Modified polyurethane carrier for immobilized enzyme as well as preparation method and application of modified polyurethane carrier

Abstract

The invention relates to the field of high polymer materials, and provides a modified polyurethane carrier for immobilized enzyme, which is prepared from the following raw materials in parts by weight: 60 to 100 parts of polyether polyol, 5 to 15 parts of isocyanate, 10 to 30 parts of activated carbon, 5 to 20 parts of diatomite, 2 to 6 parts of catalyst, 5 to 10 parts of inorganic salt, 1 to 10 parts of high-molecular polymer and 1 to 10 parts of biological active agent. The invention also provides a preparation method and application of the carrier. The carrier disclosed by the invention has the characteristics of large specific surface area, strong hydrophilicity and biological affinity, good impact resistance, high mechanical strength and the like, has relatively high bio-enzyme loading capacity, protects the catalytic activity of bio-enzyme, and is relatively simple to prepare and low in cost.

Description

Technical Field

[0001] The invention relates to the field of polymer materials, and in particular to a modified polyurethane carrier for fixing enzymes and a preparation method and application thereof. Background Art

[0002] Bioenzyme is a substance with catalytic properties. It has the advantages of high efficiency, substrate specificity, green environmental protection, and mild conditions. It is widely used in many fields such as medicine, printing and dyeing, food processing, and environmental protection treatment. However, free bioenzymes have disadvantages such as poor stability, narrow scope of application, and difficulty in recycling during use, which leads to high system operating costs and limits its application scenarios. In order to solve the above problems, enzyme immobilization technology has been proposed by scholars. By fixing bioenzymes in solid materials, the influence of the surrounding environment on bioenzymes can be avoided, thereby enhancing the stability and applicability of bioenzymes.

[0003] The characteristics of the immobilized carrier are important factors affecting the performance of the immobilized enzyme. In order to increase the immobilization capacity of biological enzymes and improve the enzyme activity, many inorganic and organic materials are used for the immobilization of biological enzymes, including Fe3O4 magnetic spheres, molecular sieves, chitosan, polyvinyl alcohol, MOFs materials, nanofibers, graphene oxide and other materials. Although there are relatively rich studies on biological enzyme immobilization carriers, these carrier materials have poor mechanical strength, complex manufacturing processes and high costs. Therefore, most of the mainstream carriers on the market are made of high molecular polymers such as polyethylene, polypropylene, and polyurethane. The biophilicity of the carrier is improved through the synthesis process or surface modification, and the shape and size can be customized according to actual needs. However, the immobilized enzyme loading capacity of these mainstream carriers on the market still needs to be further improved. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a modified polyurethane carrier for enzyme fixation and a preparation method and application thereof. The carrier has the characteristics of large specific surface area, strong hydrophilicity and biophilicity, and high mechanical strength. As a carrier, it has a high bioenzyme loading capacity and protects the catalytic activity of the bioenzyme. The preparation is relatively simple and the cost is not high.

[0005] The present invention adopts the following technical solutions to solve the above technical problems:

[0006] A modified polyurethane carrier for fixing enzymes comprises the following raw materials in parts by weight: 60-100 parts of polyether polyol, 5-15 parts of isocyanate, 10-30 parts of activated carbon, 5-20 parts of diatomaceous earth, 2-6 parts of catalyst, 5-10 parts of inorganic salt, 1-10 parts of high molecular polymer and 1-10 parts of bioactive agent.

[0007] As one of the preferred embodiments of the present invention, the polyether polyol is one or more of polyoxypropylene glycol, polytetramethylene glycol, tetramethylene glycol-propylene oxide copolymer glycol, and trihydroxy polyol.

[0008] As one of the preferred embodiments of the present invention, the isocyanate is one or more of toluene diisocyanate and diphenylmethane diisocyanate.

[0009] As one of the preferred embodiments of the present invention, the catalyst is one or more of triethanolamine, triethylenediamine, dibutyltin dilaurate, and stannous octoate.

[0010] As one of the preferred embodiments of the present invention, the inorganic salt is one or more of anhydrous ferric chloride, sodium chloride, calcium chloride and potassium chloride.

[0011] As one of the preferred embodiments of the present invention, the high molecular polymer is one or more of polyethylene, polypropylene, polycarbonate, polystyrene, polyethylene glycol, and polypropylene methyl ester.

