CN1837266A - Phospholipid modified acrylon nano fiber composite film and its preparation and immobilized enzyme thereof - Google Patents
Phospholipid modified acrylon nano fiber composite film and its preparation and immobilized enzyme thereof Download PDFInfo
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- CN1837266A CN1837266A CN 200610049955 CN200610049955A CN1837266A CN 1837266 A CN1837266 A CN 1837266A CN 200610049955 CN200610049955 CN 200610049955 CN 200610049955 A CN200610049955 A CN 200610049955A CN 1837266 A CN1837266 A CN 1837266A
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Treatments For Attaching Organic Compounds To Fibrous Goods (AREA)
Abstract
The invention discloses a phosphatide modified acrylic nanometer fibre composite film and preparing and immobilized enzyme method, which comprises the following steps: taking dimethyl sulfoxide, dimethyl formamide or dimethylacetamide as solvent; preparing acrylon copolymer with phosphatide group through electrostatic spinning method as the carrier of immobilized enzyme; immobilizing the enzyme on the nanometer fiber composite film through physical adsorptive method. The invention applies bionic property of high specific surface area, high gap rate and phosphatide property, which produces high enzyme carrying, specific activity and catalyzing rate immobilizing enzyme phosphatide modified acrylic nanometer fibre composite film.
Description
Technical field
The present invention relates to the technology of a kind of phospholipid modified acrylon nano fiber composite membrane and preparation and immobilized enzyme.
Background technology
Enzyme is the class specific proteins with efficient catalytic function, height specificity and high degree of controlled that is produced by viable cell, it can specifically be facilitated certain chemical reaction and itself not participate in reaction, has the reaction efficiency height, mild condition, reaction product is polluted little, energy consumption is low, and therefore characteristics such as the easy control of reaction are used widely.But the sex change of zymoprotein easily takes place in it under influences such as acid, alkali, heat and organic solution, thereby being lived, reduces or forfeiture enzyme, and how enzyme reaction is carried out in solution, having reacted the back is difficult for reclaiming, reaction product is separated purification difficult, and be difficult to realize industrial serialization, automatic production, so the application development of enzyme engineering has been subjected to very big restriction.Enzyme immobilization technology provides means effectively for the solution of these problems.
The Carrier Materials of Immobilized Enzyme morphological structure is of a great variety, as mould material, porous microsphere and nanofiber with vesicular structure all can be used as the carrier of enzyme immobilization.These have surface irregularity, vesicular structure, solid support material that specific surface area is big helps improving the enzyme amount of carrying of solidifying enzyme, help substrate to enzyme active center and product to conversion zone with external diffusion to improve the catalytic efficiency of immobilized enzyme.CN01139257.6 is by containing intermediary's polymkeric substance such as hydrophilic polymer of positively charged ion or amido, enzyme being fixed on polymer film surface.CN92101875.4 carries out gelation formation gel spheres with polyvinyl alcohol and the formed mixture of enzyme, thereby obtains the polyvinyl alcohol gel microballoon of immobilized enzyme; CN1320688.X has the immobilization that the suitable mode of loading of porous material surface employing has been realized penicillin acylase; CN01126606.6 adopts glutaraldehyde one step activation to have the chitosan pearl of active aldehyde, and zymoprotein is fixed on pearl chitosan surface; CN01110024.9 is to be carrier with the magnetic agarose microbeads, by physical adsorption the plain enzyme of industrial fiber is fixed on the microballoon; CN200410057257.7 is that the microballoon with multiple biological polysaccharide polymer material preparation is a carrier, by the glutaraldehyde cross-linking method beta-galactosidase enzymes is fixed on the biological polysaccharide polymer microsphere surface.
The fiber composite mould material that utilizes the method preparation of electrostatic spinning to have nano-scale more and more draws attention.This class material has advantages such as high-specific surface area and high voidage.Utilize the method for electrostatic spinning can be used for preparing various high polymer nanometer fiber composite film materials.US 20030215624; 20040013873 by electrostatic spinning with common polymkeric substance such as: polyvinyl alcohol, polyethylene, polypropylene, polystyrene, polysulfones, polycarbonate, urethane, polymethacrylate, polyvinyl chloride, polymeric amide, polyacrylic ester, polyvinylpyrrolidone etc. are prepared into the ultra-fine fibre composite membrane; CN 200410019230.9 prepares the ultra-fine fibre composite membrane with the chitosan electrostatic spinning; CN 200510014826.4 has antibiotic, anlistatig chitosan/carbon nanotube ultra-fine fibre composite membrane by the method for electrostatic spinning preparation.This fiber composite mould material with nano-scale also can be used as the carrier of enzyme immobilization, and CN200410020239.1 is prepared into polyvinyl alcohol and cellulase the mould material of immobilized cellulase by electrospinning process.
