CN100554417C - The method of Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase - Google Patents
The method of Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase Download PDFInfo
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- CN100554417C CN100554417C CNB2007100603598A CN200710060359A CN100554417C CN 100554417 C CN100554417 C CN 100554417C CN B2007100603598 A CNB2007100603598 A CN B2007100603598A CN 200710060359 A CN200710060359 A CN 200710060359A CN 100554417 C CN100554417 C CN 100554417C
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Abstract
The present invention relates to the method for a kind of Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase.Mix under sodium carbonate solution and the calcium chloride solution room temperature to stir and generate the calcium carbonate powders precipitation, leave standstill, water and washing with acetone, dry, obtain in Tutofusin tris-hydrochloric acid (Tirs-HCl) damping fluid that micron order mesopore calcium carbonate granule joins the beta-glucuronidase enzyme or rise in the aqueous solution adsorbing, centrifugation, the calcium carbonate granule and the sodium alginate soln that obtain adsorbing glucuronidase mix, be added drop-wise to again in the calcium chloride solution, aging.Preparation condition gentleness of the present invention, preparation is simple, and the gained hybridization carrier has good crystallized ability to enzyme, and the leakage rate of immobilization beta-glucuronidase is low, and swelling capacity is low, reuses good stability.
Description
Technical field
The present invention relates to the immobilization technology of enzyme, the method for particularly a kind of Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase.
Background technology
The Lalgine gel is a kind of fixed enzyme vector commonly used.At present, the gel immobilized enzyme of Lalgine mainly adopts entrapping method.The entrapping method mild condition, good biocompatibility helps keeping the structural integrity of enzyme, and then improves the active sustainment rate of enzyme, and carrier aperture is bigger, helps reactant and product transmission.
The problem that the gel immobilized enzyme of Lalgine exists usually is that the aperture of carrier is bigger, and the enzyme molecule leaks easily.Have " synersis " phenomenon in the gel formation process, a part of enzyme can be discharged carrier with water.The easy swelling of gel, bad mechanical strength, it is not high to recycle stability and stability in storage.It is crosslinked that the method for dealing with problems normally utilizes bifunctional group linking agent such as glutaraldehyde etc. to carry out, and makes gel carrier fine and close more, or utilize inorganic particulate preadsorption enzyme molecule, carries out embedding then, preparation hybrid inorganic-organic fixation support.
The most frequently used sorbent material when mesoporous silica material is immobilized enzyme studies show that, although this class mesopore material has bigger specific surface area and pore volume, but general aperture is less, and the enzyme molecule effect of absorption large-size is bad.Because synthetic method is limit, general mesoporous silica is all electronegative, has electrostatic repulsion with electronegative enzyme molecule, influences adsorption effect in addition.Mesopore lime carbonate has bigger aperture and has positive electricity, and preparation simultaneously is simple, and material is cheap and easy to get, and what be fit to immobilization and with the glucuronidase be representative has large-size and an electronegative enzyme, therefore is with a wide range of applications in the immobilized enzyme field.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing Lalgine one calcium carbonate hybrid gel fixing Beta-glucuronidase.With the prepared fixing Beta of this method-glucuronidase hybridization carrier, the leakage rate of beta-glucuronidase enzyme is low, and the carrier swelling capacity is low, and reusability is good.
The method of a kind of Lalgine provided by the invention-calcium carbonate hybrid gel fixing Beta-glucuronidase may further comprise the steps:
(1) mix stirring under isocyatic sodium carbonate solution of equal-volume and the calcium chloride solution room temperature, leave standstill, calcium carbonate powders precipitates water and washing with acetone successively, and seasoning in the air obtains micron order mesopore calcium carbonate granule;
(2) in Tutofusin tris-hydrochloric acid (Tirs-HCl) damping fluid of beta-glucuronidase enzyme or rise in the aqueous solution and add calcium carbonate granule, to adsorb under the room temperature, centrifugation obtains adsorbing the calcium carbonate granule of glucuronidase;
(3) sodium alginate soln mixes with the calcium carbonate granule of the absorption glucuronidase that makes, and is added drop-wise in the calcium chloride solution again, and is aging, obtains the particle of Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase.