[0012] As one of the preferred embodiments of the present invention, the bioactive agent is one or more of sophorolipids, rhamnolipids, trehalolipids, chitosan, and β-cyclodextrin.

[0013] A method for preparing the modified polyurethane carrier for enzyme immobilization, comprising the following steps:

[0014] S1. Take 60-100 parts of polyether polyol and 1-10 parts of high molecular polymer, mix and preheat at 60-90°C for 1-2h;

[0015] S2, adding 10-30 parts of activated carbon, 5-20 parts of diatomaceous earth, 2-6 parts of catalyst, 5-10 parts of inorganic salt and 1-10 parts of bioactive agent to the mixture of step S1, and stirring at high speed in a stirrer at a speed of 600-1800 r / min;

[0016] S3, add 5-15 parts of isocyanate and stir at high speed at a speed of 600-1800r / min;

[0017] S4. Pour the matured polyurethane foam into a sealed mold with a vacuum degree of 0.10-0.70 MPa, introduce nitrogen with a pressure of 0.08-0.12 MPa, inflate for 10-20 minutes and then release the pressure to obtain the desired modified polyurethane carrier for enzyme immobilization.

[0018] As one of the preferred embodiments of the present invention, the prepared modified polyurethane carrier has a particle size of 1 to 20 mm and a density of 0.895 to 0.945 kg / cm 3 .

[0019] An application of the modified polyurethane carrier for enzyme immobilization is to immerse the modified polyurethane carrier in an enzyme solution and allow it to stand for adsorption to obtain an immobilized enzyme.

[0020] The advantages of the present invention compared to the prior art are:

[0021] (1) The present invention changes the physical and chemical properties of the polyurethane carrier by adding inorganic, organic and biological materials; wherein the porous structure of the activated carbon improves the adsorption performance and specific surface area of ​​the carrier, the silanol group of the diatomaceous earth enhances the hydrophilicity of the carrier, the inorganic salt is used to adjust the density of the carrier, the high molecular polymer improves the porosity, specific surface area and toughness of the carrier, and the bioactive agent improves the biophilicity of the carrier; the obtained modified polyurethane carrier has the characteristics of density close to that of water, high porosity, large specific surface area, strong hydrophilicity and biophilicity, good impact resistance and high mechanical strength, and is suitable for loading biological enzymes and has a high loading amount of biological enzymes;

[0022] (2) The present invention utilizes the porosity and biophilicity of activated carbon and diatomaceous earth, and relies on the adsorption and sustained release of toxic substances to significantly reduce the toxicity of toxic substances to biological enzymes, and further improves the stability of immobilized enzymes (protecting the catalytic activity of biological enzymes);

[0023] (3) The modified polyurethane carrier of the present invention is relatively simple to prepare, has low cost and long service life. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the appearance structure diagram of the carrier of the present invention before modification in Experimental Example 1;

[0025] Figure 2 This is a structural diagram of the appearance of the modified polyurethane carrier of the present invention in Experimental Example 1. DETAILED DESCRIPTION

[0026] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the materials and reagents used in the present invention are conventional materials and reagents in the art unless otherwise specified, and can be obtained from commercial channels. The experimental conditions and experimental methods used in the present invention are conventional conditions and methods in the art unless otherwise specified, and will not be repeated.

[0027] Example 1

[0028] A modified polyurethane carrier for enzyme fixation in this embodiment includes the following raw materials in parts by weight: 60 parts of polytetrahydrofuran diol, 5 parts of toluene diisocyanate, 10 parts of activated carbon, 5 parts of diatomaceous earth, 2 parts of triethanolamine, 5 parts of sodium chloride, 1 part of polyethylene, 0.5 parts of sophorolipids, and 0.5 parts of rhamnolipids.

[0029] Preparation method:

[0030] S1. Take 60 parts of polytetrahydrofuran diol and 1 part of polyethylene, mix and preheat at 60° C. for 2 hours.

[0031] S2. Add 10 parts of activated carbon, 5 parts of diatomaceous earth, 2 parts of triethanolamine catalyst, 5 parts of sodium chloride, 0.5 parts of sophorolipids, and 0.5 parts of rhamnolipids to the mixture in step S1, and stir at high speed in a blender at a speed of 600 r / min.