Enzyme immobilization technology can effectively improve the stability and the reusability of enzyme, has obtained using widely.Yet the abiotic specific interaction of some between immobilized enzyme and carrier often causes the reduction of enzymic activity easily.Phosphatide is one of main composition of natural biological film lipid bilayer, performance to biomembranous structural integrity and every function is most important, especially in the microbial film as the activity of the chemical constitution of the lipid bilayer of main body and the various membranins of physical property remarkably influenced, thermostability etc.Studies show that, contain phosphatide or phospholipid modified polymkeric substance has good blood compatibility and biocompatibility.The biologic specificity of this excellence of material surface, for immobilized enzyme albumen provides a kind of residing microenvironment of membranin in the natural biological film that is similar to, avoided the inactivation that causes because of nonspecific effect, can reduce many abiotic specificitys effectively and interact, thereby keep the zymoprotein natural radioactivity effectively.This optimization to the immobilized enzyme protein function is necessary.
In recent years, the interaction between liposome and all kinds albumen had caused many investigators' interest, and part Study has confirmed that the chemical constitution of lipid bilayer and physical property can influence proteic activity of enzyme classes and stability [1-3] significantly.[1]Jensen T R，Balashev K，Bjrnholm T，Kjaer K.Novel methods for studying lipids and lipasesand their mutual interaction at interfaces.Part II.Surface sensitive synchrotron X-ray scattering.Biochimie 2001，83：399-408；[2]Rhoten M C，Burgess J D，Hawkridge F M.Temperature andpH effects on cytochrome c oxidase immobilized in an electrode-supported lipid bilayer membrane.Electrochimica Acta 2000，45：2855-2860；[3]Rhoten M C，Burgess J D，Hawkridge F M.Thereaction of cytochrome c from different species with cytochrome c oxidase immobilized in anelectrode supported lipid bilayer membrane.J Electroanal Chem 2002，534：143-150。
But, also do not utilize electrostatic spinning technique preparation surface to have the phosphatide of bionical characteristic or the immobilization that phospholipid modified solid support material is used for enzyme at present.So, be necessary to develop a kind of class phosphatide nano-fiber composite film material, effectively improve the catalytic efficiency of the enzyme amount of carrying, the diffusion control when eliminating enzymic catalytic reaction and the raising immobilized enzyme of immobilized enzyme with high-specific surface area, high voidage and good biocompatibility; Simultaneously, the phosphatide biomimetic modification layer on membrane fiber surface provides a kind of residing microenvironment of membranin in the natural biological film that is similar to for immobilized enzyme, thereby promotes the raising of activity of the immobilized enzyme and stability.
Summary of the invention
Cause the reduction of enzymic activity easily at abiotic specific interaction between the carrier of some being used for fixing enzymes and the enzyme, and the enzyme amount of carrying of immobilized enzyme and catalytic efficiency is low, Carrier Materials of Immobilized Enzyme is difficult to problems such as recovery, the invention provides a kind of method that has the phospholipid modified acrylon nano fiber composite film material and the preparation thereof of high-specific surface area, high voidage and good biocompatibility and be used for enzyme immobilization.
