CN103952392B - High performance polymer microballoon for bio-enzyme immobilization and preparation method thereof - Google Patents
High performance polymer microballoon for bio-enzyme immobilization and preparation method thereof Download PDFInfo
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- CN103952392B CN103952392B CN201410210936.7A CN201410210936A CN103952392B CN 103952392 B CN103952392 B CN 103952392B CN 201410210936 A CN201410210936 A CN 201410210936A CN 103952392 B CN103952392 B CN 103952392B
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Abstract
The invention discloses a kind of high performance polymer microballoon for bio-enzyme immobilization and preparation method thereof, belong to biological technical field.Specifically, with the polyfunctionality polymer performed polymer with long branched chain structure as activated centre, part substitution N, N ' methylene-bisacrylamide crosslinking agents, the porous polymer microsphere of the surface containing epoxide group of high mechanical properties and macroporous structure is prepared for using inverse suspension polymerization technology, the immobilization time was shortened to 12 hours from 72 hours, the activity and operational stability of immobilised enzymes is improve, with preferable application prospect.
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 raw
Thing technical field.Specifically, high mechanical strength and enzyme immobilization time short table are prepared for using inverse suspension polymerization technology
Porous polymer microsphere of the face containing epoxide group.
Background technology
PA ase（E.C.3.5.1.11, Penicillin G acylase, abbreviation PGA）It is semi-synthetic β-interior acyl
The enzyme of most critical in amine antibiotics production, it can be catalyzed penicillin and its ring expansion sour water solution removes side chain, produce semi-synthetic β-
The important intermediate 6-amino-penicillanic acid of lactam antibiotics（6-APA）And 7-ADCA
（7-ADCA）, 6-APA and 7-ADCA can be catalyzed again to be condensed with side chain, produce various semi-synthetic beta-lactam antibiotics（Such as
Ampicillin, Amoxicillin, Cephalexin and Cefadroxil etc.）.But resolvase is directly used for catalytic reaction
In there are many deficiencies, such as unstable in high temperature, strong acid and strong base and organic solvent, easy loss of catalytic activity；Resolvase is returned
Receive difficult, it is economically unreasonable, also result in product and be difficult to separating-purifying, have a strong impact on product quality；Production process is difficult to
Continuous operation, can only disposably intermittently operated etc..Immobilised enzymes overcomes the above-mentioned deficiency of resolvase, not only maintains resolvase
Distinctive catalysis characteristics, also improves operational stability, and production process is easily achieved continuous operation, is easy to after the completion of reaction and product
Thing is separated and can reused, and the product purity of gained is high, low production cost.Therefore, enzyme immobilizatio is always catalysisization
The study hotspot in the fields such as, biochemistry and materials chemistry, wherein the synthetic technology of high performance fixation support is crucial.
Polymer support containing epoxide group, the epoxide group of carrier surface being capable of open loop and biology enzyme at ambient temperature
Amido in molecule reacts to form covalent bond, so as to realize biological enzyme immobilizatio in a mild condition.It is this by means of epoxy
Group makes biology enzyme be fixed on carrier surface, and not only operating condition is gently easy, and immobilization process loses also very to enzyme activity
It is small, thus, it is very suitable for the industrialized production of immobilised enzymes.
We（Chinese invention patent CN1408859A）Synthesize a series of for penicillin with inverse suspension polymerization technology
The polymer support containing epoxide group of acylated enzyme immobilization, but with GMA as function in polymerization process
Property monomer, with Span-60 and calcium stearate as composite dispersing agent, has polymer support calcium stearate in last handling process
It is difficult washed removing and the shortcomings of carrier is broken during reuse.Then we（Catalysis journal, 2010,31
(5): 586-590）With Span-60 and Tween-20 as composite dispersing agent, original calcium stearate composite dispersing agent is substituted, subtracted
Time and solvent load needed for few last handling process；Simultaneously with the preferable allyl glycidyl ether part of Chain Flexibility
Rigid GMA is substituted, the mechanical strength of polymer support is improved.But the synthesis of polymer support
Condition needs to be continued optimization, further improves the pore structure and mechanical strength of polymer support, shortens the enzyme immobilization time, improves
The combination property of immobilised enzymes.
