CN113512153A - Method for preparing novel UCST type temperature-sensitive polymer immobilized enzyme - Google Patents

Method for preparing novel UCST type temperature-sensitive polymer immobilized enzyme Download PDF

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CN113512153A
CN113512153A CN202110403656.8A CN202110403656A CN113512153A CN 113512153 A CN113512153 A CN 113512153A CN 202110403656 A CN202110403656 A CN 202110403656A CN 113512153 A CN113512153 A CN 113512153A
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immobilized enzyme
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CN113512153B (en
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朱兴
田祎
贺斌
王学川
张慧洁
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Shaanxi University of Science and Technology
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    • C08F265/00—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00
    • C08F265/02—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00 on to polymers of acids, salts or anhydrides
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087—Acrylic polymers

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Abstract

The invention discloses a method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme, which comprises the following steps: firstly, dissolving a polyacrylic acid polymer and an initiator by using deionized water, transferring the polyacrylic acid polymer and the initiator into a three-neck flask, slowly adding glycidyl methacrylate, shaking up, introducing nitrogen for bubbling for half an hour, transferring the mixture into a single-neck flask, vacuumizing under the protection of nitrogen, carrying out magnetic stirring reaction, centrifuging, washing by using acetone, and drying to obtain a solid product; and dissolving the solid product by using a buffer solution, adding enzyme, carrying out constant-temperature oscillation reaction, adding collagen and hydrolysate thereof, oscillating at constant temperature, centrifuging, cleaning to obtain a precipitate, namely the immobilized enzyme carrier, and refrigerating the immobilized enzyme carrier in a refrigerator to finish the preparation of the immobilized enzyme carrier. The UCST type temperature-sensitive polymer-immobilized enzyme system can realize liquid-phase catalysis of a substrate and overcome the defect of low catalysis efficiency of the traditional immobilized enzyme solid-phase catalysis of the substrate.