Essentiality content of the present invention is as follows:
One, a kind of phospholipid modified acrylon nano fiber composite membrane is by vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer preparation, and its molecular weight is 5～300,000; 2-methacryloxypropyl-ethyl in the multipolymer-phosphatidylcholine content is 1～30mol%; Nano-fiber composite film is to be that fiber between 30 nanometers～5 micron converges long-pending forming by diameter;
Two, the preparation method of phospholipid modified acrylon nano fiber composite membrane:
1) prepares vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer earlier: join vinyl cyanide, 2-methacryloxypropyl-ethyl-phosphatidylcholine, water in the reactor successively, wherein the mol ratio of vinyl cyanide and 2-methacryloxypropyl-ethyl-phosphatidylcholine is between 99: 1～70: 30, and the add-on of water accounts for 60～80% of total mass; After 20 minutes, be warming up to 40～80 ℃ at logical nitrogen, add (NH
4)
2S
2O
8-NaHSO
3Initiator, wherein (NH
4)
2S
2O
8And NaHSO
3Mol ratio is 5: 1; Reaction after 2-6 hour leaches the white depositions that generates under nitrogen protection, and distinguishes cleaning cleaning polyalcohol three times with deionized water and ethanol; And then carry out extracting and purifying with deionized water, under 40～80 ℃, vacuumize then and carry out drying; Can obtain molecular weight is 5～300,000; 2-methacryloxypropyl-ethyl in the multipolymer-phosphatidylcholine content is vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 1～30mol%;
2) vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer is dissolved in the solvent, is prepared into the spinning solution of homogeneous;
3) above-mentioned spinning solution is injected into respectively in the multiple tracks electrostatic spinning apparatus, carries out electrostatic spinning and compile film forming, it is dry that the composite fiber membrane that makes is put into vacuum drying oven;
Three, the process for fixation of enzyme on the phospholipid modified acrylon nano fiber composite membrane:
1) enzyme fully is dissolved in makes enzyme solution in the phosphate buffer soln;
2) phospholipid modified acrylon nano fiber composite membrane is immersed in the phosphate buffer soln of above-mentioned enzyme fully, shake under constant temperature the sealing back; Take out composite fiber membrane subsequently from solution, repeated washing can obtain phospholipid modified acrylon nano fiber composite film immobilized enzyme;
3) the immobilized enzyme composite fiber membrane that makes is immersed phosphate buffer soln, store for future use.
Advantage of the present invention is:
1) this phospholipid modified acrylon nano fiber composite film material is based on the conventional material acrylic fibers is carried out the phosphatide modification, this material source abundance, and production technique is simple, and is with low cost, is suitable for suitability for industrialized production;
2) preparation of phospholipid modified acrylon nano fiber composite membrane and enzyme immobilization technology thereof are simple;
3) phospholipid modified acrylon nano fiber composite membrane has high-specific surface area and high voidage, the catalytic efficiency that helps improving the enzyme amount of carrying, the diffusion control when eliminating enzymic catalytic reaction of immobilized enzyme and improve immobilized enzyme;
4) this carrier with nano-scale composite fiber membrane enzyme immobilization is easy to reclaim from reaction system, can reuse, greatly improve enzyme utilization ratio and reduce production costs;
5) contain the acrylon nano fiber composite membrane of the bionical group of phosphatide, can provide friendly bionical environment, improve immobilized enzyme biological activity and stability effectively for its surface immobilized zymoprotein.
Description of drawings:
A among Fig. 1, b, c, d are respectively the picture that the phospholipid modified acrylon nano fiber composite membrane of example 1,6,8 and 12 is made by scanning electron microscope.
Embodiment
Phospholipid modified acrylon nano fiber composite membrane preparation method: vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer is dissolved in the solvent, being prepared into concentration is 2～16% transparent spinning solutions, be injected in the multiple tracks electrostatic spinning apparatus, at voltage is that 5kV～30kV, spinning nozzle solution flow are that 0.1mL/h～2.0mL/h, receiving range are that electrostatic spinning compiles film forming under the condition of 5cm～25cm, and the film that makes is put into vacuum drying oven in 60 ℃ of dry 24h down; Promptly obtain phospholipid modified acrylon nano fiber composite membrane solid support material, wherein Fibre diameter is between 30 nanometers～5 micron.
Vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer, its molecular weight are 5～300,000; 2-methacryloxypropyl-ethyl in the multipolymer-phosphatidylcholine content is 1～30mol%.
Being used for solvent of the present invention is: a kind of or any two kinds of mixtures wherein of dimethyl sulfoxide (DMSO), dimethylformamide, dimethyl acetyl ammonia.
Method by physical adsorption is fixed in enzyme on the phospholipid modified acrylon nano fiber composite membrane, and concrete grammar is: it is in 7.5 the phosphate buffer soln that enzyme fully is dissolved in pH, makes the enzyme solution that concentration is 1～20mg/mL; Phospholipid modified acrylon nano fiber composite membrane is immersed in the enzyme phosphate buffer soln fully, and shook 1～5 hour down in 30 ℃ constant temperature the sealing back, and concussion speed is 20～120rpm.From solution, take out film, use the deionized water repeated washing, clean altogether 3～5 times, can obtain phospholipid modified acrylon nano fiber composite film immobilized enzyme.It is in 7.5 the phosphate buffer soln that the fixed enzyme membrane that makes is immersed pH, stores for future use under 4 ℃.
