrying out step (4), in order to evaluate the method, the efficiency of whole immobilization process can be drawn compared with the work of acquisition immobilized enzyme is alive with the resolvase of loading, and carry out quantitatively to immobilized enzyme, after carrying out reductive agent treatment step, immobilized enzyme can be surveyed and lives.
In the above-described embodiment, after the immobilized heparinaseⅡ of acquisition, preservation condition is: be kept in Tris-HCl buffer system, pH 6.5-8.0, adds metal-salt CaCl
2and NaCl, storage temperature is 0-25 DEG C, and optimal conditions, for being kept at 25mM Tris-HCl(pH 7.5, wherein contains 10mM CaCl
2), temperature is in 4-10 DEG C of environment.
In use, this be fixed on enzyme on carrier can with substrate direct reaction, through being separated after having reacted, the enzyme be fixed on carrier can reuse.Immobilized heparinase can be applied to the removing of heparin in blood, for the manufacture of Low molecular heparin, for the partially or completely degraded of heparin and Low molecular heparin sample.
In the above-described embodiment, the inverse suspension method in preferred steps (2) is:
Getting commercially available Chitosan powder is dissolved in acetic acid, obtained acidic chitosan colloidal sol, dispersion agent is added in oil phase, then acidic chitosan colloidal sol is added in oil phase, after emulsification, add glutaraldehyde to be cross-linked, add the mixed solution of isopyknic NaOH and dehydrated alcohol after crosslinked, leave standstill after stirring, discard oil reservoir and water layer, and use ultrapure water repetitive scrubbing, obtain chitosan microball.
In the above-described embodiment, be the acetic acid solution of 2% for dissolving the solvent of chitosan, the massfraction of chitosan is 2%.Preferred dispersants is Span 80, and preferred oil phase is whiteruss, and preferred rotating speed is 500-800rpm; Preferred linking agent is the glutaraldehyde of 25%, and preferred crosslinking time is 1-2h, and the volume ratio of preferred 2.5M NaOH and dehydrated alcohol is 1/1.
In the above-described embodiment, the immobilized enzyme measuring method preferably used be reddish black A method [Linhardt, R.J., (1984),
appl. Biochem. Biotech.9,41-55] and 232nm method [Bernstein, H., (1987),
appl. Biochem. Biotech.16,129-143].
In the most preferred embodiment, the process for fixation of heparinaseⅡ of the present invention comprises the steps:
(1a), the preparation of chitosan microball:
Getting Chitosan powder is dissolved in the acetic acid of 2%, and obtained acidic chitosan colloidal sol, adds in whiteruss by Span 80, high-speed stirring mixes, acidic chitosan colloidal sol is dropwise added in whiteruss, after emulsification 1h, adds glutaraldehyde as linking agent, crosslinking temperature is 40 DEG C, add the mixed solution of 2.5M NaOH and dehydrated alcohol (volume ratio 1/1) after crosslinked, leave standstill after stirring, discard oil reservoir and water layer, and use ultrapure water repetitive scrubbing, obtain chitosan microball;
(1) damping fluid 25mM Tris-HCl(pH 7.0 is prepared, containing 10mM CaCl
2), it can be used as the solvent of heparinaseⅡ, obtained heparinaseⅡ solution, adjustment concentration is to enzyme activity 1-3IU/ml;
(2) get the heparinaseⅡ solution that step (1) obtains, added with 25mM Tris-HCl(pH 7.0, containing 10mM CaCl
2) in the chitosan microball that obtains of the step (1a) of abundant swelling mistake, the volume ratio of chitosan and heparinase is 1/2-2/1,4-10 DEG C of crosslinked 12-20h;
(3) 100mM acetic acid-sodium acetate (pH 7.5, wherein containing 100mM NaCl) and 50mM Tris-HCl(pH 7.5 is prepared, wherein containing 50mM CaCl
2with 100mM NaCl) damping fluid, successively the chitosan microball after crosslinked is washed, damping fluid 100mM acetic acid-sodium acetate (pH 7.5, wherein containing 100mM NaCl) elution volume is the twice of chitosan microball volume, then damping fluid 50mM Tris-HCl(pH 7.5 is used, wherein containing 50mM CaCl
2with 100mM NaCl) washing, collect elutriant survey enzyme and live, until without resolvase in elutriant;
(4) in immobilized enzyme, reductive agent is added, the remaining reductive agent of removing after reaction: add NaBH in the chitosan microball securing heparinaseⅡ
41-5mg/ml, reaction 1-3h reduces, after completion of the reaction, with 25mM Tris-HCl(pH 7.0, wherein containing 10mM CaCl
2) be damping fluid, wash chitosan microball, damping fluid volume used is 3-5 times of chitosan microball volume.
