CN103468669A - Alcohol dehydrogenase embedded gelatin-silica hybrid gel and preparation method thereof - Google Patents
Alcohol dehydrogenase embedded gelatin-silica hybrid gel and preparation method thereof Download PDFInfo
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- CN103468669A CN103468669A CN2013104254869A CN201310425486A CN103468669A CN 103468669 A CN103468669 A CN 103468669A CN 2013104254869 A CN2013104254869 A CN 2013104254869A CN 201310425486 A CN201310425486 A CN 201310425486A CN 103468669 A CN103468669 A CN 103468669A
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Abstract
The invention discloses an alcohol dehydrogenase embedded gelatin-silica hybrid gel and a preparation method thereof. According to invention, the grain diameter of the alcohol dehydrogenase embedded gelatin-silica hybrid gel is 3-4mm, and a silica coating is coated on a gelatin core. The preparation method comprises the following steps of: preparing a mixed solution of gelatin and alcohol dehydrogenase; preparing a mixed solution of sodium silicate and a glutaraldehyde solution precursor; adding the mixed solution of gelatin and alcohol dehydrogenase into deionized water at a temperature of 0-4 DEG C droplet by droplet to generate alcohol dehydrogenase embedded gelatin gel particles; stirring and incubating to obtain the alcohol dehydrogenase embedded gelatin-silica hybrid gel. The preparation method has the advantages that the preparation condition is mild, the preparation process is simple and feasible, and the alcohol dehydrogenase embedded gelatin-silica hybrid gel has a strong fixing capability; compared with gelatin gel, the hybrid gel has a lower leakage rate of immobilized alcohol dehydrogenase, and lower swelling degree and better recycling stability.
Description
Technical field
The present invention relates to a kind of gelatin of embedding alcoholdehydrogenase-silicon oxide hybrid gel and preparation method, belong to the immobilization technology of enzyme.
Background technology
Gelatin gel is a kind of fixed enzyme vector commonly used.At present, the gelatin gel immobilized enzyme mainly adopts entrapping method.The entrapping method mild condition, good biocompatibility, be conducive to maintain the structural integrity of enzyme, and then improve the active sustainment rate of enzyme, and carrier aperture is larger, is conducive to reactant and product transmission.
The problem that the gelatin gel immobilized enzyme exists usually is that the aperture of carrier is larger, and the enzyme molecule easily leaks.The easy swelling of gel, bad mechanical strength, recycle stability and stability in storage not high.The method of dealing with problems normally utilizes the bifunctional group linking agent to carry out crosslinkedly as glutaraldehyde etc., makes gel carrier finer and close, or constructs inorganic shell at the gel outside surface, prepares the hybrid inorganic-organic gel.
Silicon oxide is inorganic component common in hybrid inorganic-organic materials, can improve the mechanical property of gel for the preparation of hybridization carrier, the leakage of inhibitory enzyme molecule simultaneously, improve the tolerance that enzyme changes temperature, pH, improves and store and recycle stability simultaneously.The preparation that bionical silication is earth silicon material provides a new way, and bionical silicatization process has been avoided the use of acid base catalysator, and preparation is simple, and raw materials used all have a good biocompatibility, applied widely.
Utilizing the bionical silicatization process of gelatin to prepare the gelatin of embedding alcoholdehydrogenase-silicon oxide hybridization carrier has a good application prospect for immobilized enzyme.
Summary of the invention
The object of the present invention is to provide a kind of gelatin of embedding alcoholdehydrogenase-silicon oxide hybrid gel and preparation and method.With the gelatin of the prepared embedding alcoholdehydrogenase of the method-silicon oxide hybrid gel size homogeneous, method is simple, and the leakage rate of embedding alcoholdehydrogenase is low, and the carrier swelling capacity is low, and reusability is good.
The present invention is realized by following technical side, a kind of gelatin of embedding alcoholdehydrogenase-silicon oxide hybrid gel, it is characterized in that, the hybrid gel of this embedding alcoholdehydrogenase is particle diameter 3-4mm, wherein on gelatin core, be coated with the membranous layer of silicon oxide that thickness is 10-20 μ m, wherein one milliliter of hybrid gel embedding alcoholdehydrogenase content is 1～4mg.