The method of Lalgine provided by the invention-calcium carbonate hybrid gel fixing Beta-glucuronidase may further comprise the steps:
(1) pours the sodium carbonate solution of 100ml 0.33M into equal-volume isocyatic calcium chloride solution rapidly, high-speed stirring 30s under the room temperature, stop then stirring, left standstill 15 minutes, the calcium carbonate powders of gained is centrifugal, use deionized water and washing with acetone successively, seasoning in the air obtains micron order mesopore calcium carbonate granule;
(2) the beta-glucuronidase enzyme is dissolved in Tutofusin tris-hydrochloric acid that concentration is 0.05mol/L (Tirs-HCl) damping fluid or in the deionized water, being mixed with concentration is the beta-glucuronidase enzyme solution of 0.5mg/mL, then the 15mg calcium carbonate granule is mixed with 1ml beta-glucuronidase enzyme solution, adsorb 2h under the room temperature, centrifugation obtains adsorbing the calcium carbonate granule of glucuronidase;
(3) sodium alginate is dissolved in deionized water and makes 2.0% solution; the calcium carbonate granule of getting 5ml and the absorption glucuronidase that makes according to step (2) mixes; be added drop-wise to syringe in the calcium chloride solution of 0.2M; aging 30min obtains the particle of Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase.
The preparation method's that the present invention proposes advantage is: the preparation condition gentleness, and preparation is simple, and the gained hybridization carrier has good crystallized ability to enzyme, and the leakage rate of immobilization beta-glucuronidase is low, and swelling capacity is low, reuses good stability.
Description of drawings
Fig. 1 is particulate scanning electron microscope (SEM) photo of the Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase of embodiment 1 preparation.
Fig. 2 is the Lalgine-calcium carbonate hybrid gel of embodiment two preparations and the swelling capacity variation diagram of the blank gel of Lalgine.
Fig. 3 is the variation diagram that recycles enzyme activity of the Lalgine-calcium carbonate hybrid gel fixed beta-glucuronidase enzyme of embodiment 1 preparation.
Embodiment
Embodiment 1
Accurately take by weighing beta-glucuronidase enzyme 2.5mg, be dissolved in 0.05mol/L Tutofusin tris-hydrochloric acid (Tris-HCl) buffered soln, be settled to 5mL, obtain 0.5mg/mL beta-glucuronidase enzyme liquid.Then the 15mg calcium carbonate granule is mixed absorption 2h, centrifugation, the calcium carbonate granule of acquisition absorption glucuronidase with 1ml 0.5mg/mL beta-glucuronidase enzyme liquid.Sodium alginate is dissolved in deionized water makes 2.0% solution; the calcium carbonate granule of getting 5ml sodium alginate soln and 15mg absorption glucuronidase mixes; be added drop-wise to syringe in the calcium chloride solution of 0.2M; leave standstill 30min; obtain the particle of Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase; measure the glucuronidase content in the calcium chloride solution; obtain glucuronidase leakage rate in the gel formation process and be 9.4%. the hybrid gel particle of fixation glucose aldehyde neuraminidase is soaked in 10h in Tutofusin tris-hydrochloric acid (Tris-HCl) buffered soln; record that no glucuronidase leaks in the solution, obtaining gel, to deposit the leakage rate of glucuronidase in the process be 0.
Embodiment 2
Pour the sodium carbonate solution of 100ml 0.33M into equal-volume isocyatic calcium chloride solution rapidly, high-speed stirring 30s under the room temperature, stop then stirring, left standstill 15 minutes, after the centrifugation, the solid carbonic acid calcium of gained is used deionized water and washing with acetone successively, and seasoning in the air obtains micron order mesopore calcium carbonate granule; Sodium alginate is dissolved in deionized water makes 2.0% solution, get the 5ml sodium alginate soln and the 15mg calcium carbonate granule mixes, be added drop-wise to syringe in the calcium chloride solution of 0.2M, aging 30min obtains Lalgine-calcium carbonate hybrid gel particle.Baicalin and sodium sulphite anhydrous 99.3 are dissolved in the 0.03mol/L Tris-HCl buffered soln, being made into baicalin concentration is 0.09mol/L, sodium sulphite anhydrous 99.3 concentration is the solution of 0.1%w/v, add the hybrid gel particle, deposit 20h under the room temperature, weighing hybrid gel granular mass changes, and obtaining swelling capacity is 20%.
Embodiment 3: the mensuration that recycles stability
The hybrid gel particulate of the fixing Beta-glucuronidase of the embodiment of the invention 1 preparation is recycled stability to be measured:
Baicalin and sodium sulphite anhydrous 99.3 are dissolved in the 0.03mol/L Tris-HCl buffered soln, being made into baicalin concentration is 0.09mol/L, sodium sulphite anhydrous 99.3 concentration is the solution of 0.1%w/v, the particle that adds the immobilization beta-glucuronidase of embodiment 1 preparation, at 37 ℃, carry out the conversion reaction of baicalin under the stirring condition, determine the growing amount of scutellarin with high performance liquid chromatography, the enzyme activity of being fixed beta-glucuronidase enzyme, and be initial enzyme activity with this enzyme activity, be defined as 100%.
