Immobilization method and application of alpha-amino acid ester acyltransferase immobilized enzyme
By designing cross-linked immobilization of α-amino acid ester acyltransferase of multiple tandem glutamine residues with gelatin, the problems of poor selectivity and insufficient stability of enzymes in the prior art are solved, and the selective immobilization and purification of enzymes are synchronized, and the reaction efficiency and product yield are improved.
Patent Information
- Application Number
- CN202510682854.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-26
- Publication Date
- 2025-08-15
AI Technical Summary
The existing immobilized enzyme technology has poor selectivity and specificity, making it difficult to effectively remove hybrid enzymes, and the enzyme is easily shed from the carrier under complex reaction conditions, affecting the immobilization effect and reuse.
By designing an α-amino acid ester acyltransferase containing multiple tandem glutamine residues to cross-link and immobilize the enzyme protein with gelatin, TG enzyme is used to catalyze the specific reaction of the target protein with the gelatin carrier, avoiding the participation of hybrid enzymes, and achieving selective and specific immobilization.
The selective immobilization and purification of enzymes are achieved synchronously, which improves the stability and reusability of enzymes, reduces the interference of hybrid enzymes on the reaction, and improves the reaction efficiency and product yield.
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Abstract
Description
Technical Field
[0001] The invention relates to an immobilization method of an immobilized alpha-amino acid ester acyltransferase, belonging to the technical field of biological enzymes. Background Art
[0002] Alanine-glutamine dipeptide is a biologically active dipeptide composed of alanine and glutamine residues. It has the effects of improving intestinal cell activity, immune function, promoting protein and ester synthesis, and regulating ammonia and sugar concentrations in the blood. It can be used clinically for the prevention and treatment of intestinal diseases and lung tissue damage.
[0003] One of the methods for synthesizing alanine dipeptide is to use α-amino acid ester acyltransferase to catalyze the production using L-alanine methyl ester as a substrate. Among them, the use of immobilized enzyme technology for the synthesis of alanine dipeptide has many advantages. Immobilized enzyme technology can transform invisible enzymes into controllable visible solids that can be reused, and the composition of the reaction system is clear, which gives immobilized enzymes unparalleled advantages in the research and production of multiple industries such as food processing, medicine, and fine chemicals.
[0004] Currently, the main methods for immobilizing enzymes include adsorption, embedding, covalent bonding, and cross-linking. However, covalent bonding and cross-linking methods, such as these, use chemical synthesis to form covalent links between enzymes or between enzymes and carriers. These chemical reactions and the chemical reagents used often significantly damage the enzyme proteins. Furthermore, these chemical reactions have poor selectivity and specificity, failing to distinguish the desired enzyme (target protein) from unwanted enzymes. In industrial production, enzyme purification is a time-consuming, labor-intensive, and costly step, especially for intracellular enzymes, where the diverse and complex nature of these enzymes often significantly interferes with the reactions catalyzed by the primary enzyme.
[0005] In order to remove the influence of miscellaneous enzymes on the main enzyme, the existing method is to use histidine tag technology to achieve one-step purification and immobilization of the main enzyme. This technology uses the chelating ability of the histidine imidazole group and metal ions to separate the main enzyme from the miscellaneous enzymes. After separation, the enzyme adsorbed on the carrier can be directly used as an immobilized enzyme. However, the binding strength of this immobilized enzyme to the carrier is limited. Under some slightly complex reaction conditions, the enzyme will fall off the carrier. For example, the presence of chelating agents in the reaction system, acidic reaction systems, competitive divalent ions in the system, etc. can all lead to the breaking of the chelate bond between the enzyme protein and the carrier metal ion, or the metal ions fall off the carrier, resulting in the failure of immobilization and the failure of reuse. Summary of the Invention
[0006] The present invention aims to solve the problems existing in the above prior art and provides a method for immobilizing an α-amino acid ester acyltransferase immobilized enzyme, which solves the problem of how to effectively achieve specific immobilization and effectively remove foreign enzymes.
[0007] The objective of the present invention is achieved by the following technical solution: a method for immobilizing an immobilized α-amino acid ester acyltransferase, the method comprising the following steps:
[0008] Under the catalytic action of TG enzyme, an α-amino acid ester acyltransferase containing multiple glutamine residues is subjected to an enzyme-protein cross-linking immobilization reaction with gelatin to obtain an immobilized α-amino acid ester acyltransferase immobilized enzyme.
[0009] By designing multiple tandem glutamine residues on the target protein and using a gelatin carrier containing lysine residues, the target protein can be effectively immobilized by reacting specifically with the gelatin carrier through the catalytic action of TGase. Furthermore, the design of a tag with multiple tandem glutamine residues can distinguish the target protein from other proteins in the sample, preventing the participation of contaminating enzymes in the immobilization reaction, thereby effectively eliminating the impact of contaminating enzymes on subsequent reactions and achieving better enzyme purification. More importantly, the design of multiple tandem glutamine residue tags and the high concentration of lysine residues in the gelatin molecule preferentially allow the target protein and gelatin to react to form cross-linked products under TGase catalysis, achieving selective and specific immobilization of the enzyme protein. Other contaminating enzymes are not immobilized, effectively achieving the purpose of purification, achieving the effect of simultaneous immobilization and purification, and ensuring more stable immobilization, better preventing them from falling off the carrier during subsequent reactions, and having the advantage of good reusability. At the same time, the TG enzyme has good pH stability and thermal stability. The enzyme has high activity in the pH range of 5.0-8.0; the optimum temperature is around 50°C, and it has high activity at 45-55°C. These properties are very suitable for use as the immobilization reagent of the present invention, and it also has the advantage of low price.