The gelatin of the embedding alcoholdehydrogenase of said structure-silicon oxide hybrid gel preparation method is characterized in that comprising following process:
(1) the preparation mass concentration gelatin solution that is 15～25%, contain to adding alcoholdehydrogenase to be mixed with in this gelatin solution the mixing solutions that alcoholdehydrogenase concentration is 1～4mg/mL;
(2) the water glass precursor solution of compound concentration 40～80mM, the pH value of regulating sodium silicate solution with hydrochloric acid is 6.5～8, by the two volume ratio of sodium silicate solution and massfraction 50% glutaraldehyde solution, it is 1:0.1～0.25, glutaraldehyde solution is added to sodium silicate solution, obtain precursor mixed solution;
(3) with syringe, the mixing solutions of step (1) is dropwise joined in the deionized water of 0～4 ℃, generate embedding alcoholdehydrogenase gelatin gel particle, after filtration, use deionized water wash, gelatin particle is immersed in the precursor mixed solution that step (2) prepares, stir hatching and obtain the gelatin-silica gel of embedding alcoholdehydrogenase after 25～35 minutes.
The invention has the advantages that: the preparation condition gentleness, preparation is simple, it is strong that the hydridization gelatin-silica gel of gained embedding alcoholdehydrogenase has a crystallized ability, the leakage rate of comparing immobilization alcoholdehydrogenase enzyme with gelatin gel is low, and leakage rate of the present invention is 5%, and the gelatin gel leakage rate is 83.6%, compare swelling capacity of the present invention with gelatin gel low, swelling capacity is 7.9%, and the swelling capacity of gelatin gel is 120%, reuses good stability.
The accompanying drawing explanation
The embedding alcoholdehydrogenase gelatin that Fig. 1 is the embodiment of the present invention one preparation-silicon oxide hybrid gel particle surface scanning electron microscope (SEM) photo.
The embedding alcoholdehydrogenase gelatin that Fig. 2 is the embodiment of the present invention two preparations-silicon oxide hybrid gel particle profile scanning Electronic Speculum (SEM) photo.
The leakage rate variation diagram of the embedding alcoholdehydrogenase gelatin that Fig. 3 is the embodiment of the present invention four preparations-silicon oxide hybrid gel and gelatin gel.
The swelling capacity variation diagram of the embedding alcoholdehydrogenase gelatin that Fig. 4 is five preparations of the employing embodiment of the present invention-silicon oxide hybrid gel and gelatin gel.
The variation diagram of the vigor that recycles of the alcoholdehydrogenase that the embedding alcoholdehydrogenase gelatin that Fig. 5 is four preparations of the employing embodiment of the present invention-silicon oxide hybrid gel is fixing.
Embodiment
Embodiment mono-
The 706mg gelatin is dissolved in the deionized water of 4mL, be mixed with the gelatin solution that massfraction is 15%, upwards state clearly in sol solution and add the 4mg alcoholdehydrogenase, stir, mix, prepare the water glass precursor solution that 20 mL concentration are 40mM, the pH value of regulating sodium silicate solution with concentrated hydrochloric acid is 6.5, get massfraction 50% glutaraldehyde solution 40 μ L and add sodium silicate solution, obtain precursor mixed solution, then with syringe, the mixing solutions of gelatin and alcoholdehydrogenase dropwise is added drop-wise in 0 ℃ of deionized water, generate embedding alcoholdehydrogenase gelatin gel particle, after filtration, with after deionized water wash three times, embedding alcoholdehydrogenase gelatin gel particle is added and prepares to obtain the water glass precursor mixed solution, hatch 25 minutes, make embedding alcoholdehydrogenase gelatin-silicon oxide hybrid gel.
The gelatin that said process is made-silicon oxide hybrid gel is recycled the mensuration of stability:
Formaldehyde and reduced coenzyme Ⅰ disodium are added in 0.05mol/L Tutofusin tris-hydrochloric acid buffer solution, being made into concentration of formaldehyde is 10mM, reduced coenzyme Ⅰ two na concns are 133 μ M, the hybrid gel particle that adds the immobilization alcoholdehydrogenase of preparation, at 25 ℃, carry out the conversion reaction of formaldehyde under the condition stirred, measure the inversion quantity of reduced coenzyme Ⅰ disodium with ultraviolet spectrophotometer, the enzyme activity of being fixed alcoholdehydrogenase, and take this enzyme activity as initial enzyme activity, be defined as 100%.
By reacting liquid filtering, with the washed with de-ionized water particle to formaldehydeless and prototype cozymase disodium in supernatant liquor.Repeat above-mentioned reaction process, obtain the enzyme activity of reusable immobilization alcoholdehydrogenase for the second time, compare with initial vigor, enzyme activity now is 90.3%.