The content of the invention
A kind of high mechanical strength and enzyme immobilization are provided the invention aims to the defect for overcoming prior art to exist
The preparation method of porous polymer microsphere of the time short surface containing epoxide group, makes what is prepared as carrier with the polymer microballoon
Immobilized penicillin acylated enzyme has activity higher, operational stability and mechanical stability.
The described porous polymer microsphere containing epoxide group, is prepared using improved inverse suspension polymerization technology,
Specifically include following steps：
By polyfunctionality polymer performed polymer, N with long branched chain structure, N '-methylene-bisacrylamide, metering system
The polymer monomers such as acid amides, GMA and allyl glycidyl ether and azodiisobutyronitrile initiator
Be added in formamide solution, be then added to be dissolved with Span60 and Tween20 composite dispersing agents by normal heptane and tetrachloro
In the mixed solvent of ethene composition, inverse suspension polymerization is carried out in 45 ~ 65 DEG C under nitrogen protection and is reacted 3 ~ 6 hours, reaction knot
Shu Houyong ethanol is washed, normal heptane immersion, and 50 ~ 70 DEG C are vacuum dried 8 ~ 16 hours, obtain polymer microballoon.
The described polyfunctionality polymer performed polymer with long branched chain structure is polyether acrylate performed polymer（Three hydroxyl first
Base oxide-ethylene oxide-acrylic copolymer performed polymer）And polyurethane acrylate prepolymer（Bipentaerythrite-isophorone
Diisocyanate-hydroxyethylacrylate copolymer performed polymer）One kind or two kinds, polyfunctionality polymer performed polymer and N, N '-
The weight ratio of methylene-bisacrylamide is 0.05 ~ 0.3.
The described porous polymer microsphere containing epoxide group, can be used for PA ase, glucose isomerase, grape
Sugar turns the water-soluble biological enzyme immobilizatio such as glycosides enzyme, trypsase or amylase, is particularly well-suited to the fixation of PA ase
Change.
The immobilization of PA ase：Weigh 0.30 gram of polymer microballoon to be placed in 25 milliliters of conical flasks, add 0.93
Milliliter PA ase solution is simultaneously diluted to 5 milliliters with the phosphate buffer of the 1mol/L of pH=7.5, in 28 DEG C of water-bath
After 12 hours are fixed in shaking table, suction filtration is washed with the phosphate buffer of the 0.1mol/L of pH=7.5.
The activity and operational stability assay method of immobilized penicillin acylated enzyme use document（Catalysis journal, 2010,
31(5): 586-590）The assay method of report.
Of the invention it is critical only that uses the polyfunctionality polymer performed polymer with long branched chain structure as activated centre,
Part replaces original N, N '-methylene-bisacrylamide crosslinking agent, obtain high mechanical properties and macroporous structure containing epoxy
The porous polymer microsphere of group, the immobilization time was shortened to 12 hours from 72 hours, improve immobilised enzymes activity and
Operational stability.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail, but is in no way intended to limit protection scope of the present invention.
Comparative example
Added equipped with thermometer, condenser pipe, wireway after 90 milliliters of normal heptanes are mixed with 30 milliliters of tetrachloro-ethylenes and stirred
Mix in the reactor of oar, after being added thereto to 0.29 gram of Span-60 and 0.78 gram of Tween-20, be heated to 55 DEG C.Protected in nitrogen
Under shield and strong agitation, 17.5 milliliters are added dissolved with 3.9 grams of N, N '-methylene-bisacrylamide, 0.54 gram of methacryl
Amine, 1.35 milliliters of GMAs, 1.35 milliliters of allyl glycidyl ethers and 0.55 gram of azodiisobutyronitrile
The formamide solution of initiator.After 55 DEG C of polymerisations 4 hours, stop stirring, room temperature is cooled to, by 200 mesh sub-sieves
Sieving, obtains polymer microballoon, and obtained polymer microballoon is successively through 100 milliliters of ethanol washing by soaking 48 hours, 100 milliliters
Normal heptane washing by soaking can be used after being vacuum dried 12 hours within 48 hours, 60 DEG C.Above-mentioned polymer microballoon is used for penicillin
Acylated enzyme immobilizatio, the immobilization time is 72 hours, and the immobilised enzymes initial activity for obtaining is 320U/g (wet), by 10 times
After recycling, immobilised enzymes remains 90% initial activity, but with 280rpm's in the PBS of pH=8.0
It is just most of broken that rotating speed stirs 6 hours polymer microballoons.
