Method for aminating polystyrene resin and method for immobilizing enzymes using aminated resin

Abstract

The present invention provides a method for aminating a polystyrene resin and a method for immobilizing an enzyme using an aminated polystyrene resin. The amination method includes the steps of catalyzing a Friedel-Crafts alkylation reaction of a polystyrene resin and an enamine salt in a solvent using a Lewis acid catalyst to obtain an aminated polystyrene resin. The Friedel-Crafts alkylation reaction is used to graft an enamine salt onto a polystyrene resin, thereby completing the amination of the polystyrene resin. The Friedel-Crafts alkylation reaction is easy to control conditions and has a simple post-treatment process, and the catalyst and unreacted enamine salt only need to be removed by washing. Therefore, the above amination method of the present application has fewer steps, is simple and easy to operate, and avoids the use of a precious metal catalyst, thereby reducing production costs. When the obtained polystyrene resin is used as an enzyme immobilization support, it has good immobilization effect and reusability, greatly expanding the enzyme immobilization support library.

Description

[Technical field]

[0001] The present invention relates to the technical field of enzyme immobilization, and more particularly to a method for amminating polystyrene resins and a method for immobilizing an enzyme using an aminated polystyrene resin. [Background technology]

[0002] Compared with chemical catalysts, enzymes, which are biological catalysts, have been widely applied in industrial production due to their high activity, selectivity and substrate specificity. Biocatalysis is becoming an important component in the manufacturing program of chemicals, intermediates, fine chemicals and final drug molecules. Enzymes have high regioselectivity, high catalytic efficiency and mild reaction conditions, but free enzymes are very easy to inactivate because their catalytic activity is affected by factors such as temperature, pH and solvent. With the expansion of process demands, the requirements for enzyme use efficiency and economy are also becoming higher. Therefore, it is necessary to not only increase enzyme activity, specificity and productivity, but also to improve the recoverability and reusability of enzymes.

[0003] Immobilization of enzymes by fixing them to solid supports and obtaining heterogeneous immobilized enzyme systems provides a very good method for solving the above problems. Immobilized enzymes have a series of advantages such as high stability, easy separation and recovery, repeated operation and use, and simple process while maintaining the catalytic performance specific to enzymes. The performance of immobilized enzymes mainly depends on the immobilization method and the support material used. Depending on the binding method between the enzyme and the support, they are classified into adsorption method, covalent bonding method, crosslinking method, embedding method, etc. Among them, the adsorption method is a bond between the enzyme and the support by physical adsorption action, van der Waals force, hydrophobic action, etc., and has advantages such as simple and convenient operation, large adsorption capacity, and low industrial cost, and is the most commonly used method for enzyme immobilization. However, since the support and the enzyme are bound by physical adsorption action, the binding force is weak, and there is a problem that the enzyme falls off the support during repeated use, resulting in a decrease in the conversion rate. The covalent bonding method is a method in which the enzyme and the support are linked by a covalent bond. Therefore, the support must have a functional group that reacts with the enzyme to form a covalent bond. Compared with the adsorption method, the bond between the enzyme and the carrier is stronger, there is no problem of the enzyme falling off during use, and the stability and recyclability are better.

[0004] In the early 1970s, Davankov et al. crosslinked linear polystyrene or recrosslinked low crosslinked polystyrene by Friedel-Crafts reaction to synthesize a kind of porous polymer with unique structure and excellent performance. This reaction is called super-crosslinking reaction (also called Davankov super-crosslinking reaction) according to its manufacturing method. The porous polymer produced by the super-crosslinking reaction is called super-crosslinked adsorbent resin. Super-crosslinked adsorbent resin is considered to be the third generation adsorbent resin following gel type and macroporous type adsorbent resin. Super-crosslinked adsorbent resin usually has structural characteristics such as large specific surface area, small average pore size, narrow pore size distribution, and good mechanical strength, and currently shows the potential for wide applications in the fields of toxic organic wastewater management, herbal medicine and antibiotic extraction, gas storage and separation, etc.