Be fixed in that enzyme is on this kind material: lipase, catalase, proteolytic enzyme, peroxidase, cellulase, laccase, desaturase, oxidasic a kind of.
Polymkeric substance viscosity-average molecular weight of the present invention (M η) is to adopt the one point method in the viscosimetry to measure; In the multipolymer content of 2-methacryloxypropyl-ethyl-phosphatidylcholine be adopt the hydrogen spectrum (
1H-NMR) calculate; Solvent is to adopt N,N-dimethylacetamide; The nano-fiber composite film Fibre diameter is to adopt scanning electron microscope analysis to measure; Year enzyme amount of lipase is to detect and calculate by Bradford method [Bradford M.Anal Biochem 1976,72:248.] on the nano-fiber composite film; The activity of immobilized lipase and Michaelis often are to calculate by the method for reference literature [Chiou SH, Wu WT.Biomaterials 2004,25:197.]; The activity of immobilized catalase is to calculate by the method for reference literature [Akg l S, Kacar Y, zkara S, Yavuz H, DenizliA, Arica MY, J Mol Catal B:Enzym. 2001,15:197].
The embodiment of nano-fiber composite film solid support material preparation of the present invention:
Example 1
(viscosity-average molecular weight is 8.0 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 4wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 1.8mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 0.2mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 60～90 nanometers.
Example 2
(viscosity-average molecular weight is 8.0 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 6wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 1.8mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 0.5mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 100～150 nanometers.
Example 3
(viscosity-average molecular weight is 7.6 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 4wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 4.9mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 0.2mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 50～80 nanometers.
Example 4
(viscosity-average molecular weight is 7.6 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 6wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 4.9mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 0.5mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 90～150 nanometers.
Example 5
(viscosity-average molecular weight is 7.2 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 4wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 9.0mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 0.2mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 50～80 nanometers.
Example 6
(viscosity-average molecular weight is 7.2 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 6wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 9.0mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 1.0mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 200～300 nanometers.
Example 7
(viscosity-average molecular weight is 17.0 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 4wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 4.7mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 0.5mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 600～800 nanometers.
Example 8
(viscosity-average molecular weight is 17.0 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 6wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 4.7mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 1.0mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 1500～2000 nanometers.
Example 9
(viscosity-average molecular weight is 16.5 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 4wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 9.6mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 0.5mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 400～600 nanometers.
Example 10
(viscosity-average molecular weight is 16.5 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 6wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 9.6mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 1.0mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 900～1300 nanometers.
Example 11
(viscosity-average molecular weight is 15.8 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 4wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 17.8mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 0.2mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 200～400 nanometers.
Example 12
(viscosity-average molecular weight is 15.8 ten thousand with vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer of 6wt%, 2-methacryloxypropyl-ethyl-phosphatidylcholine content is 17.8mol%) be dissolved in the dimethylformamide, mechanical stirring under 60 ℃ of temperature, the dissolving back forms homogeneous phase, transparent, heavy-gravity solution fully; Deaeration is also removed impurity, obtain spinning solution, join in the sampler, be connected to spinning nozzle, spinning nozzle is connected with high-voltage power supply, the distance between spinning nozzle and the accepted thing is about 15 centimetres, spinning nozzle spinning solution flow velocity is 0.5mL/h, voltage is 10 kilovolts, carries out electrostatic spinning, and obtaining diameter is the phospholipid modified acrylon nano fiber composite membrane solid support material of 400～600 nanometers.
Phospholipid modified acrylic fibers thing nano-fiber composite film and each performance perameter of immobilized lipase thereof of example 1～12 preparation of the present invention, as shown in table 1.
Phospholipid modified acrylon nano fiber composite membrane and each performance perameter of immobilized catalase thereof of example 1,6,8 of the present invention and 12 preparations, as shown in table 2.