[0032] S3. Add 5 parts of toluene diisocyanate and stir at high speed at a speed of 600 r / min.

[0033] S4. Pour the matured polyurethane foam into a sealed mold with a vacuum degree of 0.10 MPa, introduce nitrogen with a pressure of 0.08 MPa, and release the pressure after inflating for 20 minutes to obtain the desired modified polyurethane carrier for enzyme immobilization.

[0034] After cutting, the particle size of the modified polyurethane carrier in this embodiment is 15 mm, and the true density is 0.918 kg / cm 3 .

[0035] Example 2

[0036] A modified polyurethane carrier for enzyme fixation in this embodiment includes the following raw materials in parts by weight: 80 parts of polyoxypropylene glycol, 10 parts of toluene diisocyanate, 20 parts of activated carbon, 18 parts of diatomaceous earth, 5 parts of triethylenediamine, 8 parts of anhydrous ferric chloride, 5 parts of polystyrene, 4 parts of chitosan, and 4 parts of β-cyclodextrin.

[0037] Preparation method:

[0038] S1. Take 80 parts of polyoxypropylene glycol and 5 parts of polystyrene, mix and preheat at 80°C for 1.5 hours.

[0039] S2. Add 20 parts of activated carbon, 18 parts of diatomaceous earth, 5 parts of triethylenediamine catalyst, 8 parts of anhydrous ferric chloride, 4 parts of chitosan and 4 parts of β-cyclodextrin to the mixture in step S1, and stir at high speed in a blender at a speed of 1000 r / min.

[0040] S3. Add 10 parts of toluene diisocyanate and stir at high speed at a speed of 1000 r / min.

[0041] S4. Pour the matured polyurethane foam into a sealed mold with a vacuum degree of 0.50 MPa, introduce nitrogen with a pressure of 0.10 MPa, inflate for 15 minutes and then release the pressure to obtain the desired modified polyurethane carrier for enzyme immobilization.

[0042] After cutting, the particle size of the modified polyurethane carrier in this embodiment is 8 mm, and the true density is 0.910 kg / cm 3 .

[0043] Example 3

[0044] A modified polyurethane carrier for enzyme fixation in this embodiment includes the following raw materials in parts by weight: 100 parts of tetrahydrofuran-propylene oxide copolymer, 10 parts of diphenylmethane diisocyanate, 15 parts of activated carbon, 10 parts of diatomaceous earth, 5 parts of dibutyltin dilaurate, 5 parts of calcium chloride, 10 parts of polypropylene, and 8 parts of trehalose lipid.

[0045] Preparation method:

[0046] S1. Take 100 parts of polyether polyol and 10 parts of high molecular polymer, mix and preheat at 80°C for 1 hour.

[0047] S2. Add 15 parts of activated carbon, 10 parts of diatomaceous earth, 5 parts of dibutyltin dilaurate catalyst, 5 parts of calcium chloride-free and 8 parts of trehalose lipid to the mixture in step S1, and stir at high speed in a mixer at a speed of 1500 r / min.

[0048] S3. Add 10 parts of diphenylmethane diisocyanate and stir at high speed at a speed of 1500 r / min.

[0049] S4. Pour the matured polyurethane foam into a sealed mold with a vacuum degree of 0.70 MPa, introduce nitrogen with a pressure of 0.12 MPa, and release the pressure after inflating for 10 minutes to obtain the desired modified polyurethane carrier for enzyme immobilization.

[0050] After cutting, the particle size of the modified polyurethane carrier in this embodiment is 10 mm, and the true density is 0.926 kg / cm 3 .

[0051] Example 4

[0052] A modified polyurethane carrier for enzyme fixation in this embodiment includes the following raw materials in parts by weight: 100 parts of trihydroxy polyol, 15 parts of diphenylmethane diisocyanate, 30 parts of activated carbon, 20 parts of diatomaceous earth, 6 parts of stannous octoate, 10 parts of potassium chloride, 10 parts of polycarbonate, and 10 parts of trehalose lipid.

[0053] Preparation method:

[0054] S1. Take 100 parts of trihydroxy polyol and 10 parts of polycarbonate, mix and preheat at 90° C. for 1 hour.

[0055] S2. Add 30 parts of activated carbon, 20 parts of diatomaceous earth, 6 parts of stannous octoate catalyst, 10 parts of potassium chloride and 10 parts of trehalose lipid to the mixture in step S1, and stir at high speed in a stirrer at a speed of 1800 r / min.