[0005] Due to the fact that amino has good reaction activity, strong electron accepting ability and is easy to protonate, aminated polystyrene resin has unpredictable future prospects in the fields of separation and purification of organic drugs, biomedical polymer materials, wastewater treatment adsorption, polymer catalyst carriers, etc. At present, there are several main methods for synthesizing aminated polystyrene resin, including the following:

[0006] Nitro compound reduction method: The reduction of nitro compounds to obtain amino compounds is an important method for synthesizing aminated polystyrene resins. There are many schemes for this, and the main methods are as follows:

[0007] Catalytic hydrogenation reduction: Catalytic hydrogenation reduction method mainly uses precious metals such as Ni, Pd, Pt or their alloys as catalysts, and when the reaction conditions reach a certain temperature and pressure, nitro is reduced to amino. This method has many advantages. First, it can reduce the input of manpower and has strong mechanical operability. Second, it has high yield, high product purity, controllable reaction steps, and is environmentally friendly and pollution-free. However, the process requires precious metal catalysts, high temperature and pressure, and high requirements for equipment. These greatly increase the synthesis cost of materials. Hydrazine hydrate reduction method: This method requires the addition of catalysts to promote the reaction, and one of the common catalysts is precious metals and their alloys, which usually include Pd / C, Pt / C, Raney Ni, etc.

[0008] Metal reduction method: This method has been used for a long time, specifically, metals and acids are made to coexist, but sometimes a salt electrolyte is dissolved in water to carry out the reduction reaction of nitro. Theoretical analysis has shown that metals with a lower electromotive force than H, such as Li, Na, K, and Mg, can be used as reducing agents under certain conditions. However, the drawback is that iron mud is generated after the reaction, causing serious environmental pollution.

[0009] Bromination amination method: Through electrophilic substitution reaction, bromine is bonded to the para position of the benzene ring, and reacted with LiN(SiMe3)2 using palladium as a catalyst, and then acid and alkali treatment are carried out for post-treatment to produce polyparaaminostyrene.

[0010] Chloromethylated polystyrene amination method: First, polystyrene microspheres are subjected to a chloromethylation reaction to bond chloromethyl to the benzene ring at the para position, and then an amination reaction is carried out with an amination reagent to produce aminopolystyrene. However, this method requires the use of chloromethyl ether and dichloromethyl ether, which are chemical reagents that are highly harmful to the human body and have a strong risk of carcinogenesis, and are currently banned in many areas internationally. In addition, this reaction involves side reactions such as multiple substitution and post-crosslinking, which complicate the chloromethyl resin structure.

[0011] Chloroacetylated polystyrene amination method: FC acylation reaction is used to introduce acyl into the benzene ring at the para position, and aminated polystyrene is produced by amination reaction. The specific reaction is as follows: [ka] Summary of the Invention [Problem to be solved by the invention]

[0012] The main objective of the present invention is to provide a new method for aminating polystyrene resins and to immobilize transaminases on the aminated polystyrene resins as enzyme immobilization supports. This method not only provides a new method for synthesizing aminated polystyrene resins, but also expands the library of amino-type enzyme immobilization supports. [Means for solving the problem]

[0013] In order to achieve the above object, one aspect of the present invention provides a method for aminating a polystyrene-based resin, comprising the steps of catalyzing a Friedel-Crafts alkylation reaction of a polystyrene-based resin with an enamine salt in a solvent using a Lewis acid catalyst to obtain an aminated polystyrene-based resin. [ka]

[0014] Furthermore, the temperature of the above Friedel-Crafts alkylation reaction is 40-80° C., and preferably, the time of the Friedel-Crafts alkylation reaction is 12-20 hours.

[0015] Furthermore, the mass ratio of the polystyrene resin to the enamine salt is 2:1 to 2:3.

[0016] Furthermore, the molar ratio of the enamine salt to the Lewis acid catalyst is 1:3 to 1:5.

[0017] Furthermore, the Lewis acid catalyst is any one of anhydrous aluminum trichloride and ferric chloride.

[0018] Further, the enamine salt is 3-butenylamine hydrochloride.

[0019] Furthermore, the solvent is 1,2-dichloroethane.

[0020] Furthermore, the polystyrene-based resin is any one selected from polystyrene resin, polystyrene-methacrylate resin, AB8 type polystyrene resin, ECR1090 type polystyrene resin, NKA9 type polystyrene resin, D101 type polystyrene resin, and SXD11 type polystyrene resin.

[0021] Furthermore, the amination method includes the steps of dispersing a polystyrene resin and an enamine salt in a solvent to form a reaction system, and mixing the reaction system with a catalyst in a nitrogen or inert atmosphere, heating the reaction system to a Friedel-Crafts alkylation reaction temperature, and performing the Friedel-Crafts alkylation reaction to obtain an aminated polystyrene resin.

