Immobilized enzymes, their preparation and use

Abstract

The present invention provides an immobilized enzyme, its preparation method and use. The immobilized enzyme includes an epoxy resin vector, which is LX-109S epoxy resin, and an enzyme linked to the epoxy resin vector by a covalent bond. The present application uses LX-109S epoxy resin as the epoxy resin vector, and based on the characteristics of the vector itself, the immobilization effect on the enzyme is more stable, and the covalent bond with the enzyme is strong, so that the activity of the enzyme itself is not affected.

Description

[Technical field]

[0001] The present invention relates to the field of enzyme immobilization technology, specifically to immobilized enzymes, their preparation and use. [Background technology]

[0002] Biocatalysis has already become an important part of the green synthesis of drugs, and is also one of the most promising technologies for catalyzing the structural units and intermediates of drugs, and in particular provides a unique alternative method for chiral synthesis. More and more biological enzymes are used as catalysts in industrial processes, but enzymes have very strict environmental requirements due to their mild use conditions and inherent instability, and are difficult to recycle, which greatly limits the industrial use of enzymes, increasing the demand for immobilized enzymes, and therefore immobilized enzymes, often called "biocatalysts", are widely used in industrial organic synthesis and biological transformation.

[0003] The methods for preparing immobilized enzymes generally include adsorption, covalent coupling, crosslinking and embedding. The covalent coupling method involves covalently bonding the inactive side groups of the enzyme with active functional groups in the vector, such as carboxylic acids, amino groups and epoxy groups. Here, the epoxy groups have very high reaction activity and plasticity, and the epoxy groups on the vector can directly react with inactive groups such as -NH2 and -HS on the enzyme molecule to perform covalent bond immobilization. Because the epoxy groups have very high reaction activity, the enzyme containing multiple inactive groups can be immobilized at multiple points to improve the bond strength between the enzyme and the vector, and therefore the epoxy group vector has a very high advantage in immobilizing the enzyme.

[0004] In recent years, synthetic chemists have been increasingly interested in continuous flow synthesis, and continuous flow synthesis is now also attracting attention in biotransformation. The emergence of new immobilization platforms allows for seamless integration of immobilized enzymes in continuous flow biocatalysis. The discovery and evolution of new efficient enzymes has enabled new retrosynthetic methods focused on biocatalysis, and the reduction in the cost of recombinant proteins and enzyme immobilization strategies have both fully prepared the way for continuous flow biocatalysis.

[0005] Epoxy resin is widely used for enzyme immobilization, but the immobilization effect of this resin on purified pure enzyme is good during the immobilization process, but the immobilization effect on crude enzyme solution is not ideal, so the cost of immobilized enzyme increases. Violent chemical reactions occurring during the covalent bonding process are often very unfavorable to the activity of biomolecules, so enzymes with active active centers will affect the activity of the enzyme during the immobilization process, leading to very low activity recovery rates. In addition, the covalent bond between the epoxy vector and the enzyme needs to be carried out at high ionic strength. If the ionic strength is not appropriate or there are many interfering factors in the environment, the bond between the enzyme and the vector will only be carried out by ion adsorption, and stable chemical bonds cannot be formed, which often results in poor reusability. Summary of the Invention [Problem to be solved by the invention]

[0006] The main object of the present invention is to provide an immobilized enzyme, its preparation method and use, so as to solve the problem that the immobilization effect of the epoxy resin immobilized enzyme in the prior art is unstable. [Means for solving the problem]

[0007] In order to achieve the above object, according to one aspect of the present invention, there is provided an immobilized enzyme, comprising an epoxy resin vector, which is an LX-109S epoxy resin, and an enzyme derived from a crude enzyme, which is covalently linked to the epoxy resin vector.

[0008] Furthermore, the enzyme is any one selected from transaminase, D-lactate dehydrogenase, cyclohexanone monooxygenase, ketoreductase, enereductase, nitrilase, ammonia lyase, amino acid dehydrogenase, imine reductase, lipase, and mutants thereof, preferably, the ammonia lyase is any one selected from phenylalanine ammonia lyase, and preferably, the amino acid dehydrogenase is any one selected from leucine dehydrogenase and phenylalanine dehydrogenase.

