CN116987215A - Preparation of novel high-strength carrier and application of novel high-strength carrier in immobilized enzyme - Google Patents

Preparation of novel high-strength carrier and application of novel high-strength carrier in immobilized enzyme Download PDF

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CN116987215A
CN116987215A CN202210436511.2A CN202210436511A CN116987215A CN 116987215 A CN116987215 A CN 116987215A CN 202210436511 A CN202210436511 A CN 202210436511A CN 116987215 A CN116987215 A CN 116987215A
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cyano
carrier
enzyme
crosslinked polymer
reaction
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阎虎生
张肆硕
崔强
李沂航
姜鹭
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Tianjin Nankai Hecheng Science & Technology Co ltd
Nankai University
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Nankai University
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Abstract

The invention provides a preparation method of a novel high-strength carrier and application of the novel high-strength carrier in immobilized enzyme. The carrier is prepared by carrying out suspension free radical copolymerization on functional monomers containing double bonds and cyano groups and crosslinking agents containing two or more double bonds in the presence of a pore-forming agent, and then carrying out functional reaction on the obtained crosslinked polymer to obtain the macroporous resin containing amino functional groups. The resin is used as an immobilized enzyme carrier and has the advantages of high enzyme carrying activity, high mechanical strength, good storage stability and the like.

Description

Preparation of novel high-strength carrier and application of novel high-strength carrier in immobilized enzyme
Technical Field
The invention relates to a macroporous amino resin carrier containing cyano, the strength of the resin carrier is obviously higher than that of the existing product, and the activity of immobilized enzyme prepared by adopting the series of resin carriers is obviously improved.
Technical Field
The enzyme catalytic reaction has the advantages of mild reaction condition, high catalytic reaction selectivity, high conversion rate and the like by taking water as a reaction medium. Meanwhile, the enzyme has the defects of poor stability, high price, difficulty in separating from products, difficulty in recycling and the like. The above problems can be well solved by immobilizing the enzyme on a carrier so that the catalytic reaction thereof occurs. The immobilized enzyme is insoluble enzyme which can be repeatedly or continuously used and is prepared by treating free enzyme by a certain chemical or physical method to limit the free enzyme to a certain space for catalytic reaction. The immobilized enzyme has various forms, and can be divided into a particle type, a linear type, a film type and an enzyme tube type according to different forms, wherein the particle type can be applied to the preparation of an enzyme column or directly applied to a reaction kettle for large-scale stirring catalytic reaction, so that the immobilized enzyme has the most widely applied form in industrial fermentation production. The performance of the immobilized enzyme is mainly dependent on the immobilization method and the carrier material used. Covalent bonding, also known as covalent coupling, is a process of immobilizing enzymes by covalent bonding of the side chain groups of the enzyme protein to the active functional groups in the carrier to form a firm and irreversible linkage. The immobilized enzyme has good stability, can be continuously used and has higher activity, and becomes the most common and active enzyme immobilization method in the current application and research. The structure and properties of the carrier material also directly determine the performance of the immobilized enzyme, and the immobilization of the enzyme has high requirements on the carrier material. Compared with natural high molecular materials and traditional inorganic materials, the synthetic organic high molecular material has the advantages of strong mechanical strength, high thermal stability and high chemical stability, good reaction polymerization capacity, excellent solvent resistance, easier regulation and control of structure and performance and the like, and is beneficial to the wide application of immobilized enzyme technology. The current synthetic polymer material carrier mainly comprises two major categories of polystyrene, poly (methyl) acrylic ester or acrylamide, and has the defects. Generally, the former has poor affinity and selectivity, and the framework has poor flexibility and is easy to be brittle after repeated use; the latter is difficult to maintain with good mechanical strength in the case of high porosity. Both ultimately result in a decrease in immobilized enzyme activity.