[0022] According to another aspect of the present invention, there is provided a method for immobilizing an enzyme with an aminated polystyrene resin, the method comprising the steps of producing an aminated polystyrene resin by any of the above-mentioned amination methods, and immobilizing an enzyme using the aminated polystyrene resin as a carrier and glutaraldehyde as a crosslinking agent.

[0023] Furthermore, the above method includes a step of modifying an aminated polystyrene resin with a buffer solution of glutaraldehyde to obtain a glutaraldehyde-modified resin, and a step of immobilizing the enzyme solution by cross-linking the glutaraldehyde-modified resin.

[0024] Furthermore, the above method includes the steps of mixing the enzyme liquid with an aminated polystyrene resin to obtain a resin having an enzyme adsorbed thereon, and immobilizing the enzyme by cross-linking the resin having the enzyme adsorbed thereon with a buffer solution of glutaraldehyde.

[0025] Furthermore, the glutaraldehyde buffer solution contains a phosphate buffer solution, the glutaraldehyde content in the glutaraldehyde buffer solution is 1 to 2 mass %, and the enzyme is a transaminase.

[0026] According to the technical solution of the present invention, the present application utilizes Friedel-Crafts alkylation reaction to graft enamine salt onto polystyrene resin to complete the amination of polystyrene resin. The Friedel-Crafts alkylation reaction is easy to control the conditions, and the post-treatment process is simple. The catalyst and unreacted enamine salt only need to be removed by washing, so the above amination method of the present application has few steps, is simple and easy to operate, and avoids the use of precious metal catalysts, reducing production costs. The aminated commercial polystyrene resin can be successfully used as an enzyme immobilization support, with good immobilization effect and reusability. This greatly expands the enzyme immobilization support library and provides more options in the pore size, specific surface area, framework structure, and polarity of the support when immobilizing various enzymes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] In addition, the embodiments and features of the embodiments of the present application can be combined with each other as long as there is no contradiction.

[0028] As analyzed in the Background of the Invention section of the present application, the conventional methods for producing aminated polystyrene resins require complicated production methods, multiple formulations, and complicated post-treatment methods, which complicate the production process. In order to solve these problems, the present application provides a method for aminating a polystyrene resin and a method for immobilizing an enzyme using an aminated polystyrene resin.

[0029] In an exemplary embodiment of the present application, there is provided a method for aminating a polystyrenic resin, comprising the steps of catalyzing a Friedel-Crafts alkylation reaction of a polystyrenic resin with an enamine salt in a solvent, utilizing a catalyst that is a Lewis acid catalyst, to obtain an aminated polystyrenic resin.

[0030] The present invention uses Friedel-Crafts alkylation to graft enamine salt onto polystyrene resin to complete amination of polystyrene resin. The Friedel-Crafts alkylation reaction is easy to control conditions and has a simple post-treatment process, and only requires the catalyst and unreacted enamine salt to be removed by washing. Therefore, the above amination method of the present invention has few steps, is simple and easy to operate, and avoids the use of precious metal catalysts, reducing production costs. The aminated commercial polystyrene resin can be successfully used as an enzyme immobilization support, with good immobilization effect and reusability. This greatly expands the enzyme immobilization support library and provides more options in the pore size, specific surface area, framework structure, and polarity of the support when immobilizing various enzymes.

[0031] In order to improve the efficiency of the Friedel-Crafts alkylation, for the above reaction raw materials, the temperature of the above Friedel-Crafts alkylation is preferably 40-80°C, and the time of the Friedel-Crafts alkylation is preferably 12-20h.

[0032] The Friedel-Crafts alkylation reaction is essentially a grafting of an alkyl group having an amino group onto the benzene ring of polystyrene. Since polystyrene resins are characterized by being large molecules, it is difficult to uniformly graft onto each benzene ring. In order to maximize the probability of amination, the molar ratio of the polystyrene resin to the enamine salt is preferably 2:1 to 2:3.

[0033] Furthermore, in order to increase the catalytic efficiency and avoid wasting the catalyst, and also to facilitate removal of the catalyst, the molar ratio of the enamine salt to the Lewis acid catalyst is preferably 1:3 to 1:5.