[0009] Furthermore, in the above immobilized enzyme, the amount of the enzyme supported per 1 g of the epoxy resin vector is 30 mg to 70 mg.

[0010] Additionally, the immobilized enzyme further comprises a cofactor.

[0011] According to another aspect of the present invention, there is provided a method for producing any one of the above immobilized enzymes, the method comprising: a step S1 of mixing a first phosphate buffer solution with an enzyme, which is a crude enzyme, to form an enzyme buffer solution; and a step S2 of mixing the enzyme buffer solution with an epoxy resin, which is an LX-109S epoxy resin, to carry out an immobilization reaction, and then washing with a second phosphate buffer solution to obtain an immobilized enzyme.

[0012] Furthermore, the volume ratio of the enzyme buffer solution to the epoxy resin is 3:1 to 5:1.

[0013] Furthermore, the pH value of the above first phosphate buffer is 7.0 to 8.0, and the first phosphate buffer contains sodium chloride, and preferably, the concentration of the sodium chloride in the first phosphate buffer is 1±0.2 mol / L, and the pH value of the second phosphate buffer is 7.0 to 8.0, and the second phosphate buffer does not contain sodium chloride.

[0014] Furthermore, the above step S2 includes mixing the enzyme buffer solution and the epoxy resin at 10 to 20°C, shaking culture for 16 to 24 hours, and then leaving and incubating at 3 to 5°C for 40 to 48 hours to obtain an immobilized system, and washing the immobilized system with a second phosphate buffer solution to obtain an immobilized enzyme.

[0015] According to another aspect of the present invention there is provided a use of any one of the immobilized enzymes described above, comprising using the immobilized enzyme as a catalyst in a continuous catalytic reaction. Effect of the Invention

[0016] Using the technical solution of the present invention, in this application, LX-109S epoxy resin is used as an epoxy resin vector, and based on the characteristics of the vector itself, the immobilization effect on the enzyme is more stable, and the covalent bond with the enzyme is stronger, so that the activity of the enzyme itself is not affected. Therefore, the crude enzyme can be used as the enzyme source directly, which greatly simplifies the enzyme immobilization process, reduces the production cost of immobilized enzyme, and shortens the process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The embodiments and features of the embodiments of the present application may be combined as long as they are not inconsistent. The present invention will now be described in detail with reference to the embodiments.

[0018] As analyzed in the background art of this application, the immobilization effect of the epoxy resin immobilized enzyme in the prior art is unstable. To solve this problem, this application provides an immobilized enzyme, its preparation method and use.

[0019] In an exemplary embodiment of the present application, an immobilized enzyme is provided, comprising an epoxy resin vector, which is an LX-109S epoxy resin, and an enzyme covalently linked to the epoxy resin vector.

[0020] In the present application, LX-109S epoxy resin is used as an epoxy resin vector, which, based on the characteristics of the vector itself, makes the immobilization effect on the enzyme more stable and strengthens the covalent bond with the enzyme, without affecting the activity of the enzyme itself. Therefore, the crude enzyme can be used as the enzyme source directly, which greatly simplifies the enzyme immobilization process, reduces the production cost of the immobilized enzyme, and shortens the process.

[0021] Since the present invention is based on the covalent bond between the epoxy resin and the enzyme, any enzyme immobilized by the covalent bond realization in the prior art can be used in the present invention, and the immobilization effect is particularly remarkable when the enzyme is any one selected from transaminase, D-lactate dehydrogenase, cyclohexanone monooxygenase, ketoreductase, enereductase, nitrilase, ammonia lyase, amino acid dehydrogenase, imine reductase, lipase, and mutants thereof. Preferably, the ammonia lyase is any one selected from phenylalanine ammonia lyase, and preferably, the amino acid dehydrogenase is any one selected from leucine dehydrogenase and phenylalanine dehydrogenase. The origin of some enzymes is exemplified in Table 1 below. [Table 1] TIFF2025514551000002.tif203154TIFF2025514551000003.tif207154TIFF2025514551000004.tif206150