Disclosure of Invention
Aiming at the problems of the existing immobilized enzyme carrier, in particular to the reduction of enzyme activity caused by the poor mechanical strength of the immobilized enzyme carrier, the invention provides a preparation method of a novel high-strength carrier and application of the novel high-strength carrier in immobilized enzyme. The invention selects cross-linked polymer microsphere containing cyano group, which has large specific surface area, high mechanical strength, high stability, low price, easy preparation AND easy separation, as the substrate of the immobilized enzyme carrier, (BOZENA N.KOLARZ et al, POLYACRYLAMIDE SORBENTS SYNTHESIS AND SORPTION PROPERTIES, reactive Polymers,11, 1989, 29-35;ANDRZEJ TROCHIMCZUK et. Al, ACRYLIC ANION EXCHANGERS AND THEIR SORPTION PROPERTIES TOWARDS COPPER (II) AND COBALT (II), reactive Polymers,7, 1988, 197-202). The cyano-containing crosslinked polymer microsphere contains a large number of active cyano groups, is easy to carry out chemical modification, has required functional groups, and can provide effective binding sites for enzyme immobilization and a simple and mild immobilization process.
Amino groups are relatively common active groups for immobilized enzyme carriers (Yu Haofeng et al, CN 107641623B). The invention provides a novel macroporous resin as an immobilized enzyme carrier, which contains general primary amino groups, has low synthesis cost, good storage stability and high mechanical strength, and has good immobilization effect on various industrial enzymes such as penicillin G acylase, D-amino acid oxidase, GL-7-ACA acylase and the like.
The preparation method of the enzyme immobilization carrier comprises the steps of preparing a cyano-containing cross-linked polymer, reacting the cyano-containing cross-linked polymer with an amination reagent to introduce amino functional groups, then reacting with excessive dialdehyde, wherein one aldehyde group of the dialdehyde reacts with amino groups on the carrier to form Schiff base so as to enable the dialdehyde to be covalently immobilized on the carrier, the other aldehyde group of the dialdehyde immobilized on the carrier which does not participate in the reaction is used as an active group, and the enzyme immobilization process is that amino groups of enzyme molecules react with active aldehyde groups on the carrier to form Schiff base so as to immobilize the enzyme on the carrier.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: and (3) carrying out suspension free radical copolymerization on the functional monomer containing double bonds and cyano groups and a cross-linking agent containing one or more double bonds in the presence of a pore-forming agent to obtain a cross-linked polymer containing cyano groups, and carrying out a functionalization reaction on the obtained cross-linked polymer containing cyano groups and an excessive amount of amination reagent containing two primary amino groups to obtain the macroporous resin containing amino groups. The preparation method of the cyano-containing crosslinked polymer is that the cyano-containing crosslinked polymer is prepared by suspension free radical copolymerization of functional monomers containing double bonds and cyano groups and crosslinking agents containing two or more double bonds in the presence of a pore-forming agent and a free radical initiator. Wherein the functional monomer, the pore-forming agent and the initiator are mixed and dissolved into an oil phase, and the dispersing agent, the salt and the water are mixed and dissolved into a water phase. The functional monomer containing double bond and cyano group comprises one or more of acrylonitrile, methacrylonitrile and 2-butenenitrile, preferably one or two to three different monomers. The crosslinking agent of two or more double bonds comprises one or more of divinylbenzene, ethylene glycol dimethacrylate, allyl itaconate, triallyl cyanurate, triallyl isocyanurate, and combinations of two or three of them are preferable. The monomers are added with proper pore forming agent and initiator, stirred and dissolved, and then added into a dispersion medium for suspension polymerization by a method well known in the art. Wherein the porogen used comprises one or more of toluene, ethylbenzene, hexane, heptane, octane, dodecane, liquid paraffin, butyl acetate, butanol, hexanol, cyclohexanol, octanol, preferably 1 or a combination of two to three. Free radical initiators include peroxides such as benzoyl peroxide, lauroyl peroxide, stearoyl peroxide, di-t-hexyl peroxide, di-t-butyl peroxide, and the like, or azo compounds such as azobisisobutyronitrile, azobisisovaleronitrile, azobisisoheptonitrile, combinations of one or more of all of the foregoing. The dispersant is not particularly limited as long as it has