Beneficial effect of the present invention:
By above-described summary of the invention, present invention achieves the immobilization of heparinaseⅡ and can reuse.The method can reach significant immobilization effect to heparinaseⅡ, and the chitosan microball of every milliliter can the heparinaseⅡ of immobilization 1.033 IU, and cross-linking efficiency can reach 60.0%, and immobilized heparinaseⅡ still keeps the activity of 43.4% after 1 week in preservation.
Embodiment
Further illustrate the present invention by the following examples, but not as limitation of the present invention.
Embodiment:
Heparinase used is that separation and purification obtains from the thalline after Flavobacterium heparinum fermentation culture, and enzyme preparation process is shown in the embodiment in application for a patent for invention number 200910039360.1 " a kind of preparation methods of heparin flavobacterium heparinase II ".
A, get the commercially available purity of 1g be 99% Chitosan powder be dissolved in the acetic acid of 50ml 2%, obtained acidic chitosan colloidal sol, by commercially available for 0.6g chemical pure Span 80(Chemical Reagent Co., Ltd., Sinopharm Group, chemical pure) add in 100ml whiteruss, be uniformly mixed with the rotating speed of 800rpm, acidic chitosan colloidal sol is dropwise added in whiteruss, after emulsification 1h, adding 5ml massfraction is that the glutaraldehyde of 25% is as linking agent, crosslinking temperature is 40 DEG C, isopyknic 2.5M NaOH of 100ml and the mixed solution of dehydrated alcohol is added after 1h, leave standstill after 10min after stirring, discard oil reservoir and water layer, with ultrapure water 5 times, discard water layer and namely obtain chitosan microball.
B, selection 25mM Tris-HCl(pH 7.0, wherein containing 10mM CaCl
2) damping fluid is as the solvent of heparinaseⅡ, obtaining and recording enzyme work by reddish black A method is 40.06 U/ml, measures the heparinaseⅡ solution that enzyme work is 1.72 IU/ml, get this heparinase liquid of 1ml and add in the pillar that 1ml chitosan microball is housed by 232nm method, shake up, crosslinked 15h in 10 DEG C of freezers.Discard clear liquid.
Chitosan microball after 100mM acetic acid-sodium acetate (pH 7.5, wherein containing 100mM NaCl) the above-mentioned cross-linked heparin enzyme of buffer solution of c, use 2ml, then use the 25mM Tris-HCl(pH 7.5 of about 10ml, wherein containing 50mM CaCl
2with 100mM NaCl) buffer solution above-mentioned crosslinked after chitosan microball, detect without enzyme work to elutriant.
D, at the above-mentioned an alkali metal salt NaBH securing the borohydride anion of the 1mg/ml adding 1ml in the chitosan microball of heparinaseⅡ
4, reaction 1h, with the 25mM Tris-HCl(pH 7.0 of 10ml, wherein containing 10mM CaCl
2) buffer solution, to remove remaining NaBH
4, being fixed enzyme 1ml.Recording activity of the immobilized enzyme by reddish black A method is 24.06 U/ml, and being scaled the international Mei Huo unit of heparinase (i.e. the enzyme of 232nm method mensuration value alive) is 1.03 IU/ml.The immobilization efficiency of heparinaseⅡ is 60.0%, and every 1ml chitosan microball can fix the heparinaseⅡ of 1.03 IU/ml.
E, above-mentioned immobilized enzyme is placed in the 25mM Tris-HCl(pH 7.5 of 2ml, wherein containing 10mM CaCl
2), temperature is preserve in the environment of 4-10 DEG C.Before the vigor of preservation immobilized enzyme after 1 week is preserve after measured 43.4%.
In an embodiment, the concrete grammar measuring enzyme activity is as follows:
1) 232nm method is surveyed enzyme and is lived: in 5ml quartz colorimetric utensil, be added in the 50mM Tris-HCl(pH 7.0 that 30 DEG C of preheated 2.5ml heparin concentrations are 1mg/ml, wherein containing 10mM CaCl
2) damping fluid, pipette 20 μ l enzyme liquid, after shaking up, measure light absorption value at 232nm, read the variable quantity of per minute.According to the mole number of the double bond that the molar extinction coefficient Units of Account time produces, and calculate the units activity (IU/ml) of enzyme liquid thus, the enzyme that the method records is lived as the international Mei Huo unit of heparinase.