Embodiment 1
Added equipped with thermometer, condenser pipe, wireway after 90 milliliters of normal heptanes are mixed with 30 milliliters of tetrachloro-ethylenes and stirred
Mix in the reactor of oar, after being added thereto to 0.29 gram of Span-60 and 0.78 gram of Tween-20, be heated to 55 DEG C.Protected in nitrogen
Under shield and strong agitation, 17.5 milliliters are added dissolved with 0.3 gram of polyether acrylate performed polymer（Trimethylolpropane-epoxy second
Alkane-acrylic copolymer performed polymer）, 3.6 grams of N, N '-methylene-bisacrylamide, 0.54 gram of Methacrylamide, 1.35 milliliters
GMA, 1.35 milliliters of allyl glycidyl ethers and 0.55 gram of formyl of azodiisobutyronitrile initiator
Amine aqueous solution.After 55 DEG C of polymerisations 4 hours, stop stirring, be cooled to room temperature, sieved by 200 mesh sub-sieves, be polymerized
Thing microballoon, obtained polymer microballoon is successively through 100 milliliters of ethanol agitator treatings 1 hour, 100 milliliters of normal heptane agitator treatings
Be can be used after being vacuum dried 12 hours within 1 hour, 60 DEG C.Above-mentioned polymer microballoon is used for the immobilization of PA ase,
The immobilization time is 12 hours, and the immobilised enzymes initial activity for obtaining is 346U/g (wet), fixed after 10 times recycle
Change the initial activity that enzyme remains 100%, but stirred 12 hours with the rotating speed of 280rpm in the PBS of pH=8.0
Polymer microballoon just starts to crush.
Embodiment 2
By polyether acrylate performed polymer, 0.3 gram is changed to 0.2 gram, and N, N ' -3.6 grams of methylene-bisacrylamide are changed to 3.7
Gram, the synthesis condition of other polymers microballoon is same as Example 1.The polymer microballoon that will synthesize is used for PA ase
Immobilization, the immobilization time is 12 hours, and the immobilised enzymes initial activity for obtaining is 337U/g (wet), by 10 recyclings
Afterwards, immobilised enzymes remains 100% initial activity, but rotating speed with 280rpm in the PBS of pH=8.0 is stirred
10 hours polymer microballoons are mixed just to start to crush.
Embodiment 3
By polyether acrylate performed polymer, 0.3 gram is changed to 0.8 gram, and N, N ' -3.6 grams of methylene-bisacrylamide are changed to 3.1
Gram, the synthesis condition of other polymers microballoon is same as Example 1.The polymer microballoon that will synthesize is used for PA ase
Immobilization, the immobilization time is 12 hours, and the immobilised enzymes initial activity for obtaining is 378U/g (wet), by 10 recyclings
Afterwards, immobilised enzymes remains 89% initial activity, but is stirred with the rotating speed of 280rpm in the PBS of pH=8.0
7 hours polymer microballoons just start to crush.
Embodiment 4
0.3 gram of polyether acrylate performed polymer is changed to 0.5 gram of polyurethane acrylate prepolymer（Bipentaerythrite-different
Isophorone diisocyanate-hydroxyethylacrylate copolymer performed polymer）, N, N ' -3.6 grams of methylene-bisacrylamide is changed to 3.4
Gram, the synthesis condition of other polymers microballoon is same as Example 1.The polymer microballoon that will synthesize is used for PA ase
Immobilization, the immobilization time is 12 hours, and the immobilised enzymes initial activity for obtaining is 268U/g (wet), by 10 recyclings
Afterwards, immobilised enzymes remains 92% initial activity, but is stirred with the rotating speed of 280rpm in the PBS of pH=8.0
16 hours polymer microballoons just start to crush.