a dispersion stabilizing effect in suspension polymerization. Including hydrophilic protective colloid agents such as one or more of polyvinyl alcohol, polyacrylic acid, gelatin, starch, and carboxymethyl cellulose. Typically, the dispersed phase also contains one or more inorganic salts, including combinations of one or more of sodium chloride, potassium chloride, ammonium chloride, calcium chloride, sodium sulfate, potassium sulfate, ammonium sulfate. The preparation method of the cyano-containing crosslinked polymer is suspension free radical copolymerization. Wherein, the proportion of the water phase to the oil phase is 3-10, and the preferable proportion is 4-8; the consumption of the dispersion stabilizer in the water phase is 0.1-2%, and the preferable consumption is 0.5-1%; the salt is used in the aqueous phase in an amount of 0 to 30%, preferably 3 to 10%; the content of the monomer containing double bond and cyano in the oil phase in the polymerizable monomer is 50-95%, preferably 60-70%; the content of the cross-linking agent in the polymerizable monomer is 5-50%, and the preferable content is 8-20%; the ratio of the pore-forming agent to the polymerizable monomer is 3/10-2/1, and the preferable ratio is 1/2-1/1; the ratio of the initiator amount to the polymerizable monomer is 0.2 to 2%, preferably 0.5 to 1%; the above contents or proportions are by weight. The cyano-containing crosslinked polymer obtained by polymerization is derivatized by reaction with an excessive amount of an amination reagent containing two primary amino groups, and a carrier for immobilized enzymes containing amino groups is prepared. The amination reagents used which contain two primary amino groups include: one or more of ethylenediamine, 1, 4-butanediamine, 1, 6-hexanediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, preferably one or a combination of one to three; the amination reagent used in the amination reaction contains 0 to 20 percent (mass ratio) of water, preferably 0 to 10 percent of water; the temperature of the amination reaction is 80-160 ℃, preferably 85-120 ℃; the amination reaction time is 5 to 24 hours, preferably 8 to 12 hours.
The amino-containing microspheres are reacted with an excess of dialdehydes, including dialdehydes of 2 to 10 carbons, preferably glutaraldehyde, to form Shan Xifu base to introduce aldehyde groups; the molar ratio of the amino groups on the resin to the dialdehyde is 1:3-1:20, preferably 1:5-1:10; the reaction temperature is from room temperature to 60 ℃, preferably room temperature; the reaction time is from 0.5 hours to 5 hours, preferably 2 hours; the pH of the reaction medium is from 4 to 10, preferably from 7 to 9.
The macroporous resin prepared by the proportion and the method has obviously higher strength than the prior product, and the enzyme activity after immobilization is also obviously improved (table 1).
Detailed Description
The invention is further illustrated by the following examples, which are only intended to provide a better understanding of the invention. It is to be understood that the inventive content is not to be limited to the scope of the embodiments, which is defined by the scope of the appended claims. And (3) detecting the water content of the carrier: measuring by using an electronic moisture meter of Shimadzu MOC-120H; and (3) detecting the content of the carrier functional groups: the measurement is carried out according to the method of weak base exchange amount in GB/T19861-2005; detection of carrier strength: in a fixed container, adding 1000mL of pure water into 100g of carrier, stirring for 5h by using a fixed stirring paddle with the rotating speed of 1000rpm, and taking liquid to carry out turbidity measurement by using a WGZ-3 type turbidity meter; detection of the most probable pore size and specific surface area of the support: the measurement was performed by means of a mercury porosimeter of the type 33/GT-17 of the United states of America Kang Da PoreMaster.
Example 1
(1) Polymerization reaction
1.5g of polyvinyl alcohol was dissolved in 300mL of distilled water, and 15g of sodium chloride was dissolved therein, which was referred to as an aqueous phase. 34.8g of acrylonitrile, 5.2g of divinylbenzene (content: 63%), 30g of toluene and 0.2g of azobisisobutyronitrile were mixed and stirred to dissolve the solid completely, called oil phase. Adding the oil phase into the water phase, starting mechanical stirring, regulating stirring speed to disperse the oil phase into small oil beads, heating to 65 ℃ and keeping the temperature of the system for 6 hours, and then heating to 75 ℃ and keeping the temperature for 4 hours. Stopping heating, cooling the system, filtering, collecting, washing with hot water for multiple times, and air drying to obtain the cyano-containing crosslinked polymer.