[0034] In the above method, the Lewis acid catalyst for Friedel-Crafts alkylation may be selected from Lewis acid catalysts commonly used in Friedel-Crafts alkylation in the prior art, but in order to ensure the efficiency of the catalyst while making it easy to separate the catalyst from the product, the Lewis acid catalyst is preferably any one or a combination of two of anhydrous aluminum trichloride and ferric chloride.

[0035] In some embodiments, the enamine salt used in the above amination method is 3-butenylamine hydrochloride, allowing for efficient amination.

[0036] The solvent used in the present application mainly dissolves the enamine salt and the Lewis acid catalyst and brings them into sufficient contact with the polystyrene resin. In order to increase the solubility of the enamine salt and the Lewis acid catalyst, the solvent is preferably 1,2-dichloroethane.

[0037] The polystyrene resin used in the present application includes, but is not limited to, any one of polystyrene resin, polystyrene-methacrylate resin, AB8 type polystyrene resin, ECR1090 type polystyrene resin, NKA9 type polystyrene resin, D101 type polystyrene resin, and SXD11 type polystyrene resin. Regardless of whether it is a pure polystyrene resin or a modified polystyrene resin, it may be amino-modified by the above-mentioned amination method.

[0038] In some embodiments of the present application, the amination method includes dispersing a polystyrene resin and an enamine salt in a solvent to form a reaction system, and mixing the reaction system with a catalyst in a nitrogen or inert atmosphere and heating to a Friedel-Crafts alkylation reaction temperature to perform the Friedel-Crafts alkylation reaction to obtain an aminated polystyrene resin. First, the enamine salt and the polystyrene resin are dispersed in a solvent, and the enamine salt is preliminarily introduced into the gaps of the polystyrene resin. Then, the catalyst is added to perform the Friedel-Crafts alkylation reaction, thereby increasing the reaction rate and improving the uniformity of the alkylation.

[0039] In another exemplary embodiment of the present application, there is provided a method for immobilizing an enzyme using an aminated polystyrene-based resin, the method including the steps of producing an aminated polystyrene-based resin by any of the amination methods described above, and immobilizing an enzyme using the aminated polystyrene-based resin as a carrier and glutaraldehyde as a crosslinker.

[0040] When aminated polystyrene resins are produced by the amination method of the present invention, the production method is simple and low cost, and the degree of amination can be flexibly adjusted by the amination method, and further the degree of subsequent enzyme immobilization can also be flexibly adjusted.

[0041] In some embodiments of the present application, the method includes the steps of modifying an aminated polystyrene resin with a buffered solution of glutaraldehyde to obtain a glutaraldehyde-modified resin, and immobilizing the enzyme solution by cross-linking the glutaraldehyde-modified resin.

[0042] In the above method, firstly, glutaraldehyde is used to modify the resin, thereby cross-linking the amino of the polystyrene resin with glutaraldehyde to form a cross-linked network, and then the amino residue of the enzyme is cross-linked with glutaraldehyde to realize cross-linking hardening for the enzyme. This process enhances the cross-linking hardening efficiency of the enzyme, and is especially suitable for enzymes that are sensitive to glutaraldehyde.

[0043] In some other embodiments of the present application, the method includes the steps of mixing the enzyme solution with an aminated polystyrene resin to obtain an enzyme-adsorbed resin, and immobilizing the enzyme-adsorbed resin by cross-linking it with a buffer solution of glutaraldehyde.

[0044] In the above method, the enzyme is first adsorbed on the inner and outer surfaces of the polystyrene resin by adsorption, and then reacted with glutaraldehyde to fix the enzyme on the polystyrene resin by cross-linking with glutaraldehyde. The above method is particularly suitable for enzymes that are not sensitive to glutaraldehyde.

[0045] The glutaraldehyde buffer solution used in the present application may refer to a common buffer solution used in crosslinking and curing, for example, the glutaraldehyde buffer solution includes a phosphate buffer solution. In addition, in order to increase the crosslinking immobilization rate of the enzyme, the glutaraldehyde content in the glutaraldehyde buffer solution is preferably 1-2 mass%, thereby increasing the utilization rate of glutaraldehyde. In the process of immobilizing the enzyme, the loading amount can be adjusted by adjusting the amount of the enzyme relative to the carrier, which will not be described in detail here.

[0046] Since the immobilization utilizes the cross-linking action between the aldehyde group of glutaraldehyde and the amino acid residue of the enzyme, the method of the present application can be applied to common enzymes, for example, the enzyme is selected from transaminases.