[0056] S3. Add 15 parts of diphenylmethane diisocyanate and stir at high speed at a speed of 1800 r / min.

[0057] S4. Pour the matured polyurethane foam into a sealed mold with a vacuum degree of 0.70 MPa, introduce nitrogen with a pressure of 0.12 MPa, and release the pressure after inflating for 10 minutes to obtain the desired modified polyurethane carrier for enzyme immobilization.

[0058] After cutting, the particle size of the modified polyurethane carrier in this embodiment is 12 mm, and the true density is 0.907 kg / cm 3 .

[0059] Comparative Example 1

[0060] A modified polyurethane carrier in this comparative example is basically the same as Example 2, the main difference being that no activated carbon is added.

[0061] Comparative Example 2

[0062] A modified polyurethane carrier in this comparative example is basically the same as Example 2, the main difference being that diatomaceous earth is not added.

[0063] Comparative Example 3

[0064] The modified polyurethane carrier of this comparative example is basically the same as that of Example 2, except that no high molecular weight polymer-polystyrene is added.

[0065] Comparative Example 4

[0066] A modified polyurethane carrier in this comparative example is basically the same as Example 2, except that the bioactive agents chitosan and β-cyclodextrin are not added.

[0067] Experimental Example 1

[0068] This experimental example is used to reveal the structural changes of the polyurethane carrier of the present invention before and after modification.

[0069] The structural changes of polyurethane carrier before and after modification are as follows: Figure 1 , 2 shown.

[0070] Depend on Figure 1 , 2It can be seen that after the polyurethane carrier is modified by the method of the present invention, the porosity is higher and the specific surface area that can be used for loading biological enzymes is larger.

[0071] Experimental Example 2

[0072] This experimental example is used to verify the effects of activated carbon, diatomaceous earth, high molecular polymer, and bioactive agent on the bioenzyme loading capacity and impact resistance of the modified polyurethane carrier of the present invention.

[0073] After dissolving cellulase in ultrapure water to prepare a 1.5 g / L enzyme solution, the present invention (using the carrier of Example 2 as an example), Comparative Examples 1 to 4, and commercially available immobilized enzyme carriers (primary amine carrier product Sepabeads EA) were immersed in the above enzyme solution, and allowed to stand for adsorption at 25°C for 48 hours to obtain the corresponding immobilized cellulase. The bioenzyme loading and impact resistance of each immobilized cellulase obtained were tested, and the data are shown in Table 1.

[0074] Table 1 Carrier performance comparison

[0075]

[0076] From the results in Table 1, we can see that:

[0077] (1) Compared with the commercially available immobilized enzyme carrier (primary amine carrier product Sepabeads EA), the carrier of the present invention is improved in terms of enzyme activity loading and impact resistance.

[0078] (1) The porous structure of activated carbon can improve the adsorption performance and specific surface area of ​​the carrier, the silicon hydroxyl group of diatomaceous earth enhances the hydrophilicity of the carrier, the high molecular polymer increases the porosity and specific surface area of ​​the carrier, and the bioactive agent improves the biophilicity of the carrier; accordingly, the prepared modified polyurethane carrier has the characteristics of high porosity, large specific surface area, strong hydrophilicity and biophilicity, good impact resistance, etc., and is suitable for loading biological enzymes and has a high biological enzyme loading capacity.

[0079] Experimental Example 3

[0080] This experimental example is used to verify the effects of activated carbon and diatomaceous earth on the bioenzyme protection function of the modified polyurethane carrier of the present invention.

[0081] This experiment takes the anaerobic fermentation of excess sludge enhanced by biological enzymes as an example. The excess sludge used is the excess sludge produced by the secondary sedimentation tank of the sewage treatment system of the refinery enterprise, which contains heavy metals, aromatic hydrocarbons, refractory organic matter, humic acid and other substances, which seriously affect the activity of biological enzymes. This experiment was carried out in an intermittent reactor, with the anaerobic fermentation reaction temperature set at 35°C and the stirring speed set at 200r / min. The activity of the carrier enzyme before and after 24h was compared, and the experimental results are shown in Table 2.