(2) Amination reaction
5g of the above cyano group-containing crosslinked polymer was suspended in 30mL of ethylenediamine, heated to 80℃with stirring and reacted for 4 hours, and then washed with deionized water several times until the washing solution was neutral. And (3) drying in vacuum to obtain the macroporous resin enzyme immobilization carrier containing amino. The water content of the carrier was found to be 67.1%, the amino content was found to be 3.60mmol/g, the stirring turbidity was found to be 98NTU, the most probable pore diameter was found to be 42nm, and the specific surface area was found to be 186m 2 /g。
(3) Immobilization of enzymes
5g of the above amino-containing carrier was added to 20mL of K containing 5% glutaraldehyde 2 HPO 4 -KH 2 PO 4 In buffer (0.2M, ph=8), after sealing, the support was filtered off and washed 5 times with buffer to give an activated support. 1g of activated carrier was added to 5mL K 2 HPO 4 -KH 2 PO 4 Adding penicillin G acylase solution into buffer solution (0.2M, pH=7.8), mixing uniformly, sealing, reacting at 20deg.C for 5h, filtering to obtain immobilized enzyme carrier, washing with pure water and 1M NaCl solution in sequence, and preserving at 4deg.C.
(4) Determination of enzyme-carrying Activity
After the penicillin G acylase is immobilized, the penicillin G potassium salt is catalytically cracked to prepare an antibiotic intermediate 6-APA, wherein the enzyme carrying activity is 280U/G, the single-batch catalytic time is 45min, and the substrate conversion rate is 98.1%. The method for measuring the enzyme-carrying activity comprises the following steps:
50mL of a 10% strength penicillin G potassium salt solution was heated to 37℃and 0.25G of the above immobilized enzyme was added with stirring and started to time, titrated with a 0.1mol/L NaOH standard solution, the pH of the reaction system was controlled to be 7.8, the volume of NaOH consumed at 10 minutes of reaction was recorded, and the activity of the immobilized enzyme was calculated as follows:
example 2
(1) Polymerization reaction
1.5g of polyvinyl alcohol was dissolved in 300mL of distilled water, and 18g of sodium chloride was dissolved therein, which was referred to as an aqueous phase. 34.8g of methacrylonitrile, 7g of divinylbenzene (content: 63%), 40g of 1, 2-dichloroethane, 0.25g of azobisisobutyronitrile were mixed and stirred to dissolve the solid completely, called oil phase. Adding the oil phase into the water phase, starting mechanical stirring, regulating stirring speed to disperse the oil phase into small oil beads, heating to 65 ℃ and keeping the temperature of the system for 6 hours, and then heating to 75 ℃ and keeping the temperature for 4 hours. Stopping heating, cooling the system, filtering and collecting the obtained resin, washing the resin with hot water for a plurality of times, and airing to obtain the cyano-containing crosslinked polymer.
(2) Amination reaction
5g of the crosslinked cyano-containing crosslinked polymer was suspended in 30mL of ethylenediamine, heated to 60℃with stirring, reacted for 8 hours, and then washed with deionized water several times until the washing solution was neutral. And (3) drying in vacuum to obtain the macroporous resin enzyme immobilization carrier containing amino. The water content of the carrier was found to be 67.3%, the amino content was found to be 3.40mmol/g, the stirring turbidity was found to be 62NTU, the most probable pore diameter was found to be 31nm, and the specific surface area was found to be 271m 2 /g。
(3) Immobilization of enzymes
5g of the above amino-containing carrier was added to 20mL of K containing 5% glutaraldehyde 2 HPO 4 -KH 2 PO 4 In buffer (0.2 m, ph=8), after sealing, the support was filtered off and washed 5 times with buffer to give an activated support. 1g of activated carrier was added to 5mL K 2 HPO 4 -KH 2 PO 4 Adding penicillin G acylase solution into buffer solution (0.2M, pH=7.8), mixing uniformly, sealing, reacting at 20deg.C for 5h, filtering to obtain immobilized enzyme carrier, washing with pure water and 1M NaCl solution in sequence, and preserving at 4deg.C.