With reacting liquid filtering, with washed with de-ionized water particle no baicalin and scutellarin to the supernatant liquor.Repeat above-mentioned reaction process, obtain the enzyme activity of reusable immobilization beta-glucuronidase for the second time, compare with initial vigor, the relative enzyme activity of this moment is 93%.
Repeated isolation and reaction process, obtaining for the third time, the relative enzyme activity of reusable immobilization beta-glucuronidase is 90%; The relative enzyme activity of the 4th reusable immobilization beta-glucuronidase is 87%; The relative enzyme activity of the 5th reusable immobilization beta-glucuronidase is 85%; The relative enzyme activity of the 6th reusable immobilization beta-glucuronidase is that the relative enzyme activity of 80%, the seven reusable immobilization beta-glucuronidase is 80%.
Comparative Examples 1
Accurately take by weighing beta-glucuronidase enzyme 2.5mg, be dissolved in 0.05mol/L Tutofusin tris-hydrochloric acid (Tris-HCl) buffered soln, be settled to 5mL, obtain 0.5mg/mL beta-glucuronidase enzyme liquid.Sodium alginate is dissolved in deionized water makes 2.5% solution, get the 4ml sodium alginate soln and 1ml glucuronidase solution mixes, be added drop-wise to syringe in the calcium chloride solution of 0.2M, aging 30min, be fixed the blank Lalgine gel particle of beta-glucuronidase enzyme, measure the glucuronidase content in the calcium chloride solution, obtain glucuronidase leakage rate in the gel formation process and be 21.8%. the hybrid gel particle of fixation glucose aldehyde neuraminidase is soaked in 10h in Tutofusin tris-hydrochloric acid (Tris-HCl) buffered soln, measure glucuronidase content in the solution, obtaining gel, to deposit the leakage rate of glucuronidase in the process be 6.2%.
Comparative Examples 2
Sodium alginate is dissolved in deionized water makes 2.0% solution, be added drop-wise to syringe in the calcium chloride solution of 0.2M, aging 30min, deionized water wash obtains blank Lalgine gel particle after the filtration.Baicalin and sodium sulphite anhydrous 99.3 are dissolved in the 0.03mol/L Tris-HCl buffered soln, being made into baicalin concentration is 0.09mol/L, sodium sulphite anhydrous 99.3 concentration is the solution of 0.1%w/v, add blank Lalgine gel particle, deposit 20h under the room temperature, weighing hybrid gel granular mass changes, and obtaining swelling capacity is 130%.
Comparative Examples 3: the mensuration that recycles stability
The stability that recycles to the blank Lalgine gel particle of the fixing Beta-glucuronidase of Comparative Examples 1 preparation is measured:
Baicalin and sodium sulphite anhydrous 99.3 are dissolved in the 0.03mol/L Tris-HCl buffered soln, being made into baicalin concentration is 0.09mol/L, sodium sulphite anhydrous 99.3 concentration is the solution of 0.1%w/v, the blank Lalgine gel particle that adds the immobilization beta-glucuronidase of Comparative Examples 1 preparation, at 37 ℃, carry out the conversion reaction of baicalin under the stirring condition, determine the growing amount of scutellarin with high performance liquid chromatography, the enzyme activity of being fixed beta-glucuronidase enzyme, and be initial enzyme activity with this enzyme activity, be defined as 100%.
With reacting liquid filtering, with washed with de-ionized water particle no baicalin and scutellarin to the supernatant liquor.Repeat above-mentioned reaction process, obtain the enzyme activity of reusable immobilization beta-glucuronidase for the second time, compare with initial vigor, the relative enzyme activity of this moment is 84%.
Repeated isolation and reaction process, obtaining for the third time, the relative enzyme activity of reusable immobilization beta-glucuronidase is 75%; The relative enzyme activity of the 4th reusable immobilization beta-glucuronidase is 73%; The relative enzyme activity of the 5th reusable immobilization beta-glucuronidase is 72%; The relative enzyme activity of the 6th reusable immobilization beta-glucuronidase is that the relative enzyme activity of 46% 7th reusable immobilization beta-glucuronidase is 39%.
Table 1 is depicted as Lalgine-calcium carbonate hybrid carrier that embodiment 1 and Comparative Examples 1 measure and the blank Lalgine carrier leakage rate comparing result at immobilization beta-glucuronidase.