[0022] Due to the above characteristics of LX-109S epoxy resin, it can not only be used to immobilize purified enzymes, but also can still obtain good immobilization effect, especially when the enzyme is derived from crude enzyme (i.e. unpurified enzyme).For example, the enzyme is derived from crude enzyme solution expressed by engineered bacteria of E. coli, the crude enzyme solution is resuspended in phosphate buffer after cell precipitation, and then disrupted by lysozyme, ultrasonic or homogenization, and then centrifuged to remove the disrupted cell debris to obtain a mixed protein solution.

[0023] In addition, the enzyme production process usually includes the following: constructing an engineered bacterium by introducing an exogenous gene into a host cell, reviving and expanding the strain, fermenting at high density, disrupting the cells, removing cell debris to collect the enzyme protein solution, purifying the target enzyme protein by column purification, and freeze-drying the purified enzyme solution to obtain an enzyme powder of pure enzyme. The crude enzyme in the present invention refers to the crude enzyme powder formed by directly freeze-drying the crude enzyme solution obtained before column purification without purification treatment. Generally, the crude enzyme contains endogenous proteins expressed in the background of the host cell, including proteases, oxidases, reductases, etc., and further includes factors, amino acids, nucleic acids, etc. used in the translation of water-soluble endogenous proteins. The proportion of the target enzyme in the crude enzyme solution is very small, usually <30%, and the viscosity of the enzyme solution is high. The crude enzyme solution is used directly to immobilize the enzyme protein on the resin vector, which is low in cost, but due to the influence of heteroproteins and other substances, the immobilization binding rate is low, and the activity of the immobilized enzyme is generally low.

[0024] Tests and research have shown that the immobilization of unrefined crude enzyme with LX-109S epoxy resin improves the enzyme activity and reusability by 20%-60% compared with the immobilization effect of crude enzyme with epoxy resins such as ECR8285 and LX120; the reaction of lipase immobilized with LX-109S epoxy resin in aqueous and organic phases improves the enzyme activity and reusability by 20%-100% compared with the immobilization effect of other types of resins; the immobilization of transaminase with LX-109S epoxy resin for the enzymatic synthesis of sitagliptin improves the enzyme activity and reusability by 20%-60% compared with the immobilization effect of other types of resins.

[0025] Based on the covalent bond between the enzyme and the epoxy resin, in addition to improving the enzyme loading amount as much as possible, the enzyme catalytic activity is ensured to be fully exerted, and preferably, in the immobilized enzyme, the loading amount of the above enzyme per 1 g of the epoxy resin vector is 30 mg to 70 mg, for example, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, or 70 mg.

[0026] In some embodiments of the present application, in order to improve the catalytic activity and catalytic stability of the immobilized enzyme, the immobilized enzyme preferably further comprises a selectable cofactor, the specific selection of which can be made according to the specific type of enzyme, for example, PLP, FAD, NAD + , NAD + etc., and no specific selections are enumerated herein.

[0027] In another exemplary embodiment of the present application, there is provided a method for producing any one of the above immobilized enzymes, the method including: a step S1 of mixing a first phosphate buffer solution with an enzyme, which is a crude enzyme, to form an enzyme buffer solution; and a step S2 of mixing the enzyme buffer solution with an epoxy resin, which is an LX-109S epoxy resin, to carry out an immobilization reaction, and then washing with a second phosphate buffer solution to obtain an immobilized enzyme.

[0028] The present application achieves the covalent bond between the enzyme and the LX-109S epoxy resin through the above preparation method, which has simple steps, is easy to operate, and is easy to popularize and use.

[0029] In some embodiments of the present application, in order to improve the immobilization rate of the enzyme in the enzyme solution, the volume ratio of the enzyme buffer to the epoxy resin is preferably 3:1 to 5:1, for example, 3:1 to 4:1, 3:1 to 3.5:1, 3.5:1 to 5:1, 3.5:1 to 4:1, or 4:1 to 5:1.