Description

Method for preparing novel UCST type temperature-sensitive polymer immobilized enzyme
Technical Field
The invention belongs to the technical field of immobilized enzymes, and particularly relates to a method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme.
Background
The enzyme is a bioactive macromolecule with high-efficiency catalytic capability, and is widely applied to the fields of industrial production, medicine and health, environmental protection and the like. However, in most cases, the specific catalytic reaction of the enzyme is carried out in an aqueous solution, remains in the solution after the reaction, is difficult to separate and recover, cannot be reused for many times, and increases the production cost, which has been a major limitation for industrial application. Immobilization of enzymes on solid supports is an effective method for recycling enzymes. However, when the immobilized enzyme carrier catalyzes the substrate, the contact sites of the enzyme with the substrate are reduced, and the catalytic efficiency is reduced, which greatly limits the use thereof.
At present, the temperature-sensitive polymer is used as a novel material with reversible solid-liquid phase conversion, and can catalyze a substrate in a liquid phase and separate and recycle the solid phase by regulating the temperature, so that the problem of low efficiency of catalyzing the substrate by a solid enzyme preparation is solved. The most widely studied (UCST) type poly (acrylic acid-co-acrylamide) compounds in the temperature sensitive materials have the high critical solution temperature of about 15 ℃ generally, and can catalyze substrates in a high-temperature liquid phase and rapidly recycle a low-temperature solid phase. However, the UCST system prepared by acrylamide monomers has certain potential safety hazard, polyacrylamide has certain toxicity, and the acrylamide series temperature-sensitive polymers can generate amine micromolecular compounds harmful to the body in the degradation/hydrolysis process, so that the application process of the acrylamide series temperature-sensitive polymers in the field of biotechnology is greatly restricted.
Disclosure of Invention
The invention aims to provide a novel method for immobilizing UCST type temperature-sensitive polymer, which can greatly improve the catalytic activity and stability of the enzyme.
The technical scheme adopted by the invention is that a method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme is implemented according to the following steps:
step 1, dissolving a polyacrylic acid polymer and an initiator by using deionized water, transferring the polyacrylic acid polymer and the initiator into a three-neck flask, slowly adding glycidyl methacrylate, shaking uniformly, introducing nitrogen for bubbling for half an hour, transferring the mixture into a single-neck flask, vacuumizing under the protection of the nitrogen, carrying out magnetic stirring reaction, centrifuging to remove supernatant after the reaction is finished, repeatedly washing by using acetone, and drying to obtain a white solid product;
and 2, dissolving the solid product by using a buffer solution, adding an enzyme, carrying out constant-temperature oscillation reaction, adding collagen and a hydrolysate thereof, oscillating for 30min at a constant temperature, centrifuging, washing by using deionized water to obtain a precipitate, namely the immobilized enzyme carrier, and refrigerating the immobilized enzyme carrier in a refrigerator to finish the preparation of the immobilized enzyme carrier.
The present invention is also characterized in that,
in the step 1, the molar ratio of the polyacrylic acid polymer, the glycidyl methacrylate, the initiator and the deionized water is 1-12: 0.01-0.024: 0.01-0.012: 20-480.
In the step 1, the reaction temperature is 50-100 ℃, and the reaction time is 4-24 h; the drying temperature is 0-40 ℃, and the drying time is 10-12 h.
In the step 1, the initiator is ammonium persulfate, potassium persulfate or sodium persulfate; the polyacrylic acid polymer has the structural formula of (C)3O2H3-R)nWherein, R is any one of methyl, ethyl, isobutyl, methoxy, ethoxy, propoxy, butoxy, formyloxy, acetoxy, propionyloxy and butyryloxy.
In the step 2, the molar ratio of the solid product, the buffer solution, the enzyme, the collagen and the hydrolysate thereof is 0.1-1: 1-10: 10-40: 0.1-1.
In the step 2, the buffer solution is any one of acetic acid buffer solution, citric acid buffer solution, phosphate buffer solution, borate buffer solution and tris buffer solution; the collagen and the hydrolysate thereof are collagen, gelatin or collagen polypeptide; the enzyme is any one or more of cellulase, lipase, protease, saccharifying enzyme, amylase, laccase and xylanase.
In the step 2, the constant temperature oscillation reaction temperature is 40-100 ℃, and the constant temperature oscillation reaction time is 2-24 h.
The invention has the beneficial effects that: the UCST-type temperature-sensitive polymer uses biomass material collagen and hydrolysate material thereof to replace acrylamide to prepare a UCST system. Endows the UCST type system with good biocompatibility and biodegradability so as to solve the defects of toxicity, harm and difficult degradation of the existing UCST type temperature-sensitive polymer. And the UCST type temperature-sensitive polymer-immobilized enzyme system can realize liquid-phase catalysis of a substrate and overcome the defect of low catalysis efficiency of the traditional immobilized enzyme solid-phase catalysis of the substrate.
Drawings
FIG. 1 is a diagram of the present invention for recycling a UCST temperature sensitive polymer-immobilized enzyme system;
FIG. 2 is a graph showing the glycation degree of free and immobilized cellulase-catalyzed filter papers according to the present invention as a function of time;
FIG. 3 is a graph showing the results of the verification of the recycling stability of the immobilized cellulase of the present invention.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
The invention relates to a method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme, which is implemented according to the following steps:
step 1, dissolving a polyacrylic acid polymer and an initiator by using deionized water at normal temperature, transferring the polyacrylic acid polymer and the initiator into a three-neck flask, slowly adding glycidyl methacrylate, shaking up, introducing nitrogen for bubbling for half an hour, transferring the mixture into a single-neck flask, vacuumizing under the protection of the nitrogen, carrying out magnetic stirring reaction, centrifuging to remove supernatant after the reaction is finished, repeatedly washing the supernatant for three times by using acetone, and drying to obtain a white solid product;
the mol ratio of the polyacrylic acid polymer to the glycidyl methacrylate to the initiator to the deionized water is 1-12: 0.01-0.024: 0.01-0.012: 20-480;
the initiator is ammonium persulfate, potassium persulfate or sodium persulfate;
the reaction temperature is 50-100 ℃, and the reaction time is 4-24 h;
the drying temperature is 0-40 ℃, and the drying time is 10-12 h;
the polyacrylic acid polymer has the structural formula of (C)3O2H3-R)nThe structural formula is as follows:
wherein, R is any one of methyl, ethyl, isobutyl, methoxy, ethoxy, propoxy, butoxy, formyloxy, acetoxyl, propionyloxy and butyryloxy;
step 2, dissolving the solid product by using a buffer solution, then adding enzyme, carrying out constant-temperature oscillation reaction at 40 ℃, then adding collagen and hydrolysate thereof, carrying out constant-temperature oscillation for 30min, cooling, centrifuging, repeatedly cleaning for 3 times by using deionized water, reserving the supernatant and measuring the protein concentration to obtain a precipitate which is immobilized enzyme, and placing the immobilized enzyme in a refrigerator for refrigeration at the refrigeration temperature of 4 ℃; thus completing the enzyme immobilization;
the molar ratio of the solid product, the buffer solution, the enzyme, the collagen and the hydrolysate thereof is 0.1-1: 1-10: 10-40: 0.1 to 1;
the buffer solution is any one of acetic acid buffer solution, citric acid buffer solution, phosphate buffer solution, borate buffer solution and tris buffer solution;
the enzyme is any one or more of cellulase, lipase, protease, saccharifying enzyme, amylase, laccase and xylanase;
the collagen and the hydrolysate thereof are collagen, gelatin or collagen polypeptide;
the constant temperature shaking reaction temperature is 40-100 ℃, and the constant temperature shaking reaction time is 2-24 h, preferably 4 h.
Example 1
The invention relates to a method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme, which specifically comprises the following steps:
dissolving 0.5g of polymethyl methacrylate and 0.0324g of Ammonium Persulfate (APS) by using 40ml of deionized water at normal temperature, transferring the solution into a three-neck flask, slowly adding 9uL of glycidyl methacrylate, shaking the solution evenly, introducing nitrogen for bubbling for about half an hour, transferring the solution into a single-neck flask, vacuumizing the flask under the protection of nitrogen, magnetically stirring the solution at the temperature of 60 ℃ for reaction for 4 hours, precipitating the solution by using acetone, centrifuging the solution, removing supernatant, repeatedly washing the solution by using acetone for three times, and drying the solution in a vacuum drying oven at the temperature of 40 ℃ for 10 hours to obtain a white solid. Dissolving 0.5g of the obtained product in 10mL of acetic acid buffer solution, adding 20mg of cellulase, carrying out constant-temperature shaking reaction at 40 ℃ for 4h, adding 0.5g of collagen (derived from pigskin), carrying out constant-temperature shaking for 30min, cooling, centrifuging, repeatedly washing for 3 times, keeping the supernatant for measuring the protein concentration, and storing the obtained immobilized cellulase in a refrigerator at 4 ℃. The phase transition temperature of the UCST type temperature-sensitive polymer system is 40 ℃, and about 86.5 percent of the original activity of the UCST type temperature-sensitive polymer system can be still reserved after 10 times of circulation.
Example 2
The invention relates to a method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme, which specifically comprises the following steps:
dissolving 0.5g of poly (2-ethyl acrylic acid) and 0.0324g of potassium persulfate by using 40mL of deionized water at normal temperature, transferring the solution into a three-neck flask, slowly adding 9uL of glycidyl methacrylate, shaking the solution evenly, introducing nitrogen, bubbling for about half an hour, transferring the solution into a single-neck flask, vacuumizing the flask under the protection of nitrogen, magnetically stirring the solution at the temperature of 60 ℃ for reaction for 4 hours, precipitating the solution by using acetone, centrifuging the solution, removing supernatant, repeatedly washing the solution by using the acetone for three times, and drying the solution overnight in a vacuum drying oven at the temperature of 40 ℃ to obtain a white solid. Dissolving 0.5g of the obtained product by using 10mL of phosphate buffer solution, adding 20mg of amylase, carrying out constant-temperature shaking reaction for 4h at 40 ℃, adding 0.5g of collagen, carrying out constant-temperature shaking for 30min, cooling, centrifuging, repeatedly cleaning for 3 times, keeping the supernatant, measuring the protein concentration, and storing the obtained immobilized amylase in a refrigerator at 4 ℃. The phase transition temperature of the UCST type temperature-sensitive polymer system is 44 ℃, and about 87.6 percent of the original activity of the UCST type temperature-sensitive polymer system can be still reserved after 10 times of circulation.
Example 3
The invention relates to a method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme, which specifically comprises the following steps:
dissolving 0.5g of polyacrylic acid and 0.0324g of Ammonium Persulfate (APS) by using 40mL of deionized water at normal temperature, transferring the solution into a three-neck flask, slowly adding 9uL of glycidyl methacrylate, shaking the solution evenly, introducing nitrogen, bubbling for about half an hour, transferring the solution into a single-neck flask, vacuumizing the flask under the protection of nitrogen, magnetically stirring the solution at the temperature of 60 ℃ for reaction for 4 hours, precipitating the solution by using acetone, centrifuging the solution, removing supernatant, repeatedly washing the solution by using the acetone for three times, and drying the solution overnight in a vacuum drying oven at the temperature of 40 ℃ to obtain a white solid. Dissolving 0.5g of the obtained product by using 10ml of phosphate buffer solution, adding 20mg of protease, carrying out constant-temperature shaking reaction at 40 ℃ for 4h, adding 0.5g of collagen, carrying out constant-temperature shaking for 30min, cooling, centrifuging, repeatedly cleaning for 3 times, keeping the supernatant, measuring the protein concentration, and storing the obtained immobilized protease material in a refrigerator at 4 ℃. The phase transition temperature of the UCST type temperature-sensitive polymer system is 36 ℃, and about 85 percent of the original activity of the UCST type temperature-sensitive polymer system can be still reserved after 10 times of circulation.
Example 4
The invention relates to a method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme, which specifically comprises the following steps:
dissolving 0.5g of polyacrylic acid and 0.0324g of ammonium persulfate by using 40ml of deionized water at normal temperature, transferring the solution into a three-neck flask, slowly adding 9uL of glycidyl methacrylate, shaking the solution evenly, introducing nitrogen for bubbling for about half an hour, transferring the solution into a single-neck flask, vacuumizing the flask under the protection of nitrogen, magnetically stirring the solution at the temperature of 60 ℃ for reaction for 4 hours, precipitating the solution by using acetone, centrifuging the solution, removing supernatant, repeatedly washing the solution by using the acetone for three times, and drying the solution overnight in a vacuum drying oven at the temperature of 40 ℃ to obtain white solid. Dissolving 0.5g of the obtained product by using 10mL of acetic acid buffer solution, adding 20mg of protease, carrying out constant-temperature shaking reaction at 40 ℃ for 4h, adding 0.5g of gelatin, carrying out constant-temperature shaking for 30min, cooling, centrifuging, repeatedly cleaning for 3 times, keeping the supernatant, measuring the protein concentration, and storing the obtained immobilized glucoamylase material in a refrigerator at 4 ℃. The phase transition temperature of the UCST type temperature-sensitive polymer system is 38 ℃, and about 83.2 percent of the original activity of the UCST type temperature-sensitive polymer system can be still reserved after 10 times of circulation.
Example 5
The invention relates to a method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme, which specifically comprises the following steps:
dissolving 0.5g of polyacrylic acid and 0.0324g of ammonium persulfate by using 40ml of deionized water at normal temperature, transferring the solution into a three-neck flask, slowly adding 9uL of glycidyl methacrylate, shaking the solution evenly, introducing nitrogen for bubbling for about half an hour, transferring the solution into a single-neck flask, vacuumizing the flask under the protection of nitrogen, magnetically stirring the solution at the temperature of 60 ℃ for reaction for 4 hours, precipitating the solution by using acetone, centrifuging the solution, removing supernatant, repeatedly washing the solution by using the acetone for three times, and drying the solution overnight in a vacuum drying oven at the temperature of 40 ℃ to obtain white solid. Dissolving 0.5g of the obtained product by using 10mL of citric acid buffer solution, adding 20mg of xylanase, carrying out constant-temperature shaking reaction for 4h at the temperature of 40 ℃, adding 0.5g of collagen polypeptide, carrying out constant-temperature shaking for 30min, cooling, centrifuging, repeatedly washing for 3 times, keeping the measured protein concentration of the supernatant, and storing the obtained immobilized xylanase in a refrigerator at the temperature of 4 ℃. The phase transition temperature of the UCST type temperature-sensitive polymer system is 41 ℃, and about 84.4 percent of the original activity of the UCST type temperature-sensitive polymer system can be still reserved after 10 times of circulation.
FIG. 1 is a diagram of the recycling of a UCST temperature sensitive polymer-immobilized enzyme system. The figure shows that the UCST temperature sensitive polymer-immobilized enzyme system can efficiently catalyze the substrate in a high-temperature liquid phase, and the solid phase can be rapidly recycled.
FIG. 2 is a graph showing the glycation degree of free and immobilized cellulase-catalyzed filter paper as a function of time. Whether the immobilized enzyme catalysis substrate can achieve the same catalytic activity as the free enzyme or not is an important index for evaluating the commercial value of the immobilized enzyme. As shown in FIG. 2, after 52h of catalytic hydrolysis of the filter paper, the immobilized enzyme was able to achieve the same saccharification yield as the free enzyme.
FIG. 3 is a graph showing the results of the verification of the recycling stability of the immobilized cellulase. The recycling stability is an important reference standard for measuring whether the enzyme immobilization is successful and effective. As shown in FIG. 3, after the immobilized cellulase of the catalytic decomposition filter paper is repeatedly used for 10 times, the relative enzyme activity still reaches 86.5 percent. When part of enzyme activity is lost, the loss inevitably occurs in the separation process, and the hydrogen bond action is weakened along with the increase of the cycle number, so that the immobilized enzyme is difficult to completely separate. However, the immobilized cellulase of the experiment has potential broad application prospect in view of the non-reusability of the free enzyme.