[0047] The beneficial effects of the present invention will be further illustrated below by examples and comparative examples.

[0048] Amination Example Example 1-1 Synthesis of styrene resin 150mL of an aqueous solution containing 0.5% PVA was added to a 500mL four-neck flask and heated to 40°C. 16g of styrene, 5g of divinylbenzene (80%), 10mL of toluene, 20mL of n-heptane, and 100mg of benzoyl peroxide were mixed uniformly and added to the four-neck flask. After stirring was started and the droplets were adjusted to an appropriate particle size, the temperature was raised to 80°C and kept at that temperature for 2 hours, and then the temperature was raised to 88°C and kept at that temperature for 4 hours. After the reaction was completed, the mixture was cooled to room temperature and filtered to obtain the desired beads, which were washed in the order of hot water, ethanol, and deionized water, sieved, and baked to obtain polystyrene resin.

[0049] Polystyrene Resin Amination 1g of the baked polystyrene resin and 1g of 3-butenylamine hydrochloride were dispersed in 100mL of 1,2-dichloroethane and stirred at room temperature for 1h to form a reaction waiting system. Then, 2g of anhydrous aluminum trichloride was weighed and added to the reaction waiting system, stirred for another 10min, heated to 80℃, and reacted for 20h under N2 protection. After the reaction was completed, it was cooled to room temperature and washed with methanol and deionized water in that order to obtain aminated styrene resin: PS-NH2.

[0050] Example 1-2 Synthesis of polystyrene-methacrylate resin 240mL of an aqueous solution containing 0.5% PVA and 10% NaCl was added to a 500mL four-neck flask and heated to 40°C. 6g of methyl methacrylate, 15g of divinylbenzene (80%), 20mL of 1,2-dichloroethane, 40mL of methylcyclohexane, and 100mg of azobisisobutyronitrile were mixed uniformly and added to a four-neck flask. After stirring was started and the droplets were adjusted to an appropriate particle size, the temperature was raised to 70°C and kept for 2h, then raised to 80°C and kept for 4h, and kept at 85°C for 1h. After the reaction was completed, the mixture was cooled to room temperature and filtered to obtain the desired beads, which were washed in the order of hot water, ethanol, and deionized water, sieved, and baked to obtain polystyrene-methacrylate resin (PDMA).

[0051] PDMA resin modification 1 g of the baked PDMA resin and 0.7 g of 3-butenylamine hydrochloride were dispersed in 100 mL of 1,2-dichloroethane and stirred at room temperature for 1 h to form a reaction waiting system. Then, 2 g of anhydrous aluminum trichloride was weighed and added to the reaction waiting system, and after stirring for another 10 min, the temperature was raised to 80°C and the reaction was carried out for 20 h under N2 protection. After the reaction was completed, the mixture was cooled to room temperature and washed with methanol and deionized water in that order to obtain aminated resin: PDMA-NH2.

[0052] Examples 1-3 1 g of baked ECR1090 type polystyrene resin (purchased from Purlite) and 1 g of 3-butenylamine hydrochloride were dispersed in 100 mL of 1,2-dichloroethane and stirred at room temperature for 1 h to form a reaction waiting system. Then, 2 g of anhydrous aluminum trichloride was weighed and added to the reaction waiting system, stirred for another 10 min, heated to 80°C, and reacted for 20 h under N2 protection. After the reaction was completed, the resin was cooled to room temperature and washed with methanol and deionized water in that order to obtain aminated ECR1090 resin: ECR1090-NH2.

[0053] Examples 1-4 1g of baked AB8 resin (purchased from Nankai Synthetic Co., Ltd.) and 1g of 3-butenylamine hydrochloride were dispersed in 100mL of 1,2-dichloroethane and stirred at room temperature for 1h to form a reaction waiting system. Then, 2g of anhydrous aluminum trichloride was weighed and added to the reaction waiting system, stirred for another 10min, heated to 80℃, and reacted for 20h under N2 protection. After the reaction was completed, it was cooled to room temperature and washed with methanol and deionized water in that order to obtain aminated AB8 resin: AB8-NH2.

[0054] Examples 1-5 1g of baked NKA9 resin (purchased from Nankai Synthetic Co., Ltd.) and 1g of 3-butenylamine hydrochloride were dispersed in 100mL of 1,2-dichloroethane and stirred at room temperature for 1h to form a reaction waiting system. Then, 2g of anhydrous aluminum trichloride was weighed and added to the reaction waiting system, stirred for another 10min, heated to 80℃, and reacted for 20h under N2 protection. After the reaction was completed, it was cooled to room temperature and washed with methanol and deionized water in that order to obtain aminated NKA9 resin: NKA9-NH2.

