CN103952392A - High-performance polymer microsphere for biologic enzyme immobilization and preparation method of high-performance polymer microsphere - Google Patents
High-performance polymer microsphere for biologic enzyme immobilization and preparation method of high-performance polymer microsphere Download PDFInfo
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- CN103952392A CN103952392A CN201410210936.7A CN201410210936A CN103952392A CN 103952392 A CN103952392 A CN 103952392A CN 201410210936 A CN201410210936 A CN 201410210936A CN 103952392 A CN103952392 A CN 103952392A
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Abstract
The invention discloses a high-performance polymer microsphere for biologic enzyme immobilization and a preparation method of the of high-performance polymer microsphere, belonging to the field of biotechnology. Specifically, N, N'-methylene-bisacrylamide cross-linking agent is partially substituted by a polyfunctional polymer prepolymer having a long-branched chain structure as an active center, porous polymeric microspheres containing epoxy groups, which has high mechanical strength and a macroporous structure, is prepared by reverse phase suspension polymerization techniques, the immobilization time is shortened from 72 hours to 12 hours, the activity and operation stability of the immobilized enzyme are improved, and good prospects are achieved.
Description
Technical field
The present invention relates to a kind of high performance polymer microballoon for bio-enzyme immobilization and preparation method thereof, belong to biological technical field.Specifically, adopt inverse suspension polymerization technology to prepare the porous polymer microsphere of the surface that physical strength is high and the enzyme immobilization time is short containing epoxide group.
Background technology
Penicillin acylase (E.C.3.5.1.11, Penicillin G acylase, be called for short PGA) be the enzyme of most critical during semi-synthetic β-lactam antibitics is produced, it can catalysis penicillin and ring expansion acid hydrolysis remove side chain, produce important intermediate 6-amino-penicillanic acid (6-APA) and the 7-ADCA (7-ADCA) of semi-synthetic β-lactam antibitics, again can catalysis 6-APA and 7-ADCA and side chain condensation, produce multiple semi-synthetic β-lactam antibitics (as Ampicillin, Amoxicillin, Cephalexin and Cefadroxil etc.).But directly be there are to many deficiencies for catalyzed reaction in resolvase, as unstable in high temperature, strong acid and strong base and organic solvent, easily loss of catalytic activity; Resolvase reclaims difficulty, unreasonable economically, also causes product to be difficult to separating-purifying, has a strong impact on quality product; Production process is difficult to realize operate continuously, can only disposable periodical operation etc.Immobilized enzyme has overcome the above-mentioned deficiency of resolvase, has not only kept the distinctive catalysis characteristics of resolvase, has also improved operational stability, production process is easy to realize operate continuously, after having reacted, be easy to product separation and can reuse, the product purity of gained is high, and production cost is low.Therefore, the immobilization of enzyme is the study hotspot in the fields such as catalytic chemistry, biological chemistry and materials chemistry always, and wherein the synthetic technology of high performance fixation support is crucial.
Containing the polymer support of epoxide group, the epoxide group of carrier surface at ambient temperature open loop reacts formation covalent linkage with the amido in biological enzyme molecule, thereby under mild conditions, realizes the immobilization of biological enzyme.Thisly make biological enzyme be fixed on carrier surface by means of epoxide group, not only operational condition gentleness is easy, and immobilization process is also very little to the enzyme loss of living, thereby, be very suitable for the suitability for industrialized production of immobilized enzyme.
We (Chinese invention patent CN1408859A) synthesized a series of polymer supports containing epoxide group for immobilized penicillin amidase by inverse suspension polymerization technology; but in polymerization process taking glycidyl methacrylate as functional monomer; taking Span-60 and calcium stearate as composite dispersing agent, exist polymer support calcium stearate in last handling process and be difficult for being washed and remove and reusing in process the easily shortcoming such as broken of carrier.Then we (catalysis journal, 2010,31 (5): 586-590) taking Span-60 and Tween-20 as composite dispersing agent, substitute original calcium stearate composite dispersing agent, reduce required time and solvent load in last handling process; Substitute the glycidyl methacrylate of rigidity simultaneously by the good glycidyl allyl ether part of Chain Flexibility, improve the physical strength of polymer support.But the synthesis condition of polymer support needs to continue to optimize, and further improves pore structure and the physical strength of polymer support, shortens the enzyme immobilization time, improves the over-all properties of immobilized enzyme.