[0082] Table 2 Comparison of carrier enzyme activity

[0083] Carrier Type Initial activity U / g Activity after 24h U / g Enzyme activity retention The vector of the present invention 316 281 88.9％ Comparative Example 1 Carrier 285 216 75.7％ Comparative Example 2 Carrier 293 233 79.5％

[0084] From the results in Table 2, we can see that:

[0085] Activated carbon and diatomaceous earth can adsorb and slowly release toxic substances, significantly reduce the toxicity of toxic substances to biological enzymes, and further improve the stability of immobilized enzymes.

[0086] In summary, the present invention changes the physical and chemical properties of the polyurethane carrier by adding inorganic, organic and biological materials, and the prepared modified polyurethane carrier has the characteristics of density close to that of water, high porosity, large specific surface area, strong hydrophilicity and biophilicity, good impact resistance, etc., is suitable for loading biological enzymes, and has a high biological enzyme loading capacity; at the same time, by utilizing the porosity and biophilicity of activated carbon and diatomaceous earth, relying on the adsorption and sustained release of toxic substances, the toxicity of toxic substances to biological enzymes can be significantly reduced, and the stability of immobilized enzymes can be further improved.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A modified polyurethane carrier for enzyme fixation, characterized in that: The invention comprises the following raw materials in parts by weight: 60-100 parts of polyether polyol, 5-15 parts of isocyanate, 10-30 parts of activated carbon, 5-20 parts of diatomaceous earth, 2-6 parts of catalyst, 5-10 parts of inorganic salt, 1-10 parts of high molecular polymer and 1-10 parts of bioactive agent.

2. The modified polyurethane carrier for enzyme immobilization according to claim 1, characterized in that: The polyether polyol is one or more of polyoxypropylene glycol, polytetramethylene glycol, tetramethylene glycol-oxypropylene copolymer glycol and trihydroxy polyol.

3. The modified polyurethane carrier for enzyme immobilization according to claim 1, characterized in that: The isocyanate is one or more of toluene diisocyanate and diphenylmethane diisocyanate.

4. The modified polyurethane carrier for enzyme immobilization according to claim 1, characterized in that: The catalyst is one or more of triethanolamine, triethylenediamine, dibutyltin dilaurate, and stannous octoate.

5. The modified polyurethane carrier for enzyme immobilization according to claim 1, characterized in that: The inorganic salt is one or more of anhydrous ferric chloride, sodium chloride, calcium chloride and potassium chloride.

6. The modified polyurethane carrier for enzyme immobilization according to claim 1, characterized in that: The high molecular polymer is one or more of polyethylene, polypropylene, polycarbonate, polystyrene, polyethylene glycol, and polypropylene methyl ester.

7. The modified polyurethane carrier for enzyme immobilization according to claim 1, characterized in that: The bioactive agent is one or more of sophorolipids, rhamnolipids, trehalolipids, chitosan, and β-cyclodextrin.

8. A method for preparing a modified polyurethane carrier for enzyme immobilization as claimed in any one of claims 1 to 7, characterized in that: Here are the steps: S1. Take 60-100 parts of polyether polyol and 1-10 parts of high molecular polymer, mix and preheat at 60-90°C for 1-2h; S2, adding 10-30 parts of activated carbon, 5-20 parts of diatomaceous earth, 2-6 parts of catalyst, 5-10 parts of inorganic salt and 1-10 parts of bioactive agent to the mixture of step S1, and stirring at high speed in a stirrer at a speed of 600-1800 r / min; S3, add 5-15 parts of isocyanate and stir at high speed at a speed of 600-1800r / min; S4. Pour the matured polyurethane foam into a sealed mold with a vacuum degree of 0.10-0.70 MPa, introduce nitrogen with a pressure of 0.08-0.12 MPa, inflate for 10-20 minutes and then release the pressure to obtain the desired modified polyurethane carrier for enzyme immobilization.

9. The method for preparing a modified polyurethane carrier for enzyme immobilization according to claim 8, characterized in that: The obtained modified polyurethane carrier has a particle size of 1 to 20 mm and a density of 0.895 to 0.945 kg / cm 3 .

10. Use of a modified polyurethane carrier for enzyme immobilization as claimed in any one of claims 1 to 7, characterized in that: The modified polyurethane carrier is immersed in an enzyme solution and allowed to stand for adsorption to obtain an immobilized enzyme.

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