The phospholipid modified acrylon nano fiber composite membrane of table 1. and each performance perameter of immobilized lipase thereof
| Example/sample number | Molecular weight of copolymer * 10 -4g/mol | Phospholipids content (mol%) | Electrospinning membrane fiber diameter (nm) | Carry enzyme amount (mg/g) | The immobilized enzyme specific activity a(U/mg) | Activity of the immobilized enzyme retention rate (%) | Michaelis-Menton constant K m b (mM) |
| 1 | 8.0 | 1.8 | 60～90 | 24.0±1.1 | 22.1 | 52.6±0.8 | 0.75 |
| 2 | 8.0 | 1.8 | 100～150 | 22.2±0.9 | 21.6 | 51.4±0.7 | 0.82 |
| 3 | 7.6 | 4.9 | 50～80 | 27.2±0.7 | 30.7 | 72.9±0.8 | 0.74 |
| 4 | 7.6 | 4.9 | 90～150 | 24.9±1.2 | 31.1 | 73.8±0.6 | 0.80 |
| 5 | 7.2 | 9.0 | 50～80 | 26.0±1.4 | 33.0 | 78.5±0.9 | 0.76 |
| 6 | 7.2 | 9.0 | 200～300 | 22.2±0.6 | 32.2 | 76.4±0.7 | 0.88 |
| 7 | 17.0 | 4.7 | 600～800 | 19.2±0.7 | 30.0 | 71.3±0.8 | 0.97 |
| 8 | 17.0 | 4.7 | 1500～ 2000 | 17.9±0.8 | 29.8 | 70.7±0.7 | 1.18 |
| 9 | 16.5 | 9.6 | 400～600 | 21.4±1.1 | 31.9 | 75.7±0.6 | 0.93 |
| 10 | 16.5 | 9.6 | 900～ 1300 | 20.2±1.2 | 31.5 | 74.9±0.7 | 1.06 |
| 11 | 15.8 | 17.8 | 200～400 | 22.2±1.2 | 35.7 | 84.9±0.9 | 0.92 |
| 12 | 15.8 | 17.8 | 400～600 | 20.9±1.1 | 35.3 | 83.8±0.6 | 0.96 |
A. the specific activity of used lipase is a 42.1U/mg albumen;
De Shi constant K during b. free lipase-catalyzed reaction
mBe 0.44.
The phospholipid modified acrylon nano fiber composite membrane of table 2. and each performance perameter of immobilized catalase thereof
| Example/sample number | Molecular weight of copolymer * 10 -4 g/mol | Phospholipids content (mol%) | Electrospinning membrane fiber diameter (nm) | Carry enzyme amount (mg/g) | The immobilized enzyme specific activity a(μmol H 2O 2/mg.min) | Activity of the immobilized enzyme retention rate (%) |
| 1 | 8.0 | 1.8 | 60～90 | 27.0±0.9 | 888.05±113.4 | 36.11 |
| 6 | 7.2 | 9.0 | 200～300 | 24.3±1.0 | 1330.48±93.5 | 54.10 |
| 8 | 17.0 | 4.7 | 1500～2000 | 19.6±0.6 | 1173.09±129.8 | 47.70 |
| 12 | 15.8 | 17.8 | 400～600 | 22.4±1.3 | 1448.53±124.4 | 58.90 |
A. used catalatic specific activity is 2459.3 (μ mol H
2O
2/ mg.min)
To obtain the carrier of phospholipid modified acrylon nano fiber composite membrane in the above-mentioned example, carry out the immobilized implementation method of enzyme as enzyme immobilization:
Method by physical adsorption is fixed on lipase or catalase on the nano-fiber composite film.In the lipase or catalase phosphate buffer soln with the 5mg/mL that is submerged into fully of the phospholipid modified acrylon nano fiber composite membrane of 1 gram, the sealing back is in the concussion 4 hours down of 30 ℃ constant temperature, and concussion speed is 60rpm.From solution, take out film, use the deionized water repeated washing, clean altogether 3～5 times, can obtain phospholipid modified acrylon nano fiber composite film immobilized enzyme.The fixed enzyme membrane that makes is immersed in the phosphate buffer soln, under 4 ℃, store for future use.