[0030] In some embodiments of the present application, the first phosphate buffer and the second phosphate buffer may be the same or different, and preferably, the compositions of the two are different to perform the functions at each stage, and preferably, the pH value of the first phosphate buffer is 7.0-8.0, and the pH value of the second phosphate buffer is 7.0-8.0. The first phosphate buffer and the second phosphate buffer have a basic composition similar to that of phosphate buffers commonly used in the art, and the first phosphate buffer contains sodium chloride, and preferably, the concentration of sodium chloride in the first phosphate buffer is 1±0.2 mol / L, that is, phosphate is used as a buffer pair and the ionic strength is adjusted with sodium chloride. The second phosphate buffer does not contain sodium chloride.

[0031] In order to improve the immobilization efficiency and the stability of the immobilization as much as possible, the above step S2 preferably includes mixing the enzyme buffer solution and the epoxy resin at 10 to 20° C., shaking culture for 16 to 24 hours, and then leaving and incubating at 3 to 5° C. for 40 to 48 hours to obtain an immobilized system, and washing the immobilized system with a second phosphate buffer solution to obtain an immobilized enzyme. For the above shaking culture, a shaker device generally used in the prior art, such as an orbital shaker, can be used.

[0032] In some embodiments, the mixing temperature is 10°C, 20°C, 15°C, 12°C, 18°C, 16°C, or 14°C, the shaking incubation time is 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, or 24h, and the standing time is 40h, 41h, 42h, 43h, 44h, or 45h.

[0033] In another exemplary embodiment of the present application, there is further provided a use of any of the above immobilized enzymes, including the use of the immobilized enzyme as a catalyst in a continuous catalytic reaction. Due to the high stability of the immobilized enzyme of the present application, the catalytic activity in the continuous catalytic reaction is well maintained, the service life is extended, and the reaction efficiency of the continuous catalytic reaction is ensured.

[0034] The beneficial effects of the present invention will be described below with reference to examples and comparative examples.

[0035] In the examples below, PB stands for phosphate buffer, and the amount of enzyme used in preparing immobilized enzyme is higher than that of free enzyme, taking into account some loss of enzyme during immobilization.

[0036] (Example 1) Immobilization process of transaminase (TA IV-Ss) on epoxy resin Take 1g of epoxy resin, wash with 0.2M PB8.0 and 0.8M NaCl buffer, remove the buffer, and keep the resin for use. Then add 4mL of enzyme solution (prepared enzyme powder of crude enzyme in 0.2M PB8.0 and 0.8M NaCl buffer so that the protein content is 25mg / mL, correspondingly with cofactor PLP) and incubate with epoxy resin on an orbital shaker at 20℃ for 20h, remove and leave to stand at 4℃ for 48h. Wash three times with 20mM PB8.0 to obtain immobilized transaminase carrying 30-80mg protein (protein loading amount is obtained by microplate reader Coomassie brilliant blue method or BCA (bicinchoninic acid) protein measurement method, and protein loading amount is different because epoxy resin is different).

[0037] Testing the activity and reusability of immobilized transaminases (TA IV-Ss-1, TA IV-Ss-2) [ka]

[0038] Add 0.5mL of MeOH to a 20mL reaction flask, dissolve 0.1g of main raw material 1, and add 15eq of isopropylamine hydrochloride and 15.0mg of PLP (5'-pyridoxal 5'-phosphate). Add 0.1M PB8.0 until the final volume of the reaction solution is 5mL, then add 0.1g of TA IV-Ss-1, 0.1g of TA IV-Ss-2 crude enzyme powder, or the above immobilized transaminase prepared with 0.2g of TA IV-Ss-1, 0.2g of TA IV-Ss-2 crude enzyme powder, and stir at 47℃ for 20h. The conversion rate of the system was detected by HPLC, and the immobilized enzyme was separated after each reaction and used repeatedly in the next reaction, thereby considering the number of times of repeated use. The transaminase reaction data is shown in Table 2. [Table 2] TIFF2025514551000007.tif156156

[0039] (Example 2) Immobilization process of lipase (CALB) on epoxy resin The immobilization method was the same as in Example 1.