2) reddish black A method survey enzyme is lived: get immobilized enzyme and add certain density heparin substrate, react 5min in 45 DEG C of water-baths, sampling, measure the concentration of residue heparin, measuring method is: by the heparin substrate 50mM Tris-HCl(pH 7.0 of unknown concentration, wherein containing 10mM CaCl
2) damping fluid dilution certain multiple, the concentration adding 2.5ml in 5ml glass cuvette is the reddish black solution A of 20mg/L, add the heparin solution after 25 μ l dilutions again, shake up the light absorption value at rear mensuration 620nm place, remaining heparin concentration after trying to achieve enzymolysis according to the typical curve of reddish black A method mensuration heparin concentration, be a Ge Meihuo unit with the enzyme amount needed for degraded 1mg heparin per hour, calculate the units activity (U/ml) of immobilized enzyme thus.
232nm method is the most universal method measuring heparinaseⅡ activity, can be used for the mensuration of unbound heparin enzymic activity, but when measuring for activity of the immobilized enzyme, microballoon brings very large interference and is difficult to use; Reddish black A method can be used for the mensuration of resolvase and Immobilized heparinase activity.By measuring respectively the reddish black A method of the unbound heparin enzyme of same concentrations and 232nm method in the present invention, obtain the conversion relation of two kinds of measuring methods, the enzyme that reddish black A method records numerical value alive is 23.3 times that 232nm method measures numerical value, converts to immobilized heparinaseⅡ enzyme international unit activity of carrying out alive with this.
Claims (8)
1. a process for fixation for heparinaseⅡ, comprises the steps:
(1) preparation of heparinaseⅡ solution: select Tris-HCl damping fluid as the solvent of heparinaseⅡ, the pH of damping fluid is 7.0, containing CaCl
2, heparinaseⅡ is dissolved in this damping fluid, obtained enzyme solution;
(2) heparinaseⅡ and chitosan microball is crosslinked: by fully swelling for chitosan microball Tris-HCl damping fluid, the pH of damping fluid is 7.0, containing 10mM CaCl
2, then get the heparinaseⅡ solution that step (1) obtains, added in this chitosan microball, be cross-linked;
(3) wash the chitosan microball after above-mentioned being cross-linked respectively with acetic acid-sodium acetate and Tris-HCl two kinds of damping fluids, to elutriant non-enzymatic activity, wherein acetic acid-sodium acetate pH 6.5-8.0, containing NaCl, Tris-HCl pH 6.5-8.0, containing CaCl
2and NaCl;
(4) utilize the chitosan microball securing heparinaseⅡ obtained in reductive agent treatment step (3), subsequently, the reductive agent that removing is remaining, is secured to the heparinaseⅡ on chitosan microball thus;
Wherein in above-mentioned steps (2), the preparation method of chitosan microball is as follows: get commercially available Chitosan powder and be dissolved in acetic acid, obtained acidic chitosan colloidal sol, dispersion agent is added in oil phase, then acidic chitosan colloidal sol is added in oil phase, after emulsification, add glutaraldehyde to be cross-linked, add the mixed solution of isopyknic NaOH and dehydrated alcohol after crosslinked, leave standstill after stirring, discard oil reservoir and water layer, and use ultrapure water repetitive scrubbing, obtain chitosan microball.
2. process for fixation according to claim 1, is characterized in that, is 10-50mM, CaCl for dissolving the concentration of the solvent Tris-HCl of heparinaseⅡ in step (1)
2concentration is 5-20mM.
3. process for fixation according to claim 2, is characterized in that, the Tris-HCl solvent used in step (1) is 25mM Tris-HCl, pH 7.0, containing 10mM CaCl
2.
4. according to the process for fixation of any one of claims 1 to 3, it is characterized in that, the chitosan microball used in step (2) and the volume ratio of heparinaseⅡ solution are 1/10-10/1.
5. process for fixation according to claim 1, is characterized in that, the acetic acid-sodium acetate buffer solution used in step (3) is 50-150mM acetic acid-sodium acetate buffer solution, and pH 6.5-8.0, containing 50-200mM NaCl.