[0055] Examples 1-6 1g of baked SXD-11 resin (purchased from Anhui Samsung Co., Ltd.) and 1g of 3-butenylamine hydrochloride were dispersed in 100mL of 1,2-dichloroethane and stirred at room temperature for 1h to form a reaction waiting system. Then, 2g of anhydrous aluminum trichloride was weighed and added to the reaction waiting system, stirred for another 10min, heated to 80℃, and reacted for 20h under N2 protection. After the reaction was completed, it was cooled to room temperature and washed with methanol and deionized water in that order to obtain aminated SXD-11 resin: SXD-11-NH2.

[0056] Examples 1-7 1g of baked D101 resin (purchased from Anhui Samsung Co., Ltd.) and 1g of 3-butenylamine hydrochloride were dispersed in 100mL of 1,2-dichloroethane and stirred at room temperature for 1h to form a reaction waiting system. Then, 2g of anhydrous aluminum trichloride was weighed and added to the reaction waiting system, stirred for another 10min, heated to 80℃, and reacted for 20h under N2 protection. After the reaction was completed, it was cooled to room temperature and washed with methanol and deionized water in that order to obtain aminated D101 resin: D101-NH2.

[0057] The amino content of the aminated styrene resin obtained in each of the above Examples was detected by the ninhydrin coloring method and acid-base titration method, and the specific surface area and pore size were measured by the nitrogen adsorption / desorption method. The detection results are shown in Table 1. [Table 1]

[0058] Examples of enzyme immobilization The transaminase from Chromobacterium violaceum DSM30191 (CVTA) was taken as a model and its sequence SEQ ID NO:1 is as follows: MQKQRTCSQWRELDAAHHLHPFTDTASLNQAGARVMTRGEGVYLWDCEGNKIIDGMAGLWCVNVGYGRKDFAEAAARRQMEELPFYNTFFGTTHPPVVELSSLLAEVTPAGFDRVF YTNSGSESVDTMIRMVRRYWDVQGKPEKKTLIGRWNGYHGSTIGGASLGGMKYMHEQGDLPIPGMAHIEQPWWYKHGKDMTPDEFGVVAARWLEEKILEIGADKVAAFVGEPIQG AGGVIVPPATYWPEIERICRKYDVLLVADEVICGFGRTGEWFGHQHFGFQPDLFTAAKGLSSGYLPLGAVFVGDRVAEGLIAGGDFNHGFTYSGHPVCAAVAHANVAALRDEGIV QRVKDDIGPYMQKRWRETFSRFEHVDDVRGVGMMLAFTLVKNKAKRELFPDFGEIGTLCEDIFFRNNLIMTAQGDHIVSAPPLVMTRAEVDEMLAVAERCLEEFEQTLKARGLA.

[0059] Example 2-1 A 1% glutaraldehyde solution was prepared using 20 mM pH 7.0 phosphate buffer, and 1 g each of the aminated styrene resin and styrene resin of Example 1-1 were weighed and dispersed in the above solution. The mixture was incubated at 30°C for 1 h, and then washed three times with deionized water to obtain red glutaraldehyde-modified aminated styrene resin and glutaraldehyde-modified styrene resin. The resin modified with glutaraldehyde turned reddish brown, so the color change could be used to determine whether the glutaraldehyde modification was successful.

[0060] The prepared enzyme solution (4 mL) was added to two types of resins (1 g) modified with glutaraldehyde to obtain the aminated styrene-immobilized enzyme and the styrene resin-immobilized enzyme.

[0061] The difference in protein content before and after immobilization of the system was measured to calculate the amount of enzyme supported. The amount of immobilized enzyme supported on the aminated styrene-immobilized enzyme was 26.44 mg / g, and the amount of immobilized enzyme supported on the styrene resin-immobilized enzyme was 11.09 mg / g.

[0062] Example 2-2 A 1% glutaraldehyde solution was prepared using 20 mM pH 7.0 phosphate buffer, and 1 g each of PDMA resin and PDMA-NH2 resin was weighed and dispersed in the above solution. The resin was incubated at 30°C for 1 h, and then washed three times with deionized water to obtain red glutaraldehyde-modified PDMA resin and glutaraldehyde-modified PDMA-NH2 resin.