[0040] Testing the activity and reusability of immobilized lipase (CALB) [ka]

[0041] Add 1.8mL of MTBE to a 10mL reaction flask, dissolve 0.1g of main material 2, add 0.8eq of HMDS (hexamethyldisilazane), add 0.1g of CALB crude enzyme powder or immobilized enzyme made with 0.1g of CALB crude enzyme powder, and stir at 40℃ for 20h. HPLC was used to detect the conversion rate of the system, and the immobilized enzyme was separated after each reaction and used repeatedly in the next reaction, and the number of times of use was considered and recorded in Table 3.

[0042] Testing the activity and reusability of immobilized lipases (CALB-1, CALB-2) [ka]

[0043] Add 2mL of MTBE to a 10mL reaction flask, dissolve 0.1g of main raw material 3, add 0.1M KPB7.5 until the final volume of the reaction solution is 4mL, then add 0.1g of CALB-1, CALB-2 crude enzyme powder, or immobilized enzyme made with 0.1g of CALB-1, 0.1g of CALB-2 crude enzyme powder, and stir at 47℃ for 16~20h. The conversion rate of the system was detected by HPLC, and the immobilized enzyme was separated after each reaction and used repeatedly in the next reaction, and the number of times of repeated use was considered, and raw material 3 was recorded in Table 3.

[0044] Testing the activity and reusability of immobilized lipases (CALB-1, CALB-2) [ka]

[0045] Add 2mL of MTBE to a 10mL reaction flask, dissolve 0.1g of main raw material 4, add 0.275g of vinyl acetate, add 0.1g of CALB-1, CALB-2 crude enzyme powder, or immobilized enzyme made from 0.1g of CALB-1, 0.1g of CALB-2 crude enzyme powder, and stir at 30℃ for 16~20h. HPLC was used to detect the conversion rate of the system, and the immobilized enzyme was separated after each reaction and used repeatedly in the next reaction, and the number of times of use was considered and recorded in Table 3. [Table 3] TIFF2025514551000012.tif171154TIFF2025514551000013.tif175163TIFF2025514551000014.tif168151TIFF2025514551000015.tif170155

[0046] Example 3: Testing the conversion rate and reusability of immobilized ketoreductase (CR-Ac) The immobilization method is the same as in Example 1.

[0047] Detection of activity and reusability of immobilized enzyme (CR-Ac) [ka]

[0048] Add 0.2 mL of isopropyl alcohol (IPA) to a 10 mL reaction flask, dissolve 0.1 g of main raw material 5, and add 1 mL of 0.1 M PB7.0 and NAD. + 10mg of CR-Ac crude enzyme was added, followed by 0.1g of CR-Ac crude enzyme powder or 0.2g of immobilized enzyme made from CR-Ac crude enzyme powder, and stirred at 30℃ for 20h. The conversion rate of the system was detected by HPLC, and the immobilized enzyme was separated after each reaction and reused in the next reaction, so that the number of times of reuse was considered. The reaction data is shown in Table 4. [Table 4]

[0049] Example 4: Testing the conversion rate and reusability of immobilized monooxygenase (CHMO-Rr) The immobilization method was the same as in Example 1.

[0050] The activity of the CHMO-Rr epoxy group vector immobilized enzyme was detected by reaction with the following main raw material 6. [ka]

[0051] Add 0.3 mL of isopropyl alcohol to a 10 mL reaction flask, then add 50 mg of the main raw material 6 and 5 mg of NADP +3mL of 0.1M PB (pH8.0) containing was added, followed by 5mg of dry powder of alcohol dehydrogenase (ABY93890.1) as a coenzyme, and then 0.1g of CHMO-Rr crude enzyme powder or immobilized enzyme made with 0.2g of CHMO-Rr crude enzyme powder was added. The reaction was carried out at 30℃ for 20h to test the conversion rate. After each reaction, the immobilized enzyme was separated and used repeatedly in the next reaction, thereby considering the number of times it was used repeatedly. The results can be seen in the table below. [Table 5]

[0052] Example 5: Testing the conversion rate and reusability of immobilized ene-reductase (ERED-Sc) The immobilization method was the same as in Example 1.