Claims (5)
1, a kind of phospholipid modified acrylon nano fiber composite membrane feature is as follows:
(1) phospholipid modified acrylon nano fiber composite membrane is by vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer preparation, and its molecular weight is 5～300,000; 2-methacryloxypropyl-ethyl in the multipolymer-phosphatidylcholine content is 1～30mol%;
(2) nano-fiber composite film is to be that fiber between 30 nanometers～5 micron converges long-pending forming by diameter;
2, the preparation method of the described a kind of phospholipid modified acrylon nano fiber composite membrane of claim 1 is characterized in that:
(1) preparation method of vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer is: join vinyl cyanide, 2-methacryloxypropyl-ethyl-phosphatidylcholine, water in the reactor successively, wherein the mol ratio of vinyl cyanide and 2-methacryloxypropyl-ethyl-phosphatidylcholine is between 99: 1～70: 30, and the add-on of water accounts for 60～80% of total mass; After 20 minutes, be warming up to 40～80 ℃ at logical nitrogen, add (NH
4)
2S
2O
8-NaHSO
3Initiator, wherein (NH
4)
2S
2O
8And NaHSO
3Mol ratio is 5: 1; Start and stir, under nitrogen protection, carry out polyreaction; After polymerization 2-6 hour, the white depositions that generates is leached, and distinguish cleaning cleaning polyalcohol three times with deionized water and ethanol; And then carry out extracting and purifying with deionized water, to remove unreacted monomer and possible solubility homopolymer; The white solid polymkeric substance that obtains vacuumizes under 40～80 ℃ and carries out drying;
(2) adopt method of electrostatic spinning among the nano-fiber composite film preparation method: vinyl cyanide/2-methacryloxypropyl-ethyl-phosphatidyl choline copolymer is dissolved in the solvent, is made into spinning solution, wherein the shared weight ratio of multipolymer is 2～16%; Above-mentioned spinning solution is injected into respectively in the multiple tracks electrostatic spinning apparatus, at voltage is that 5kV～30kV, spinning nozzle solution flow are that 0.1mL/h～2.0mL/h, receiving range are to carry out electrostatic spinning and accumulate composite fiber membrane under the condition of 5cm～25cm, and the composite fiber membrane that makes is put into vacuum drying oven in 60 ℃ of dry 24h down.
3, a kind of phospholipid modified acrylon nano fiber composite membrane preparation method according to claim 2, it is characterized in that said solvent is a kind of or any two kinds of mixtures wherein in dimethyl sulfoxide (DMSO), dimethylformamide, the dimethyl acetyl ammonia.
4, the method for described a kind of phospholipid modified being used for fixing of the acrylon nano fiber composite membrane enzyme of claim 1 is characterized in that: it is in 7.5 the phosphate buffer soln that enzyme fully is dissolved in pH, makes the enzyme solution that concentration is 1～20mg/mL; Nano-fiber composite film is immersed in the enzyme phosphate buffer soln fully, and shook 1～5 hour down in 30 ℃ constant temperature the sealing back, and concussion speed is 20～120rpm; From solution, take out film, use the deionized water repeated washing, clean altogether 3～5 times, can obtain phospholipid modified acrylon nano fiber composite film immobilized enzyme; It is in 7.5 the phosphate buffer soln that the nano-fiber composite film of immobilized enzyme is immersed pH, stores for future use under 4 ℃.
5, the method for enzyme immobilization according to claim 4 is characterized in that, the enzyme that is fixed on this kind material is: a kind of in lipase, catalase, proteolytic enzyme, peroxidase, cellulase, laccase, desaturase, the oxydase.
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| CN101285791B (en) * | 2008-05-23 | 2010-12-08 | 浙江大学 | Ampere type biosensor electrode and method for making same |
| CN101265448B (en) * | 2008-04-03 | 2011-01-26 | 浙江大学 | Grease catalysis separation biphasic enzyme-film bioreactor and its preparation and application |
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| CN108034651A (en) * | 2017-12-18 | 2018-05-15 | 天津现代职业技术学院 | A kind of method for cooperateing with immobilized L-arabinose isomerase and ionic liquid |
| CN112480458A (en) * | 2020-11-05 | 2021-03-12 | 武汉轻工大学 | High-enzyme-activity modified membrane and preparation method thereof |
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| CN1539868A (en) * | 2003-08-26 | 2004-10-27 | 南阳师范学院 | Method for preparing composite membrane of high polymer Nano materal |
| CN100340663C (en) * | 2004-08-06 | 2007-10-03 | 天津大学 | Nano superfine fiber membrane of polyvinyl alcohol with immovable cellulase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101265448B (en) * | 2008-04-03 | 2011-01-26 | 浙江大学 | Grease catalysis separation biphasic enzyme-film bioreactor and its preparation and application |
| CN101285791B (en) * | 2008-05-23 | 2010-12-08 | 浙江大学 | Ampere type biosensor electrode and method for making same |
| CN101285792B (en) * | 2008-05-23 | 2011-05-18 | 浙江大学 | Mediator electrochemical enzyme electrode and method for making same |
| CN108034651A (en) * | 2017-12-18 | 2018-05-15 | 天津现代职业技术学院 | A kind of method for cooperateing with immobilized L-arabinose isomerase and ionic liquid |
| CN112480458A (en) * | 2020-11-05 | 2021-03-12 | 武汉轻工大学 | High-enzyme-activity modified membrane and preparation method thereof |
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