(4) Determination of enzyme-carrying Activity
After the penicillin G acylase is immobilized, the penicillin G potassium salt is catalytically cracked to prepare an antibiotic intermediate 6-APA, wherein the enzyme carrying activity is 295U/G, the single-batch catalytic time is 35min, and the substrate conversion rate is 98.9%.
Example 3
(1) Polymerization reaction
3g of gelatin was dissolved in 300mL of distilled water, and 20g of sodium chloride was dissolved therein, which was referred to as an aqueous phase. 16g of 2-butenenitrile, 4.5g of divinylbenzene (content: 63%), 2.5g of triallyl cyanurate, 15g of toluene, 10g of n-octane and 0.25g of benzoyl peroxide were mixed and stirred to dissolve the solids completely, called oil phase. Adding the oil phase into the water phase, stirring mechanically, regulating stirring speed to disperse the oil phase into small oil beads, heating to 80deg.C, and maintaining for 12 hr. Stopping heating, cooling the system, filtering and collecting the obtained resin, washing the resin with hot water for a plurality of times, and airing to obtain the cyano-containing crosslinked polymer.
(2) Amination reaction
5g of the crosslinked cyano-containing crosslinked polymer described above was suspended in 30mL of diethylenetriamine, heated to 120℃with stirring, reacted for 4 hours, and then washed with deionized water several times until the washing solution was neutral. And (3) drying in vacuum to obtain the macroporous resin enzyme immobilization carrier containing amino. The water content of the carrier was 75.6%, the amino content was 3.83mmol/g, the stirring turbidity was 115NTU, the most probable pore diameter was 38nm, and the specific surface area was 345m 2 /g。
(3) Immobilization of enzymes
5g of the above amino-containing carrier was added to 20mL of K containing 5% glyoxal 2 HPO 4 -KH 2 PO 4 In buffer (0.2M, ph=8), after sealing, the support was filtered off and washed 5 times with buffer to give an activated support. 1g of activated carrier was added to 5mL K 2 HPO 4 -KH 2 PO 4 Adding desalted D-amino acid oxidase solution into buffer solution (0.2M, pH=7.8), mixing well, sealing, reacting at 20deg.C for 20 hr, filtering to obtain immobilized enzyme carrier, washing with pure water and 1M NaCl solution sequentially, and preserving at 4deg.C.
(4) Determination of enzyme-carrying Activity
After the D-amino acid oxidase is immobilized, the cephalosporin C sodium salt is catalyzed and oxidized to prepare an antibiotic intermediate GL-7-ACA, the enzyme activity is 201U/g, the single batch catalysis time is 46min, and the substrate conversion rate is 98.5%. The detection method of the enzyme activity of the D-amino acid oxidase comprises the following steps:
50mL of 5% strength cephalosporin C sodium salt solution was heated to 20℃and 0.25g of the above immobilized enzyme was added under stirring, oxygen was introduced, and 3mol/L NH was used 3 ·H 2 Dripping O standard solution, controlling pH=7.2 of the reaction system, sampling after 10min of reaction, and detecting cephalosporin in the reaction system before and after the reaction by using a high performance liquid chromatographThe change of the C content calculates the activity of the immobilized enzyme according to the following formula:
example 4
(1) Polymerization reaction
2g of gelatin, 1g of polyvinyl alcohol were dissolved in 300mL of distilled water, and 18g of sodium chloride was dissolved therein, which was called an aqueous phase. 30g of acrylonitrile, 4.5g of divinylbenzene (content: 63%), 15g of toluene, 10g of n-heptane and 0.25g of benzoyl peroxide were mixed and stirred to dissolve the solids completely, called oil phase. Adding the oil phase into the water phase, stirring mechanically, regulating stirring speed to disperse the oil phase into small oil beads, heating to 60deg.C, and maintaining for 5 hr. Stopping heating, cooling the system, filtering and collecting the obtained resin, washing the resin with hot water for a plurality of times, and airing to obtain the cyano-containing crosslinked polymer.