[0053] The activity of the ERED-Sc epoxy group vector immobilized enzyme was detected by reaction with the following main raw material 7. [ka]

[0054] Add 3 mL of 0.1 M PB (pH 7.0-8.0) to a 10 mL reaction flask, then add 0.1 g of the main ingredient 7, followed by 10 mg of NAD(P) as a coenzyme. + , 80mg of ammonium formate, 20mg of glucose dehydrogenase (ACR78513.1) were added, and 0.1g of EREC-Sc crude enzyme powder or 0.2g of immobilized enzyme made with ERED-Sc crude enzyme powder was added. The reaction was carried out at 30℃ for 20h to test the conversion rate. After each reaction, the immobilized enzyme was separated and reused in the next reaction, and the number of times it could be reused was considered. The test results can be seen in the table below. [Table 6]

[0055] Example 6: Testing the conversion rate and reusability of immobilized imine reductase (IRED) The immobilization method was the same as in Example 1.

[0056] The activity of the IRED-1 and IRED-2 epoxy group vector immobilized enzymes was detected by reaction with the following main raw material 8. [ka]

[0057] Add 2 mL of 0.1 M PB buffer (pH 7.0-8.0) to a 10 mL reactor, followed by 100 mg of the above main raw material 8 and 10 mg of NAD as a coenzyme. + , 60mg ammonium formate, 5mg dry powder of formate ammonia dehydrogenase (AIY34662.1) were added. Then, 0.1g IRED-1, 0.1g IRED-2 crude enzyme powder, or immobilized enzyme made with 0.2g IRED-1, 0.2g IRED-2 crude enzyme powder were added. After reacting for 20h at 30℃, the conversion rate was detected, and after each reaction, the immobilized enzyme was separated and used repeatedly in the next reaction, so that the number of times of repeated use was considered. The test results are as follows: [Table 7] TIFF2025514551000024.tif164151

[0058] Example 7: Testing the conversion rate and reusability of immobilized amino acid dehydrogenase (AADH-Ti) The immobilization method was the same as in Example 1.

[0059] The activity of the AADH-Ti epoxy group vector immobilized enzyme was detected by reaction with the following main raw material 9. [ka]

[0060] Add 5 mL of 0.1 M Tris-Cl (pH 8.0-9.0) to a 10 mL reaction flask, then add 0.1 g of main ingredient 9, add 108 mg of ammonium chloride, adjust the pH value to 7.5-8.0, and then add 10 mg of NAD as a coenzyme. + 50mg of GDH was added, along with 0.1g of crude AADH-Ti enzyme powder or 0.2g of immobilized enzyme made with crude AADH-Ti enzyme powder. After reacting at 30℃ for 20h, it was used for conversion rate test. The test results can be seen in the table below. [Table 8]

[0061] Example 8: Use of transaminase epoxy group vector immobilized enzyme in a packed bed continuous reaction In Example 1, transaminase TA IV-Ss-2 was immobilized on vector LX-109S, and the resulting immobilized enzyme was packed into a column reactor with a column volume of 120 mL, and the dosage of the immobilized enzyme was 70 g.

[0062] 100 g of the main raw material 1 was dissolved in 0.5 L of methanol, 15 eq of isopropylamine hydrochloride (0.6 L of 6 M isopropylamine hydrochloride aqueous solution) and 5 g of PLP were added, and the solution was adjusted to 5 L with PB buffer (0.1 M, pH 8.0).

[0063] The flow rate was set to 0.4mL / min, i.e., the retention time was set to 300min, and a continuous reaction was carried out. The conversion rate of the liquid flowing out from the outlet was detected, and the conversion rate was >98%, and after 280h of continuous operation, the conversion rate decreased to 86%. For details, please refer to the table below. [Table 9]

[0064] Example 9: Use of transaminase epoxy vector immobilized enzyme in a continuous stirred tank reaction The same immobilized enzyme TA IV-Ss-2 as in Example 1 was used, and the vector was LX-109S. 60 g of the immobilized enzyme of transaminase TA IV-Ss was added to a 1 L reactor, and 300 mL of phosphate buffer was added.