[0063] The prepared enzyme solution (4 mL of 1V enzyme solution plus 3V phosphate buffer) was added to two types of resin (1 g) activated with glutaraldehyde and immobilized to obtain PDMA-NH2 immobilized enzyme and PDMA immobilized enzyme.

[0064] The immobilized enzyme loading amount of the prepared PDMA-NH2 immobilized enzyme is 28.35 mg / g, and the immobilized enzyme loading amount of the prepared PDMA immobilized enzyme is 9.17 mg / g.

[0065] Example 2-3 A 1% glutaraldehyde solution was prepared using 20 mM pH 7.0 phosphate buffer, and 1 g each of ECR1090 and ECR1090-NH2 was weighed and dispersed in the above solution. The mixture was incubated at 30°C for 1 h, and then washed three times with deionized water to obtain red glutaraldehyde-modified ECR1090 resin and glutaraldehyde-modified ECR1090-NH2 resin.

[0066] The prepared enzyme solution (4 mL) was added to two types of resins (1 g) activated with glutaraldehyde to obtain the immobilized ECR1090-NH2 immobilized enzyme and the ECR1090 immobilized enzyme.

[0067] The immobilized enzyme loading amount of the ECR1090-NH2 immobilized enzyme is 21.82 mg / g, and the immobilized enzyme loading amount of the ECR1090 immobilized enzyme is 21.09 mg / g.

[0068] Examples 2-4 A 1% glutaraldehyde solution was prepared using 20 mM pH 7.0 phosphate buffer, and 1 g each of AB8 and AB8-NH2 was weighed and dispersed in the above solution. The mixture was incubated at 30°C for 1 h, and then washed three times with deionized water to obtain red glutaraldehyde-modified AB8 resin and glutaraldehyde-modified AB8-NH2 resin.

[0069] The prepared enzyme solution (4 mL of 1 V enzyme solution plus 3 V phosphate buffer) was added to two types of resin (1 g) activated with glutaraldehyde and immobilized to prepare AB8-NH2 immobilized enzyme and AB8 immobilized enzyme.

[0070] The immobilized enzyme loading of the AB8-NH2 immobilized enzyme is 28.37 mg / g, and the immobilized enzyme loading of the AB8 immobilized enzyme is 12.91 mg / g.

[0071] Examples 2-5 A 1% glutaraldehyde solution was prepared using 20 mM pH 7.0 phosphate buffer, and 1 g each of NKA9 and NKA9-NH2 was weighed and dispersed in the above solution. The mixture was incubated at 30°C for 1 h, and then washed three times with deionized water to obtain red glutaraldehyde-modified NKA9 resin and glutaraldehyde-modified NKA9-NH2 resin.

[0072] The prepared enzyme solution (1V enzyme solution plus 3V phosphate buffer) (4mL) was added to two types of resin (1g) activated with glutaraldehyde to prepare NKA9-NH2 immobilized enzyme and NKA9 immobilized enzyme.

[0073] The immobilized enzyme loading amount of the NKA9-NH2 immobilized enzyme is 44.56 mg / g, and the immobilized enzyme loading amount of the NKA9 immobilized enzyme is 42.31 mg / g.

[0074] Examples 2-6 A 1% glutaraldehyde solution was prepared using 20 mM pH 7.0 phosphate buffer, and 1 g each of SXD-11 and SXD-11-NH2 was weighed and dispersed in the above solution. The mixture was incubated at 30°C for 1 h, and then washed three times with deionized water to obtain red glutaraldehyde-modified SXD-11 resin and glutaraldehyde-modified SXD-11-NH2 resin.

[0075] The prepared enzyme solution (4 mL of 1 V enzyme solution plus 3 V phosphate buffer) was added to two types of resin (1 g) activated with glutaraldehyde and immobilized to prepare SXD-11-NH2 immobilized enzyme and SXD-11 immobilized enzyme.

[0076] The immobilized enzyme loading amount of the SXD-11-NH2 immobilized enzyme is 13.31 mg / g, and the immobilized enzyme loading amount of the SXD-11 immobilized enzyme is 13.41 mg / g.