6. process for fixation according to claim 1, is characterized in that, the Tris-HCl damping fluid used in step (3) is 50mM Tris-HCl damping fluid, and pH 6.5-8.0, containing 10-100mM CaCl
2, 50-200mM NaCl.
7. process for fixation according to claim 1, is characterized in that, the reductive agent used in step (4) is alkali metal borohydride.
8. process for fixation according to claim 1, is characterized in that, said method comprising the steps of:
(1a), the preparation of chitosan microball:
Getting Chitosan powder is dissolved in the acetic acid of 2%, and obtained acidic chitosan colloidal sol, adds in whiteruss by Span 80, high-speed stirring mixes, and is dropwise added in whiteruss by acidic chitosan colloidal sol, after emulsification 1h, add glutaraldehyde as linking agent, crosslinking temperature is 40 DEG C, and add the mixed solution of 2.5M NaOH and dehydrated alcohol after crosslinked, the volume ratio of NaOH and dehydrated alcohol is 1/1, leave standstill after stirring, discard oil reservoir and water layer, and use ultrapure water repetitive scrubbing, obtain chitosan microball;
(1) prepare damping fluid 25mM Tris-HCl, pH 7.0, containing 10mM CaCl
2, it can be used as the solvent of heparinaseⅡ, obtained heparinaseⅡ solution, adjustment concentration is to enzyme activity 1-3IU/ml;
(2) get the heparinaseⅡ solution that step (1) obtains, added in the chitosan microball obtained by the step (1a) of the abundant swelling mistake of 25mM Tris-HCl, Tris-HCl pH 7.0, containing 10mM CaCl
2, the volume ratio of chitosan and heparinase is 1/2-2/1,4-10 DEG C of crosslinked 12-20h;
(3) 100mM acetic acid-sodium acetate and 50mM Tris-HCl damping fluid is prepared, successively the chitosan microball after crosslinked is washed, damping fluid 100mM acetic acid-sodium acetate elution volume is the twice of chitosan microball volume, then with damping fluid 50mM Tris-HCl washing, collect elutriant survey enzyme and live, until without resolvase in elutriant, wherein acetic acid-sodium acetate pH 7.5, containing 100mM NaCl, Tris-HCl pH 7.5, containing 50mM CaCl
2, 100mM NaCl;
(4) in the chitosan microball securing heparinaseⅡ, NaBH is added
41-5mg/ml, reaction 1-3h, after completion of the reaction, with 50mM Tris-HCl for damping fluid, pH 7.0, containing 10mM CaCl
2, wash chitosan microball, damping fluid volume used is 3-5 times of chitosan microball volume, and the reductive agent that removing is remaining, is secured to the heparinaseⅡ on chitosan microball thus.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1405914A1 (en) * | 1994-06-10 | 2004-04-07 | Ibex Technologies | Nucleic acid sequences and expression systems for heparinase II and heparinase III derived from flavobacterium heparinum |
| CN101942024A (en) * | 2010-08-20 | 2011-01-12 | 清华大学 | Heparanase II fusion protein and coding gene and expression method thereof |
-
2012
- 2012-10-30 CN CN201210423324.7A patent/CN102888391B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1405914A1 (en) * | 1994-06-10 | 2004-04-07 | Ibex Technologies | Nucleic acid sequences and expression systems for heparinase II and heparinase III derived from flavobacterium heparinum |
| CN101942024A (en) * | 2010-08-20 | 2011-01-12 | 清华大学 | Heparanase II fusion protein and coding gene and expression method thereof |
Non-Patent Citations (3)
| Title |
|---|
| Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum;Xiaolai Ma et al.;《Journal of Chromatography B》;20060717;摘要 * |
| 固定化内切肝素酶的研究;钞亚鹏 等;《微生物学报》;20041231;第44卷(第5期);第190页第2.1-2.3节 * |
| 壳聚糖微球固定化脂肪酶的制备工艺及应用性质研究;孙培龙 等;《中国粮油学报》;20081231;第23卷(第4期);第689页摘要 * |
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Address after: 518057 Guangdong city of Shenzhen province Nanshan District Song Ping Lang Road No. 21 Patentee after: Shenzhen Hepalink Pharmaceutical Group Limited by Share Ltd Address before: 518057 Guangdong city of Shenzhen province Nanshan District Song Ping Lang Road No. 21 Patentee before: Shenzhen Hepalink Pharmaceutical Co., Ltd. |