Transaminase immobilization method

Abstract

The invention relates to a transaminase immobilization method, and belongs to the technical field of enzyme immobilization. In order to solve the problem of improving the enzyme immobilization activity, the invention provides a transaminase immobilization method, the transaminase immobilization method is a covalent organic framework immobilized transaminase, and the transaminase immobilization method comprises the following steps: constructing a covalent organic framework and carrying out immobilization screening on the transaminase, the covalent organic framework is prepared from the following raw materials: benzenetricarboxaldehyde and diamine and diacid monomers, the diamine and diacid monomers are selected from one of 1, 4-phenylenediamine, p-phenylenediamine and terephthalic acid, a solvent used in the process of constructing the covalent organic framework is one of DMF (Dimethyl Formamide), THF (Tetrahydrofuran) and dioxane, and a catalyst used in the process of constructing the covalent organic framework is glacial acetic acid; the method has the advantage of high enzyme activity recovery rate after enzyme immobilization on the whole.

Description

Technical Field

[0001] The invention relates to a transaminase immobilization method and belongs to the field of enzyme immobilization. Background Art

[0002] The transaminase family is an important green biocatalyst that can catalyze the asymmetric amination reaction of ketones to produce chiral amines and is widely used in the synthesis of pharmaceutically active molecules, fine chemicals and pesticides.

[0003] In the common process of transaminase immobilization, amino-modified multi-walled carbon nanotubes are used to prepare immobilized transaminase with stronger stability, which is of great significance for the application of transaminase. However, the multi-walled carbon nanotube carrier has a good effect on improving the enzyme activity, but cannot achieve a higher enzyme activity recovery rate. When used in the production process of drug intermediates, it has a poor effect on reducing production costs. Summary of the invention

[0004] In view of the defects existing in the above prior art, the present invention provides a method for immobilizing a transaminase, and solves the problem that the preparation method of the traditional transaminase immobilization carrier has a poor effect on improving the recovery rate of enzyme activity.

[0005] The objective of the present invention is achieved through the following technical scheme: the transaminase immobilization method is a covalent organic framework immobilization transaminase, the transaminase immobilization method comprises constructing a covalent organic framework and performing immobilization screening on the transaminase, and the raw materials of the covalent organic framework comprise trimesic acid, diamine and diacid monomers.

[0006] Furthermore, the diamine and diacid monomers are selected from one of 1,4-phenylenediamine, p-phenylenediamine and terephthalic acid.

[0007] Furthermore, the solvent used in the process of constructing the covalent organic framework is one of DMF, THF and dioxane.

[0008] Furthermore, the catalyst used in the process of constructing the covalent organic framework is glacial acetic acid.

[0009] Furthermore, the feed ratio of trimesaldehyde to catalyst is 1:0.05-0.25.

[0010] Furthermore, the enzyme loading amount is 270-390 mg 酶 / g 载体 .

[0011] Furthermore, the pH of the transaminase immobilization screening stage is 4-8.

[0012] In summary, compared with the prior art, the present invention has the following advantages: 1. In the scheme of the present invention, the covalent organic framework has a good protective effect on the immobilized transaminase enzyme under different conditions and during the recovery process; 2. In the scheme of the present invention, the diamine and diacid monomers can affect the pore structure and size of the final immobilized enzyme particles, thereby affecting the activity, stability and substrate contact efficiency of the enzyme. Through immobilization, the enzyme is fixed on a solid carrier, which can significantly improve its stability under specific reaction conditions. DETAILED DESCRIPTION

[0013] The technical solution of the present invention is further specifically described below through specific embodiments, but the present invention is not limited to these embodiments.

[0014] Dissolve 8.11g of trimesaldehyde in 125mL of DMF, transfer the solution to 10mL of deionized water, ultrasonicate for 3min, take it out and add 0.45mL of glacial acetic acid and 5mL of crude transaminase solution (10mg / mL). Prepare a solution of 5.81g of 1,4-phenylenediamine and 25mL of DMF and add it to the reaction system, and react at room temperature for 10min. After the reaction is completed, adjust the pH of the solution to 4 with NaOH. Stir the mixture at 25°C for 10min. Wash the precipitate three times with deionized water to obtain a particle suspension of immobilized enzyme.