Table 1
| Example | Glucuronidase leakage rate (%) in the gel formation process | Gel is deposited glucuronidase leakage rate (%) in the process | The total leakage rate of glucuronidase (%) |
| Embodiment 1 | 9.4 | 0 | 9.4 |
| Comparative Examples 1 | 21.8 | 6.2 | 28.0 |
Table 2 is depicted as embodiment 2 and the Lalgine-calcium carbonate hybrid carrier of Comparative Examples 2 mensuration and the swelling capacity comparing result of blank Lalgine carrier.
Table 2
| Example | Swelling capacity |
| Embodiment 2 | 20％ |
| Comparative Examples 2 | 130％ |
Table 3 is depicted as the comparing result that recycling of Lalgine-calcium carbonate hybrid carrier that embodiment three and Comparative Examples three measure and the carrier immobilized glucuronidase of blank Lalgine is used for the conversion reaction of baicalin for seven times
Table 3
| Example | For the first time | For the second time | For the third time | The 4th time | The 5th time | The 6th time | The 7th time |
| Embodiment 3 | 100％ | 93％ | 90％ | 87％ | 85％ | 80％ | 80％ |
| Comparative Examples 3 | 100％ | 84％ | 75％ | 73％ | 72％ | 46％ | 39％ |
From comparing result as seen, Lalgine-calcium carbonate hybrid gel immobilization beta-glucuronidase leakage rate is low, the carrier swelling capacity is low. and circulate when being used for the conversion reaction of baicalin for seven times, each reacting phase all is higher than blank calcium alginate gel immobilization beta-glucuronidase to enzyme activity.
Claims (2)
1, the method for a kind of Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase is characterized in that may further comprise the steps:
(1) pours the sodium carbonate solution of 100ml 0.33M into equal-volume isocyatic calcium chloride solution rapidly, high-speed stirring 30s under the room temperature, stop then stirring, left standstill 15 minutes, the calcium carbonate powders of gained is centrifugal, use deionized water and washing with acetone successively, seasoning in the air obtains micron order mesopore calcium carbonate granule;
(2) the beta-glucuronidase enzyme is dissolved in the tris-HCI buffer that concentration is 0.05mol/L or in the deionized water, being mixed with concentration is the beta-glucuronidase enzyme solution of 0.5mg/mL, then the 15mg calcium carbonate granule is mixed with 1ml beta-glucuronidase enzyme solution, adsorb 2h under the room temperature, centrifugation obtains adsorbing the calcium carbonate granule of glucuronidase;
(3) sodium alginate is dissolved in deionized water and makes 2.0% solution, the calcium carbonate granule of getting 5ml and the absorption glucuronidase that makes according to step (2) mixes, and is added drop-wise in the calcium chloride solution of 0.2M aging 30min with syringe.
2, the described method of claim 1 obtains the particle of Lalgine-calcium carbonate hybrid gel fixing Beta-glucuronidase.
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| CN113527579B (en) * | 2021-07-14 | 2022-02-22 | 暨南大学 | Polymer nano gel particle/calcium carbonate composite material and preparation method thereof |
| CN113750039B (en) * | 2021-10-26 | 2023-08-18 | 浙江中医药大学 | Baicalin-calcium alginate temperature-sensitive nanogel, preparation method and application thereof |
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Non-Patent Citations (7)
| Title |
|---|
| Calcium phosphate-alginate microspheres as enzyme deliverymatrices. C. C. Ribeiro et. al.Biomaterials,Vol.25 No.18. 2004 |
| Calcium phosphate-alginate microspheres as enzyme deliverymatrices. C. C. Ribeiro et. al.Biomaterials,Vol.25 No.18. 2004 * |
| Encapsulation of β-Glucuronidase in Biomimetic AlginateCapsules for Bioconversion of Baicalin to Baicalein. Zhongyi Jiang et.al.Industrial & Engineering Chemistry Research,Vol.46 No.7. 2007 |
| Encapsulation of β-Glucuronidase in Biomimetic AlginateCapsules for Bioconversion of Baicalin to Baicalein. Zhongyi Jiang et.al.Industrial & * |
| Engineering Chemistry Research,Vol.46 No.7. 2007 * |
| Protein encapsulation via porous CaCO3 microparticlestemplating. Dmitry V. Volodkin et.al.Biomacromolecules,Vol.5 No.5. 2004 |
| Protein encapsulation via porous CaCO3 microparticlestemplating. Dmitry V. Volodkin et.al.Biomacromolecules,Vol.5 No.5. 2004 * |
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