Summary of the invention
The present invention seeks to the defect in order to overcome prior art existence and provide a kind of physical strength height and short surface of enzyme immobilization time to contain the preparation method of the porous polymer microsphere of epoxide group, make the immobilized penicillin acylated enzyme of preparing taking this polymer microballoon as carrier there is higher activity, operational stability and mechanical stability.
The described porous polymer microsphere containing epoxide group, adopts improved inverse suspension polymerization technology to be prepared, and specifically comprises the steps:
By the polyfunctionality polymkeric substance performed polymer with long branched chain structure, N, N '-methylene-bisacrylamide, Methacrylamide, the polymer monomer such as glycidyl methacrylate and glycidyl allyl ether and Diisopropyl azodicarboxylate initiator join in formamide soln, then join in the mixed solvent being formed by normal heptane and zellon that is dissolved with Span60 and Tween20 composite dispersing agent, under nitrogen protection, carry out inverse suspension polymerization reaction 3 ~ 6 hours in 45 ~ 65 DEG C, reaction finishes rear by washing with alcohol, normal heptane soaks, 50 ~ 70 DEG C of vacuum-drying 8 ~ 16 hours, obtain polymer microballoon.
The described polyfunctionality polymkeric substance performed polymer with long branched chain structure is a kind of of polyether acrylate performed polymer (TriMethylolPropane(TMP)-oxyethane-acrylic copolymer performed polymer) and polyurethane acrylate prepolymer (dipentaerythritol-different sym.-diisopropylideneacetone Er Yi Qing Suan Zhi – Hydroxyethyl acrylate multipolymer performed polymer) or two kinds, polyfunctionality polymkeric substance performed polymer and N, the weight ratio of N '-methylene-bisacrylamide is 0.05 ~ 0.3.
The described porous polymer microsphere containing epoxide group, can be used for the immobilization of the water-soluble biological enzymes such as penicillin acylase, glucose isomerase, glucose transglucosidase, trypsinase or amylase, is specially adapted to the immobilization of penicillin acylase.
The immobilization of penicillin acylase: take 0.30 gram of polymer microballoon and be placed in 25 milliliters of Erlenmeyer flasks; add 0.93 ml penicillin acylase solution and be diluted to 5 milliliters with the phosphate buffered saline buffer of the 1mol/L of pH=7.5; in the shaking bath of 28 DEG C, fix after 12 hours, use the phosphate buffered saline buffer washing suction filtration of the 0.1mol/L of pH=7.5.
The activity of immobilized penicillin acylated enzyme and operational stability measuring method adopt the measuring method of document (catalysis journal, 2010,31 (5): 586-590) report.
Key of the present invention is to use with the polyfunctionality polymkeric substance performed polymer of long branched chain structure as active centre, part replaces original N, N '-methylene-bisacrylamide linking agent, obtain the porous polymer microsphere containing epoxide group of high mechanical strength and macroporous structure, the immobilization time was shortened to 12 hours from 72 hours, improved activity and the operational stability of immobilized enzyme.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail, but never limit the scope of the invention.
Comparative example
After 90 milliliters of normal heptanes are mixed with 30 milliliters of zellons, add in the reactor that thermometer, prolong, airway and stirring rake are housed, add wherein after 0.29 gram of Span-60 and 0.78 gram of Tween-20, be heated to 55 DEG C.Under nitrogen protection and violent stirring; add again 17.5 milliliters to be dissolved with 3.9 grams of N, the formamide soln of N '-methylene-bisacrylamide, 0.54 gram of Methacrylamide, 1.35 milliliters of glycidyl methacrylate, 1.35 milliliters of glycidyl allyl ethers and 0.55 gram of Diisopropyl azodicarboxylate initiator.55 DEG C of polyreactions after 4 hours, stop stirring, be cooled to room temperature, sieve by 200 order sub-sieves, obtain polymer microballoon, prepared polymer microballoon can use after 12 hours through 48 hours, 60 DEG C vacuum-dryings of 100 milliliters of alcohol immersion 48 hours, 100 milliliters normal heptane washing by soaking of washing successively.Immobilization by above-mentioned polymer microballoon for penicillin acylase; the immobilization time is 72 hours; the immobilized enzyme initial activity obtaining is 320U/g (wetting); after 10 times recycle; immobilized enzyme has retained 90% initial activity, but it is just most of broken in the phosphate buffer soln of pH=8.0, to stir 6 hours polymer microballoons with the rotating speed of 280rpm.