Repeated isolation and reaction process, obtaining the enzyme activity of reusable immobilization alcoholdehydrogenase for the third time is 84%; The enzyme activity of the 4th reusable immobilization alcoholdehydrogenase is 75.7%; The enzyme activity of the 5th reusable immobilization alcoholdehydrogenase is 66.5%; The enzyme activity of the 6th reusable immobilization alcoholdehydrogenase is 59.5%.
Embodiment bis-
The 1g gelatin is dissolved in the deionized water of 3mL, be mixed with the gelatin solution that massfraction is 25%, upwards state clearly in sol solution and add the 12mg alcoholdehydrogenase, stir, mix, prepare the water glass precursor solution that 20 mL concentration are 80mM, the pH value of regulating sodium silicate solution with concentrated hydrochloric acid is 8, get massfraction 50% glutaraldehyde solution 100 μ L glutaraldehyde and add sodium silicate solution, obtain precursor mixed solution, then with syringe, the mixing solutions of gelatin and alcoholdehydrogenase dropwise is added drop-wise in 4 ℃ of deionized waters, generate embedding alcoholdehydrogenase gelatin gel particle, after filtration, with after deionized water wash three times, embedding alcoholdehydrogenase gelatin gel particle is added and prepares to obtain the water glass precursor mixed solution, hatch 35 minutes, make embedding alcoholdehydrogenase gelatin-silicon oxide hybrid gel.
Embodiment tri-
The 1g gelatin is dissolved in the deionized water of 3mL, be mixed with the gelatin solution that massfraction is 25%, upwards state clearly in sol solution and add the 12mg alcoholdehydrogenase, making its concentration is 4mg/mL, stir, mix, prepare the water glass precursor solution that 20 mL concentration are 50mM, the pH value of regulating sodium silicate solution with concentrated hydrochloric acid is 7.0, getting massfraction 50% glutaraldehyde solution makes 40 μ L glutaraldehyde add sodium silicate solution, obtain precursor mixed solution, then with syringe, the mixing solutions of gelatin and alcoholdehydrogenase dropwise is added drop-wise in 4 ℃ of deionized waters, generate embedding alcoholdehydrogenase gelatin gel particle, after filtration, with after deionized water wash three times, embedding alcoholdehydrogenase gelatin gel particle is added and prepares to obtain the water glass precursor mixed solution, hatch 30 minutes, made the gelatin of embedding alcoholdehydrogenase-silicon oxide hybrid gel, it is in 7 Tutofusin triss-hydrochloric acid buffer solution that the gelatin of embedding alcoholdehydrogenase-silicon oxide hybrid gel is soaked in to pH, record solution in first three hour in without the leakage of alcoholdehydrogenase, leakage rate is 0, after ten hours, leakage rate is 5%.
Embodiment tetra-
The 706mg gelatin is dissolved in the deionized water of 4mL, be mixed with the gelatin solution that massfraction is 15%, upwards state clearly in sol solution and add the 4mg alcoholdehydrogenase, making its concentration is 1mg/mL, stir, mix, prepare the water glass precursor solution that 20 mL concentration are 50mM, the pH value of regulating sodium silicate solution with concentrated hydrochloric acid is 7.0, get massfraction 50% glutaraldehyde solution 40 μ L glutaraldehyde and add sodium silicate solution, obtain precursor mixed solution, then with syringe, the mixing solutions of gelatin and alcoholdehydrogenase dropwise is added drop-wise in 0 ℃ of deionized water, generate embedding alcoholdehydrogenase gelatin gel particle, after filtration, with after deionized water wash three times, embedding alcoholdehydrogenase gelatin gel particle is added and prepares to obtain the water glass precursor mixed solution, hatch 30 minutes, made the gelatin of embedding alcoholdehydrogenase-silicon oxide hybrid gel, this hybrid gel is soaked in the buffered soln that pH is 7 Tutofusin triss-hydrochloric acid, under room temperature, deposit 30 hours, weighing hybrid gel granular mass changes, calculating swelling capacity is 7.9%.