[0065] 4 L of PB (0.1 M, pH 8.0), 0.6 L of an aqueous solution of isopropylamine hydrochloride (6 M) and 5 g of PLP were added to 100 g of the main raw material 1, and the mixture was beaten to produce a suspension.

[0066] The substrate suspension was continuously added to the reaction flask at a rate of 0.4mL / min (i.e., retention time 500min), and at the same time, the reaction system was extracted from the outlet at the same flow rate (a filter head was attached to the end of the tube to prevent extraction of the immobilized enzyme). Under these conditions, the conversion rate reached more than 90%, and the system could be operated continuously for 350 hours with almost no decrease in the conversion rate. The results are shown in the table below. [Table 10]

[0067] (Example 10) Immobilization process of transaminase (TA IV-Ss) on epoxy resin 1 g of epoxy resin was taken and washed with 0.2 M PB8.0 and 0.8 M NaCl buffer, the buffer was removed, and the resin was kept for use. Then, 4 mL of crude enzyme solution (enzyme powder of crude enzyme was dissolved in 0.2 M PB8.0 and 0.8 M NaCl buffer to prepare an enzyme solution with a protein content of 20 mg / mL, correspondingly with the cofactor PLP) was added, and the mixture was incubated with epoxy resin in an orbital shaker at 20 °C for 20 h, removed, and incubated at 4 °C for 48 h. After washing three times with 20 mM PB8.0, immobilized transaminase containing 44 mg / g protein was obtained (protein loading was calculated by the microplate reader Coomassie Brilliant Blue method or BCA (bicinchoninic acid)).

[0068] (Example 11) Immobilization process of transaminase (TA IV-Ss) on epoxy resin 1 g of epoxy resin was taken and washed with 0.2 M PB8.0 and 0.8 M NaCl buffer, the buffer was removed, and the resin was kept for use. Then, 4 mL of crude enzyme solution (enzyme powder of crude enzyme was dissolved in 0.2 M PB8.0 and 0.8 M NaCl buffer to prepare an enzyme solution with a protein content of 30 mg / mL, correspondingly with the cofactor PLP) was added, and the mixture was incubated with epoxy resin in an orbital shaker at 20 °C for 20 h, removed, and incubated at 4 °C for 48 h. After washing three times with 20 mM PB8.0, immobilized transaminase containing 51 mg / g protein (protein loading was calculated by the microplate reader Coomassie Brilliant Blue method or BCA (bicinchoninic acid)) was obtained.

[0069] (Example 12) Immobilization process of transaminase (TA IV-Ss) on epoxy resin 1 g of epoxy resin was taken and washed with 0.2 M PB8.0 and 0.8 M NaCl buffer, the buffer was removed, and the resin was kept for use. Then, 4 mL of crude enzyme solution (enzyme powder of crude enzyme was dissolved in 0.2 M PB8.0 and 0.8 M NaCl buffer to prepare an enzyme solution with a protein content of 25 mg / mL, correspondingly with the cofactor PLP) was added, and the mixture was incubated with epoxy resin in an orbital shaker at 10 °C for 24 h, removed, and incubated at 4 °C for 48 h. After washing three times with 20 mM PB8.0, immobilized transaminase containing 55 mg / g protein (protein loading was calculated by the microplate reader Coomassie Brilliant Blue method or BCA (bicinchoninic acid)) was obtained.

[0070] (Example 13) Immobilization process of transaminase (TA IV-Ss) on epoxy resin 1 g of epoxy resin was taken and washed with 0.2 M PB8.0 and 0.8 M NaCl buffer, the buffer was removed, and the resin was kept for use. Then, 4 mL of crude enzyme solution (enzyme powder of crude enzyme was dissolved in 0.2 M PB8.0 and 0.8 M NaCl buffer to prepare an enzyme solution with a protein content of 25 mg / mL, correspondingly with the cofactor PLP) was added, and the mixture was incubated with epoxy resin in an orbital shaker at 20 °C for 16 h, removed, and incubated at 4 °C for 48 h. After washing three times with 20 mM PB8.0, immobilized transaminase containing 48 mg / g protein was obtained (protein loading was calculated by the microplate reader Coomassie Brilliant Blue method or BCA (bicinchoninic acid)).