Embodiment 1
After 90 milliliters of normal heptanes are mixed with 30 milliliters of zellons, add in the reactor that thermometer, prolong, airway and stirring rake are housed, add wherein after 0.29 gram of Span-60 and 0.78 gram of Tween-20, be heated to 55 DEG C.Under nitrogen protection and violent stirring; add again 17.5 milliliters to be dissolved with 0.3 gram of polyether acrylate performed polymer (TriMethylolPropane(TMP)-oxyethane-acrylic copolymer performed polymer), 3.6 grams of N, the formamide soln of N '-methylene-bisacrylamide, 0.54 gram of Methacrylamide, 1.35 milliliters of glycidyl methacrylate, 1.35 milliliters of glycidyl allyl ethers and 0.55 gram of Diisopropyl azodicarboxylate initiator.55 DEG C of polyreactions after 4 hours, stop stirring, be cooled to room temperature, sieve by 200 order sub-sieves, obtain polymer microballoon, prepared polymer microballoon can use after 12 hours through 1 hour, 60 DEG C vacuum-dryings of 1 hour, 100 milliliters normal heptane agitator treatings of 100 milliliters of ethanol agitator treatings successively.Immobilization by above-mentioned polymer microballoon for penicillin acylase; the immobilization time is 12 hours; the immobilized enzyme initial activity obtaining is 346U/g (wetting); after 10 times recycle; immobilized enzyme has retained 100% initial activity, just starts fragmentation but stir 12 hours polymer microballoons with the rotating speed of 280rpm in the phosphate buffer soln of pH=8.0.
Embodiment 2
Change 0.3 gram of polyether acrylate performed polymer into 0.2 gram, N, 3.6 grams of N '-methylene-bisacrylamides change 3.7 grams into, and the synthesis condition of other polymer microballoons is identical with embodiment 1.Immobilization by synthetic polymer microballoon for penicillin acylase; the immobilization time is 12 hours; the immobilized enzyme initial activity obtaining is 337U/g (wetting); after 10 times recycle; immobilized enzyme has retained 100% initial activity, just starts fragmentation but stir 10 hours polymer microballoons with the rotating speed of 280rpm in the phosphate buffer soln of pH=8.0.
Embodiment 3
Change 0.3 gram of polyether acrylate performed polymer into 0.8 gram, N, 3.6 grams of N '-methylene-bisacrylamides change 3.1 grams into, and the synthesis condition of other polymer microballoons is identical with embodiment 1.Immobilization by synthetic polymer microballoon for penicillin acylase; the immobilization time is 12 hours; the immobilized enzyme initial activity obtaining is 378U/g (wetting); after 10 times recycle; immobilized enzyme has retained 89% initial activity, just starts fragmentation but stir 7 hours polymer microballoons with the rotating speed of 280rpm in the phosphate buffer soln of pH=8.0.
Embodiment 4
Change 0.3 gram of polyether acrylate performed polymer into 0.5 gram of polyurethane acrylate prepolymer (dipentaerythritol-different sym.-diisopropylideneacetone Er Yi Qing Suan Zhi – Hydroxyethyl acrylate multipolymer performed polymer), N, 3.6 grams of N '-methylene-bisacrylamides change 3.4 grams into, and the synthesis condition of other polymer microballoons is identical with embodiment 1.Immobilization by synthetic polymer microballoon for penicillin acylase; the immobilization time is 12 hours; the immobilized enzyme initial activity obtaining is 268U/g (wetting); after 10 times recycle; immobilized enzyme has retained 92% initial activity, just starts fragmentation but stir 16 hours polymer microballoons with the rotating speed of 280rpm in the phosphate buffer soln of pH=8.0.
Embodiment 5
Change 0.3 gram of polyether acrylate performed polymer into 0.2 gram of polyurethane acrylate prepolymer, N, 3.6 grams of N '-methylene-bisacrylamides change 3.7 grams into, and the synthesis condition of other polymer microballoons is identical with embodiment 1.Immobilization by synthetic polymer microballoon for penicillin acylase; the immobilization time is 12 hours; the immobilized enzyme initial activity obtaining is 295U/g (wetting); after 10 times recycle; immobilized enzyme has retained 95% initial activity, just starts fragmentation but stir 11 hours polymer microballoons with the rotating speed of 280rpm in the phosphate buffer soln of pH=8.0.