Claims (7)

1. A method for preparing a novel UCST type temperature-sensitive polymer immobilized enzyme is characterized by comprising the following steps:
step 1, dissolving a polyacrylic acid polymer and an initiator by using deionized water, transferring the polyacrylic acid polymer and the initiator into a three-neck flask, slowly adding glycidyl methacrylate, shaking uniformly, introducing nitrogen for bubbling for half an hour, transferring the mixture into a single-neck flask, vacuumizing under the protection of the nitrogen, carrying out magnetic stirring reaction, centrifuging to remove supernatant after the reaction is finished, repeatedly washing by using acetone, and drying to obtain a white solid product;
and 2, dissolving the solid product by using a buffer solution, adding enzyme, carrying out constant-temperature oscillation reaction, adding collagen and hydrolysate thereof, oscillating for 30min at constant temperature, centrifuging, washing by using deionized water to obtain a precipitate, namely immobilized enzyme, and refrigerating the immobilized enzyme in a refrigerator to finish the immobilization.
2. The method for immobilizing a novel UCST-type thermo-sensitive polymer according to claim 1, wherein in the step 1, the molar ratio of the polyacrylic acid polymer, the glycidyl methacrylate, the initiator and the deionized water is 1-12: 0.01-0.024: 0.01-0.012: 20-480.
3. The method for preparing a novel UCST-type temperature-sensitive polymer immobilized enzyme according to claim 1, wherein in the step 1, the reaction temperature is 50-100 ℃, and the reaction time is 4-24 h; the drying temperature is 0-40 ℃, and the drying time is 10-12 h.
4. The method for immobilizing a novel UCST-type thermo-sensitive polymer according to claim 1, wherein in step 1, the initiator is ammonium persulfate, potassium persulfate or sodium persulfate; the polyacrylic acid polymer has the structural formula of (C)3O2H3-R)nWherein, R is any one of methyl, ethyl, isobutyl, methoxy, ethoxy, propoxy, butoxy, formyloxy, acetoxy, propionyloxy and butyryloxy.
5. The method for immobilizing a novel UCST-type thermo-sensitive polymer according to claim 1, wherein the molar ratio of the solid product, the buffer, the enzyme, the collagen and the hydrolysate thereof in step 2 is 0.1-1: 1-10: 10-40: 0.1-1.
6. The method for immobilizing a novel UCST-type thermo-sensitive polymer according to claim 1, wherein in step 2, the buffer is any one of an acetic acid buffer, a citric acid buffer, a phosphate buffer, a borate buffer, and a tris buffer; the collagen and the hydrolysate thereof are collagen, gelatin or collagen polypeptide; the enzyme is any one or more of cellulase, lipase, protease, saccharifying enzyme, amylase, laccase and xylanase.
7. The method for preparing a novel UCST-type temperature-sensitive polymer immobilized enzyme according to claim 1, wherein in the step 2, the reaction temperature is controlled to be 40-100 ℃ by constant temperature oscillation, and the reaction time is controlled to be 2-24 h by constant temperature oscillation.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114350647A (en) * 2021-12-23 2022-04-15 江苏大学 Application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase

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US3764477A (en) * 1967-12-27 1973-10-09 Roehm Gmbh Resin bound protein like active agents prepared by reacting a proteinwith a water insoluble polymer of acrylic or methacrylic acid anhydride
EP0008498A1 (en) * 1978-08-15 1980-03-05 Imperial Chemical Industries Plc Immobilized enzyme preparation and process for its production
US20160101190A1 (en) * 2013-04-22 2016-04-14 Alan J. Russell POLYMER-BASED PROTEIN ENGINEERING METHODS TO RATIONALLY TUNE ENZYME ACTIVITY, pH-DEPENDENCE AND STABILITY
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114350647A (en) * 2021-12-23 2022-04-15 江苏大学 Application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase
CN114350647B (en) * 2021-12-23 2024-05-10 江苏大学 Application of UCST type nonionic water-soluble polymer in immobilized glucose isomerase

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