[0015] The reaction was carried out in PBS buffer (50 mM, pH 8.0), and 5 g of immobilized enzyme, 12.24 mL of 1-acetonapthone, 9.7 mL of 1-phenylethylamine, and 0.32 mL and 0.12 g of pyridoxal phosphate were added. The reaction mixture was shaken at 30 °C and 500 rpm / min for 15 min. 1 mL of the mixture was diluted with 50% acetonitrile and analyzed for enzyme activity by HPLC; The chromatographic column was EC-C18 column, the column box temperature was 30°C, the detection wavelength was 340nm, the mobile phase was acetonitrile and ultrapure water, the volume ratio was 60:4, the flow rate was 1mL / min, and the running time was 8min. The retention time of (R)-NEA was 4.8min. Each measurement was repeated at least 3 times, and the standard deviation was less than 5%; The enzyme activity after fixation was: 247.6U / g; Enzyme activity recovery rate: 79.36%; Residual enzyme activity after 10 batches: 81.2%.

[0016] This example is a comparative example of Example 1, and the reaction conditions are modified as follows: 1,4-phenylenediamine is replaced by p-phenylenediamine, and other conditions are the same. The modified immobilized enzyme obtained by this example scheme is used to perform enzyme activity detection, and the results are as follows: The enzyme activity after fixation was: 242.4 U / g; Enzyme activity recovery rate: 71.1%; Residual enzyme activity after 10 batches: 77.4%.

[0017] This example is a comparative example of Example 1, and the reaction conditions are modified as follows: 1,4-phenylenediamine is replaced by terephthalic acid, and other conditions are the same. The modified immobilized enzyme obtained by the scheme of this example is used to perform enzyme activity detection, and the results are as follows: The enzyme activity after fixation was: 238.6U / g; Enzyme activity recovery rate: 71.3%; Residual enzyme activity after 10 batches: 78.2%.

[0018] This embodiment is a comparative example of embodiment 1, and the reaction conditions are modified as follows: the feed ratio of trimesaldehyde to catalyst is adjusted to 1:0.05 and 1:0.25, and other conditions are the same. The modified immobilized enzyme obtained by using the scheme of this embodiment is used for enzyme activity detection. As the concentration of glacial acetic acid gradually increases, the enzyme activity recovery decreases accordingly. After reducing the concentration of glacial acetic acid to a lower concentration, the enzyme activity recovery fluctuates less.

[0019] This example is a comparative example of Example 1, and the reaction conditions are modified as follows: the solvents used in the process of constructing the covalent organic framework are changed to THF and dioxane in sequence, and other conditions are the same. The modified immobilized enzyme obtained by using the scheme of this example is used for enzyme activity detection, and the results are very different, and the enzyme activity recovery rates are all around 78%.

[0020] This example is a comparative example of Example 1, and the reaction conditions are modified as follows: the enzyme loading amount is adjusted to 270 mg 酶 / g 载体 With 390mgmg 酶 / g 载体 , other conditions being the same, the modified immobilized enzyme obtained by the scheme of this embodiment was used to detect the enzyme activity. As the amount of enzyme gradually increased, the unit enzyme activity gradually increased. The enzyme loading amount was 390 mg mg 酶 / g 载体 , the unit enzyme activity is the highest.

[0021] This example is a comparative example of Example 1, and the reaction conditions are modified as follows: the pH of the transaminase immobilization screening stage is adjusted to 4, 5, 6, 7, and 8. Other conditions are the same. The modified immobilized enzyme obtained by using the scheme of this example is used for enzyme activity detection. The test results show that under acidic conditions, the enzyme will not be destroyed for at least 1 hour after immobilization.

[0022] The implementation of the present invention is not limited to the above-mentioned embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and detail, and these are considered to fall within the protection scope of the present invention.

Claims

1. A method for immobilizing a transaminase, characterized in that: The transaminase immobilization method is to immobilize the transaminase in a covalent organic framework, and the transaminase immobilization method includes constructing a covalent organic framework and performing immobilization screening on the transaminase, and the raw materials of the covalent organic framework include trimesic acid, diamine and diacid monomers.

2. A method for immobilizing a transaminase according to claim 1, characterized in that: The diamine and diacid monomers are selected from one of p-phenylenediamine, 1,4-phenylenediamine and terephthalic acid.

3. A method for immobilizing a transaminase according to claim 2, characterized in that: The solvent used in the process of constructing the covalent organic framework is one of DMF, THF and dioxane.

4. A method for immobilizing a transaminase according to claim 3, characterized in that: The catalyst used in the process of constructing the covalent organic framework is glacial acetic acid.

5. A method for immobilizing a transaminase according to claim 4, characterized in that: The feed ratio of trimesaldehyde to catalyst is 1:0.05-0.

25.

6. A method for immobilizing a transaminase according to claim 5, characterized in that: The enzyme loading amount is 270~390mg 酶 / g 载体 .

7. The method for immobilizing a transaminase according to claim 1, characterized in that: The pH value of the transaminase immobilization screening stage is 4-8.