(2) Amination reaction
5g of the crosslinked cyano-containing crosslinked polymer described above was suspended in 30mL of diethylenetriamine, heated to 150℃with stirring, reacted for 2 hours and then washed with deionized water several times until the washing solution was neutral. And (3) drying in vacuum to obtain the macroporous resin enzyme immobilization carrier containing amino. The water content of the carrier was 75.3%, the amino content was 3.95mmol/g, the stirring turbidity was 89NTU, the most probable pore diameter was 36nm, and the specific surface area was 268m 2 /g。
(3) Immobilization of enzymes
5g of the above amino-containing carrier was added to 20mL of K containing 5% glutaraldehyde 2 HPO 4 -KH 2 PO 4 In buffer (0.2M, ph=8), after sealing, the support was filtered off and washed 5 times with buffer to give an activated support. 1g of activated carrier was added to 5mL K 2 HPO 4 -KH 2 PO 4 Adding 250U desalted GL-7-ACA acylase solution into buffer solution (0.2M, pH=7.8), mixing uniformly, sealing, reacting at 25 ℃ for 40h, filtering out immobilized enzyme carrier, washing with pure water and 1M NaCl solution in sequence, and preserving at 4 ℃.
(4) Determination of enzyme-carrying Activity
The immobilized enzyme carrier obtained in example 4 is used for catalyzing and cracking GL-7-ACA by immobilized GL-7-ACA acylase to prepare an antibiotic intermediate 7-ACA, and the enzyme activity is 150U/g, and the single batch catalysis time is 59min. The method for measuring the GL-7-ACA acylase enzyme activity comprises the following steps:
50mL of 2% GL-7-ACA solution is heated to 37 ℃, 0.25g of the immobilized enzyme is added under stirring, timing is started, 0.1mol/L NaOH standard solution is used for titration, the pH=8.0 of the reaction system is controlled, the volume of NaOH consumed during the reaction for 10min is recorded, and the activity of the immobilized enzyme is calculated according to the following formula:
the preparation of 6-APA by immobilized penicillin G acylase is taken as an example of a plurality of immobilized enzyme carrier samples prepared by the method of the patent, and compared with a commercial carrier (a primary amino carrier product Sepabeads EC-EA of Mitsubishi corporation of Japan). The enzyme activity, the catalytic rate and the like are improved, the mechanical strength is obviously improved, and the comparison data are shown in table 1.
TABLE 1 Activity and Strength of novel vector immobilized enzyme
Wherein, the shorter the single batch of catalytic time is, the higher the activity of the immobilized enzyme is, and the better the reaction kinetics is; the lower the turbidity of the medium after rubbing, the higher the mechanical strength of the support.
Description: the above embodiments are only for illustrating the present invention and not for limiting the technical solution described in the present invention; thus, while the invention has been described in detail with reference to the various embodiments described above, it will be understood by those skilled in the art that the invention may be modified or equivalents; all technical solutions and modifications thereof that do not depart from the spirit and scope of the present invention are intended to be included in the scope of the appended claims.

Claims (10)

1. An aldehyde group-containing high-strength carrier (macroporous resin) for enzyme immobilization, characterized in that: the macroporous resin is prepared by reacting a cyano-containing crosslinked polymer with an excessive amount of an amination reagent containing two primary amino groups to obtain an amino-containing resin, and then reacting with an excessive amount of dialdehyde.
2. The method for preparing macroporous resin according to claim 1, wherein: the preparation method of the cyano-containing crosslinked polymer comprises the step of carrying out suspension radical copolymerization on functional monomers containing double bonds and cyano groups and crosslinking agents containing two or more double bonds in the presence of a pore-forming agent and a radical initiator, wherein the functional monomers, the pore-forming agent and the initiator are miscible as an oil phase, and the dispersing agent, the salt and the water are miscible as a water phase.