Embodiment 6
Change 0.3 gram of polyether acrylate performed polymer into 0.8 gram of polyurethane acrylate prepolymer, N, 3.6 grams of N '-methylene-bisacrylamides change 3.1 grams into, and the synthesis condition of other polymer microballoons is identical with embodiment 1.Immobilization by synthetic polymer microballoon for penicillin acylase; the immobilization time is 12 hours; the immobilized enzyme initial activity obtaining is 198U/g (wetting); after 10 times recycle; immobilized enzyme has retained 84% initial activity, just starts fragmentation but stir 25 hours polymer microballoons with the rotating speed of 280rpm in the phosphate buffer soln of pH=8.0.
Claims (4)
1. the preparation method for the polymer microballoon of bio-enzyme immobilization, it is characterized in that, use polyfunctionality polymkeric substance performed polymer with long branched chain structure as active centre, part replaces N, N '-methylene-bisacrylamide linking agent, adopts inverse suspension polymerization technology to prepare the porous polymer microsphere of the surface that physical strength is high and the enzyme immobilization time is short containing epoxide group.
2. the preparation method of the polymer microballoon for bio-enzyme immobilization according to claim 1, is characterized in that, for improved inverse suspension polymerization technology, specifically comprises the steps:
By the polyfunctionality polymkeric substance performed polymer with long branched chain structure, N, N '-methylene-bisacrylamide, Methacrylamide, the polymer monomer such as glycidyl methacrylate and glycidyl allyl ether and Diisopropyl azodicarboxylate initiator join in formamide soln, then join in the mixed solvent being formed by normal heptane and zellon that is dissolved with Span60 and Tween20 composite dispersing agent, under nitrogen protection, carry out inverse suspension polymerization reaction 3 ~ 6 hours in 45 ~ 65 DEG C, reaction finishes rear by washing with alcohol, normal heptane soaks, 50 ~ 70 DEG C of vacuum-drying 8 ~ 16 hours, obtain polymer microballoon.
3. according to the preparation method of the polymer microballoon for bio-enzyme immobilization described in claim 1 and 2, it is characterized in that, the described polyfunctionality polymkeric substance performed polymer with long branched chain structure is a kind of of polyether acrylate performed polymer (TriMethylolPropane(TMP)-oxyethane-acrylic copolymer performed polymer) and polyurethane acrylate prepolymer (dipentaerythritol-different sym.-diisopropylideneacetone Er Yi Qing Suan Zhi – Hydroxyethyl acrylate multipolymer performed polymer) or two kinds, polyfunctionality polymkeric substance performed polymer and N, the weight ratio of N '-methylene-bisacrylamide is 0.05 ~ 0.3.
4. according to the preparation method of the polymer microballoon for bio-enzyme immobilization described in claim 1 and 2; it is characterized in that; the described porous polymer microsphere containing epoxide group; can be used for the immobilization of the water-soluble biological enzymes such as penicillin acylase, glucose isomerase, glucose transglucosidase, trypsinase or amylase, be specially adapted to the immobilization of penicillin acylase.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104874615A (en) * | 2015-04-22 | 2015-09-02 | 首钢总公司 | Rod-wire roll-pass low-temperature controlled-rolling motor power load distribution design method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408859A (en) * | 2002-09-06 | 2003-04-09 | 华东理工大学 | Immobilized penicillin amidase carrier and its preparing method |
| CN103012673A (en) * | 2013-01-21 | 2013-04-03 | 华东理工大学 | Biological enzyme immobilizing core-shell type superparamagnetic polymeric microsphere and preparing method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1408859A (en) * | 2002-09-06 | 2003-04-09 | 华东理工大学 | Immobilized penicillin amidase carrier and its preparing method |
| CN103012673A (en) * | 2013-01-21 | 2013-04-03 | 华东理工大学 | Biological enzyme immobilizing core-shell type superparamagnetic polymeric microsphere and preparing method thereof |
Non-Patent Citations (3)
| Title |
|---|
| HAITAO YU ET AL.: "Immobilization of glucose isomerase onto GAMM support for isomerization of glucose to fructose", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》 * |
| 蒋昱: "亲水性聚氨酯泡沫固定化青霉素酰化酶制备和应用的研究", 《抗生素》 * |
| 薛屏 等: "高分子载体材料对青霉素酰化酶的固定化作用", 《功能高分子学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104874615A (en) * | 2015-04-22 | 2015-09-02 | 首钢总公司 | Rod-wire roll-pass low-temperature controlled-rolling motor power load distribution design method |
| CN104874615B (en) * | 2015-04-22 | 2016-11-23 | 首钢总公司 | Bar Wire Product pass low temperature controlled rolling power of motor sharing of load method for designing |
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