[0071] (Example 14) Immobilization process of transaminase (TA IV-Ss) on epoxy resin 1 g of epoxy resin was taken and washed with 0.2 M PB8.0 and 0.8 M NaCl buffer, the buffer was removed, and the resin was kept for use. Then, 4 mL of crude enzyme solution (prepared by dissolving the crude enzyme powder in 0.2 M PB8.0 and 0.8 M NaCl buffer so that the protein content is 25 mg / mL, and correspondingly with the cofactor PLP) was added, incubated with epoxy resin in an orbital shaker at 25 °C for 20 h, removed, and incubated at 4 °C for 48 h. After washing three times with 20 mM PB8.0, immobilized transaminase containing 61 mg / g protein was obtained.

[0072] The test was carried out using the same test means as in Example 1, and the test results can be seen in Table 11. [Table 11]

[0073] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects.

[0074] In the present application, LX-109S epoxy resin is used as the epoxy resin vector, and based on the characteristics of the vector itself, the immobilization effect on the enzyme is more stable, and the covalent bond with the enzyme is strengthened, without affecting the activity of the enzyme itself.

[0075] 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. Any modifications, equivalent replacements, improvements, etc. made within the scope of the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. an epoxy resin vector, which is LX-109S epoxy resin; and an enzyme derived from a crude enzyme, which is covalently linked to the epoxy resin vector; 1. An immobilized enzyme comprising:

2. The enzyme is any one selected from transaminase, D-lactate dehydrogenase, cyclohexanone monooxygenase, ketoreductase, enereductase, nitrilase, ammonia lyase, amino acid dehydrogenase, imine reductase, lipase, and mutants thereof; 2. The immobilized enzyme according to claim 1 .

3. The ammonia lyase is any one selected from phenylalanine ammonia lyases.

3. The immobilized enzyme according to claim 2.

4. The amino acid dehydrogenase is any one selected from leucine dehydrogenase and phenylalanine dehydrogenase.

3. The immobilized enzyme according to claim 2.

5. In the immobilized enzyme, the amount of the enzyme supported per 1 g of the epoxy resin vector is 30 mg to 70 mg.

2. The immobilized enzyme according to claim 1 .

6. The immobilized enzyme further comprises a cofactor.

2. The immobilized enzyme according to claim 1 .

7. Step S1 of mixing a first phosphate buffer solution with an enzyme, which is a crude enzyme, to form an enzyme buffer solution; and step S2 of mixing the enzyme buffer with an epoxy resin, which is an LX-109S epoxy resin, to carry out an immobilization reaction, and then washing with a second phosphate buffer to obtain the immobilized enzyme. The method for producing the immobilized enzyme according to any one of claims 1 to 6.

8. The volume ratio of the enzyme buffer to the epoxy resin is 3:1 to 5:

1. The method according to claim 7 .

9. The first phosphate buffer has a pH value of 7.0 to 8.0 and contains sodium chloride, preferably, the concentration of sodium chloride in the first phosphate buffer is 1±0.2 mol / L, and the second phosphate buffer has a pH value of 7.0 to 8.0 and does not contain sodium chloride; The method according to claim 7 .

10. The step S2 is mixing the enzyme buffer and the epoxy resin at 10 to 20° C., shaking the mixture for 16 to 24 hours, and then allowing the mixture to stand and incubate at 3 to 5° C. for 40 to 48 hours to obtain an immobilized system; and washing the immobilization system with the second phosphate buffer to obtain the immobilized enzyme. The method according to claim 7 .

11. The immobilized enzyme is used as a catalyst in a continuous catalytic reaction. Use of the immobilized enzyme according to any one of claims 1 to 6.

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