3. The cyano-containing crosslinked polymer of claim 1 wherein: the functional monomer containing double bond and cyano group comprises one or more of acrylonitrile, methacrylonitrile and 2-butenenitrile.
4. The cyano-containing crosslinked polymer of claim 1 wherein: the crosslinking agent with two or more double bonds comprises one or more of divinylbenzene, ethylene glycol dimethacrylate, allyl itaconate, triallyl cyanurate and triallyl isocyanurate.
5. The method for producing a cyano-containing crosslinked polymer according to claim 2, characterized in that: the pore-forming agent used in the preparation method comprises one or a combination of more of toluene, ethylbenzene, hexane, heptane, octane, dodecane, liquid paraffin, butyl acetate, butanol, hexanol, cyclohexanol and octanol.
6. The method for producing a cyano-containing crosslinked polymer according to claim 2, characterized in that: the dispersant is not particularly limited as long as it has a dispersion stabilizing effect in suspension polymerization, and includes a hydrophilic protective colloid agent such as a combination of one or more of polyvinyl alcohol, polyacrylic acid, gelatin, starch and carboxymethyl cellulose; the salt comprises one or more of sodium chloride, potassium chloride, ammonium chloride, calcium chloride, sodium sulfate, potassium sulfate, and ammonium sulfate.
7. The method for producing a cyano-containing crosslinked polymer according to claim 2, characterized in that: in the suspension free radical copolymerization, the proportion of the water phase to the oil phase is 3-10, and the preferable proportion is 4-8; the consumption of the dispersion stabilizer in the water phase is 0.1-2%, and the preferable consumption is 0.5-1%; the salt is used in the aqueous phase in an amount of 0 to 30%, preferably 3 to 10%; the content of the monomer containing double bond and cyano in the oil phase in the polymerizable monomer is 50-95%, preferably 60-70%; the content of the cross-linking agent in the polymerizable monomer is 5-50%, and the preferable content is 8-20%; the ratio of the pore-forming agent to the polymerizable monomer is 3/10-2/1, and the preferable ratio is 1/2-1/1; the ratio of the initiator amount to the polymerizable monomer is 0.2 to 2%, preferably 0.5 to 1%; the above contents or proportions are by weight.
8. The reaction of a cyano-containing crosslinked polymer with an excess of an amination reagent comprising two primary amino groups according to claim 1, wherein: the amination reagent containing two primary amino groups comprises: one or more of ethylenediamine, 1, 4-butanediamine, 1, 6-hexanediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, preferably one or a combination of one to three; the amination reagent used in the amination reaction contains 0 to 20 percent (mass ratio) of water, preferably 0 to 10 percent of water; the temperature of the amination reaction is 80-160 ℃, preferably 85-120 ℃; the amination reaction time is 5 to 24 hours, preferably 8 to 12 hours.
9. Reaction of an amino-containing resin according to claim 1 with an excess of a dialdehyde, characterized in that the dialdehyde comprises a dialdehyde of 2 to 10 carbons, preferably glutaraldehyde; the molar ratio of the amino groups on the resin to the dialdehyde is 1:3-1:20, preferably 1:5-1:10; the reaction temperature is from room temperature to 60 ℃, preferably room temperature; the reaction time is from 0.5 hours to 5 hours, preferably 2 hours; the pH of the reaction medium is from 4 to 10, preferably from 7 to 9.
10. The macroporous resin of the invention is applied to the field of immobilized enzymes, wherein the enzyme which can be immobilized can be any enzyme compatible with the resin, including but not limited to penicillin G acylase, D-amino acid oxidase, GL-7-ACA acylase and the like.
CN202210436511.2A 2022-04-25 2022-04-25 Preparation of novel high-strength carrier and application of novel high-strength carrier in immobilized enzyme Pending CN116987215A (en)

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