Comparative Examples one
The 1g gelatin is dissolved in the deionized water of 3mL, be mixed with the gelatin solution that massfraction is 25%, upwards state clearly in sol solution and add the 12mg alcoholdehydrogenase, making its concentration is 4mg/mL, stir, mix, then syringe dropwise is added drop-wise to gelatin and alcoholdehydrogenase mixing solutions in 4 ℃ of deionized waters, generate embedding alcoholdehydrogenase gelatin gel particle, filter, with after deionized water wash three times, it is 5h in 7 Tutofusin triss-hydrochloric acid buffer solution that the gel particle of embedding alcoholdehydrogenase is soaked in to pH, measure alcoholdehydrogenase content in solution, obtaining gelatin gel, to deposit the leakage rate of alcoholdehydrogenase in process be 83.6%
Comparative Examples two
The 706mg gelatin is dissolved in the deionized water of 4mL, be mixed with the gelatin solution that massfraction is 15%, upwards state clearly in sol solution and add the 4mg alcoholdehydrogenase, making its concentration is 1mg/mL, stir, mix, then syringe dropwise is added drop-wise to gelatin and alcoholdehydrogenase mixing solutions in 0 ℃ of deionized water, generate embedding alcoholdehydrogenase gelatin gel particle, filter, with after deionized water wash three times, it is 7 Tutofusin triss-hydrochloric acid buffer solution that the gel particle of embedding alcoholdehydrogenase is soaked in to pH, under room temperature, deposit 30 hours, weighing gelatin gel granular mass changes, calculating swelling capacity is 120%.
Claims (2)
1. the gelatin of an embedding alcoholdehydrogenase-silicon oxide hybrid gel, it is characterized in that, the hybrid gel of this embedding alcoholdehydrogenase is particle diameter 3-4mm, wherein on gelatin core, is coated with the membranous layer of silicon oxide that thickness is 10-20 μ m, and wherein one milliliter of hybrid gel embedding alcoholdehydrogenase content is 1～4mg.
2. one kind by the gelatin of embedding alcoholdehydrogenase claimed in claim 1-silicon oxide hybrid gel preparation method, it is characterized in that comprising following process:
(1) the preparation mass concentration gelatin solution that is 15～25%, contain to adding alcoholdehydrogenase to be mixed with in this gelatin solution the mixing solutions that alcoholdehydrogenase concentration is 1～4mg/mL;
(2) the water glass precursor solution of compound concentration 40～80mM, the pH value of regulating sodium silicate solution with hydrochloric acid is 6.5～8, by the two volume ratio of sodium silicate solution and massfraction 50% glutaraldehyde solution, it is 1:0.1～0.25, glutaraldehyde solution is added to sodium silicate solution, obtain precursor mixed solution;
(3) with syringe, the mixing solutions of step (1) is dropwise joined in the deionized water of 0～4 ℃, generate embedding alcoholdehydrogenase gelatin gel particle, after filtration, use deionized water wash, gelatin particle is immersed in the precursor mixed solution that step (2) prepares, stir hatching obtains embedding alcoholdehydrogenase gelatin after 25～35 minutes-silicon oxide hybrid gel.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762289A (en) * | 2015-04-29 | 2015-07-08 | 天津科技大学 | Preparing method for gelatin microsphere of fixed alcohol dehydrogenase by micro-porous membrane permeation and emulsification |
| CN111333200A (en) * | 2020-03-18 | 2020-06-26 | 运城学院 | Embedded immobilized microorganism particles, preparation method and sewage treatment method |
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| CN101974510A (en) * | 2010-11-09 | 2011-02-16 | 厦门大学 | Method for carrying out coupling immobilization on coenzyme and coenzyme dependent enzyme |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101974510A (en) * | 2010-11-09 | 2011-02-16 | 厦门大学 | Method for carrying out coupling immobilization on coenzyme and coenzyme dependent enzyme |
Non-Patent Citations (4)
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| 姜燕: "仿生卡拉胶-二氧化硅杂化凝胶固定化醇脱氢酶", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762289A (en) * | 2015-04-29 | 2015-07-08 | 天津科技大学 | Preparing method for gelatin microsphere of fixed alcohol dehydrogenase by micro-porous membrane permeation and emulsification |
| CN104762289B (en) * | 2015-04-29 | 2018-09-21 | 天津科技大学 | The method that microporous membrane permeation emulsification prepares the gelatine microsphere of fixed alcohol dehydrogenase |
| CN111333200A (en) * | 2020-03-18 | 2020-06-26 | 运城学院 | Embedded immobilized microorganism particles, preparation method and sewage treatment method |
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Application publication date: 20131225 |