[0077] The reaction scheme for enzyme catalysis is as follows: [ka]

[0078] 0.5 mL of methanol was added to a 20 mL reaction flask, 0.1 g of carbonyl substrate was dissolved therein, 15 eq of isopropylamine hydrochloride and 25.0 mg of PLP (pyridoxal 5'-phosphate) were added, and 0.1 M phosphate buffer (pH 8.0) was added until the final volume of the reaction solution reached 5 mL to form a reaction waiting system.

[0079] 0.1g of transaminase enzyme powder or immobilized enzyme produced with 0.1g of transaminase enzyme powder was added to the reaction system independently and stirred at 47℃ for 20h. The conversion rate of the system was detected by HPLC, and each time the reaction was completed, the immobilized enzyme was separated and added to the next reaction for repeated use. The number of times of repeated use was counted and the results were recorded in Table 2. [Table 2]

[0080] From the above description, the above-described embodiment of the present invention provides the following technical effects.

[0081] The present invention uses Friedel-Crafts alkylation to graft enamine salt onto polystyrene resin to complete amination of polystyrene resin. The Friedel-Crafts alkylation reaction is easy to control conditions and has a simple post-treatment process, and only requires the catalyst and unreacted enamine salt to be removed by washing. Therefore, the above amination method of the present invention has few steps, is simple and easy to operate, and avoids the use of precious metal catalysts, reducing production costs. The aminated commercial polystyrene resin can be successfully used as an enzyme immobilization support, with good immobilization effect and reusability. This greatly expands the enzyme immobilization support library and provides more options in the pore size, specific surface area, framework structure, and polarity of the support when immobilizing various enzymes.

[0082] The above is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art may make various modifications and changes to the present invention. All modifications, equivalent replacements, improvements, etc. made without departing from the spirit and principles of the present invention shall be included in the patent scope of the present invention.

Claims

1. A method for amminating a polystyrene-based resin, comprising the steps of: catalyzing a Friedel-Crafts alkylation reaction of a polystyrene resin and an enamine salt in a solvent using a catalyst that is a Lewis acid catalyst to obtain an aminated polystyrene resin; The method for amminating a polystyrene resin, wherein the enamine salt is 3-butenylamine hydrochloride.

2. 2. The amination process according to claim 1, wherein the temperature of the Friedel-Crafts alkylation reaction is 40°C to 80°C.

3. The amination method according to claim 1, characterized in that the time of the Friedel-Crafts alkylation reaction is 12 hours to 20 hours.

4. 2. The method for amination according to claim 1, wherein a mass ratio of the polystyrene resin to the enamine salt is from 2:1 to 2:

3.

5. 2. The amination process according to claim 1, wherein the molar ratio of the enamine salt to the Lewis acid catalyst is from 1:3 to 1:

5.

6. 2. The method according to claim 1, wherein the Lewis acid catalyst is any one or a combination of two of anhydrous aluminum trichloride and ferric chloride.

7. 2. The amination process according to claim 1, wherein the solvent is 1,2-dichloroethane.

8. 2. The method according to claim 1, wherein the polystyrene-based resin is any one selected from the group consisting of polystyrene resins and polystyrene-methacrylate resins.

9. dispersing the polystyrene resin and the enamine salt in the solvent to form a reaction system; and mixing the reaction system with the catalyst in a nitrogen or inert atmosphere, heating the reaction system to a Friedel-Crafts alkylation reaction temperature, and conducting the Friedel-Crafts alkylation reaction to obtain the aminated polystyrene resin.

10. A method for immobilizing an enzyme using an aminated polystyrene resin, comprising the steps of: A step of producing an aminated polystyrene-based resin by the amination method according to any one of claims 1 to 9; and immobilizing an enzyme using the aminated polystyrene resin as a carrier and glutaraldehyde as a crosslinking agent.

11. modifying the aminated polystyrene resin with a buffer solution of glutaraldehyde to obtain a glutaraldehyde-modified resin; The method according to claim 10, further comprising a step of cross-linking an enzyme solution with the glutaraldehyde-modified resin to achieve the immobilization.

12. mixing the enzyme solution with the aminated polystyrene resin to obtain an enzyme-adsorbed resin; and a step of cross-linking the enzyme-adsorbed resin with a buffer solution of glutaraldehyde to achieve the immobilization.

13. 13. The method of claim 11 or 12, wherein the glutaraldehyde buffer solution comprises a phosphate buffer solution, the glutaraldehyde content in the glutaraldehyde buffer solution is 1% by weight to 2% by weight, and the enzyme is a transaminase.

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