Embodiment 5
0.3 gram of polyether acrylate performed polymer is changed to 0.2 gram of polyurethane acrylate prepolymer, N, N '-di-2-ethylhexylphosphine oxide
3.6 grams of acrylamide is changed to 3.7 grams, and the synthesis condition of other polymers microballoon is same as Example 1.The polymer that will synthesize is micro-
Ball is used for the immobilization of PA ase, and the immobilization time is 12 hours, and the immobilised enzymes initial activity for obtaining is 295U/g
(wet), after 10 times recycle, immobilised enzymes remains 95% initial activity, but molten in the phosphate-buffered of pH=8.0
11 hours polymer microballoons are stirred in liquid with the rotating speed of 280rpm just to start to crush.
Embodiment 6
0.3 gram of polyether acrylate performed polymer is changed to 0.8 gram of polyurethane acrylate prepolymer, N, N '-di-2-ethylhexylphosphine oxide
3.6 grams of acrylamide is changed to 3.1 grams, and the synthesis condition of other polymers microballoon is same as Example 1.The polymer that will synthesize is micro-
Ball is used for the immobilization of PA ase, and the immobilization time is 12 hours, and the immobilised enzymes initial activity for obtaining is 198U/g
(wet), after 10 times recycle, immobilised enzymes remains 84% initial activity, but molten in the phosphate-buffered of pH=8.0
25 hours polymer microballoons are stirred in liquid with the rotating speed of 280rpm just to start to crush.
Claims (3)
1. the preparation method of a kind of polymer microballoon for bio-enzyme immobilization, it is characterised in that with carrying long branched chain structure
Polyfunctionality polymer performed polymer as activated centre, part substitution N, N/- methylene-bisacrylamide crosslinking agent, using anti-
Phase suspension polymerization technique is prepared for the porous polymer that epoxide group is contained on high mechanical strength and enzyme immobilization time short surface
Microballoon, the polyfunctionality polymer performed polymer with long branched chain structure is selected from polyether acrylate performed polymer and polyurethane
One kind in acrylate prepolymer body or two kinds, the polyether acrylate performed polymer are trimethylolpropane-oxirane-the third
Olefin(e) acid copolymer performed polymer, the polyurethane acrylate prepolymer is bipentaerythrite-IPDI-propylene
Sour hydroxyl ethyl ester copolymer performed polymer；The polyfunctionality polymer performed polymer and N, N/The weight ratio of-methylene-bisacrylamide
It is 0.05-0.3；
The inverse suspension polymerization technology specifically includes following steps：
By polyfunctionality polymer performed polymer, N, N with long branched chain structure/- methylene-bisacrylamide, Methacrylamide,
GMA and allyl glycidyl ether polymer monomer and azodiisobutyronitrile initiator are added to
In formamide solution, be then added to be dissolved with Span60 and Tween20 composite dispersing agents by normal heptane and tetrachloro-ethylene group
Into mixed solution in, carried out in 45-65 DEG C under nitrogen protection inverse suspension polymerization react 3-6 hour, react terminate after use
Ethanol is washed, normal heptane immersion, and 50-70 DEG C is vacuum dried 8-16 hours, obtains polymer microballoon.
2. the preparation method of the polymer microballoon for bio-enzyme immobilization according to claim 1, it is characterised in that institute
The porous polymer microsphere containing epoxide group stated, glycosides enzyme, pancreas are turned for PA ase, glucose isomerase, glucose
Protease or starch enzyme immobilizatio.
3. the preparation method of the polymer microballoon for bio-enzyme immobilization according to claim 2, it is characterised in that institute
The porous polymer microsphere containing epoxide group stated is applied to the immobilization of PA ase.
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