[0010] In the above-mentioned immobilization method of the α-amino acid ester acyltransferase immobilized enzyme, preferably, the α-amino acid ester acyltransferase carries 5 connected glutamine residues at the N-terminus or C-terminus. It can better enable the TG enzyme to catalyze the cross-linking reaction between the glutamine residue and the lysine residue of the target protein, distinguish other miscellaneous enzymes and thus avoid the reaction of miscellaneous enzymes with gelatin, have better selectivity and specificity, and facilitate the purpose of purification. The design of the residues here may be achieved by conventional molecular biological operations, by introducing corresponding codons into DNA, such as by expressing in cells such as Escherichia coli and yeast through conventional genetic engineering techniques. Preferably, the gene sequence of the α-amino acid ester acyltransferase is shown in SEQ ID NO.1, and the amino acid sequence of the α-amino acid ester acyltransferase is shown in SEQ ID NO.2. It can be more conducive to the subsequent catalytic performance for the synthesis of alanine dipeptide.
[0011] In the above-mentioned immobilization method for α-amino acid ester acyltransferase, the enzyme protein cross-linking immobilization reaction is preferably carried out in a PBS buffer with a pH of 7.5-8.5. This can better ensure enzyme activity, facilitate the cross-linking reaction, and improve reaction efficiency and product quality.
[0012] The second object of the present invention is achieved through the following technical scheme: an immobilized α-amino acid ester acyltransferase is used to synthesize alanine dipeptide using glutamine and alanine methyl ester as raw materials under the catalytic action of the immobilized α-amino acid ester acyltransferase.
[0013] Conventional, unpurified free enzymes or immobilized enzymes often hydrolyze the raw material alanine methyl ester during the reaction due to the presence of impurities, resulting in reduced reaction yield and increased manufacturing costs. Compared to conventional, unpurified free enzymes or immobilized enzymes, the immobilized α-amino acid ester acyltransferase of the present invention utilizes an AET enzyme tagged with multiple tandem glutamine residues (e.g., QQQQ) to form an immobilized enzyme with gelatin under the catalysis of TG. This effectively removes impurities that are not the target protease, thereby better catalyzing the reaction between glutamine and alanine methyl ester to form alanine-glutathione dipeptide, avoiding the hydrolysis of alanine methyl ester and improving production yield and reducing costs.
[0014] In the application of the above-mentioned immobilized α-amino acid ester acyltransferase, the synthesis route of alanylglutathione catalyzed by the free enzyme or the immobilized enzyme of the present invention can be represented by the following route:
[0015]
[0016] In summary, compared with the prior art, the present invention has the following advantages:
[0017] 1. By designing multiple serially connected glutamine residue tags and the presence of a large number of lysine residues in the gelatin molecule, cross-linking products between the target protein and gelatin can be preferentially formed under the catalysis of TG enzyme, thereby achieving selective and specific immobilization of the enzyme protein; while other miscellaneous enzymes will not be immobilized, thereby better achieving the purpose of purification and realizing the effect of simultaneous immobilization and purification.
[0018] 2. By designing the glutamine residues with QQQQQ tags, the TG enzyme can better catalyze the cross-linking reaction between the glutamine residues and lysine residues of the target protein, distinguishing other miscellaneous enzymes and thus avoiding the reaction between miscellaneous enzymes and gelatin, with better selectivity and specificity, which is conducive to achieving the purpose of purification. DETAILED DESCRIPTION
[0019] 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.
[0020] Example 1
[0021] Preparation of QQQQQ-AET enzyme
[0022] The gene sequence shown in SEQ ID NO. 1 was synthesized using conventional gene synthesis techniques and cloned into plasmid pET30a. The clones were then inoculated into Escherichia coli BL21(DE3) and Rosetta™ 2(DE3), respectively. Single colonies were selected and cultured in LB medium containing 50 μg / ml kanamycin at 37°C with shaking. After the OD value reached 0.8, 0.5 mM IPTG was added and induced at 15°C for 16 hours. The cells were harvested by centrifugation and further disrupted to obtain a crude enzyme solution containing α-amino acid ester acyltransferase. The amino acid sequence of the α-amino acid ester acyltransferase is shown in SEQ ID NO. 2, and the α-amino acid ester acyltransferase carries a QQQQQQQ tag. The crude enzyme solution was further purified by centrifugation to obtain a supernatant (T195 supernatant), which contains the QQQQQQ-AET enzyme.
[0023] Example 2
[0024] Immobilization of QQQQQ-AET enzyme
[0025] Immobilization with amino resin
[0026] To a 50 mL reaction flask, 29 mL of pH 8.0 PBS buffer and 4 g of amino resin (Model 703, Manufacturer: Xi'an Lanxiao Technology New Materials Co., Ltd.) were added. The reaction temperature was controlled at 15°C. 1 mL of the supernatant enzyme solution obtained in Example 1 (T195 supernatant enzyme solution) was added and adsorbed for 4 hours with stirring at 100 rpm. The mixture was then filtered to obtain 3.9 g of amino-immobilized enzyme immobilized on the amino resin.
[0027] Example 3
[0028] Immobilized enzymes immobilized with gelatin
[0029] To a 50 mL reaction flask, add 9 mL of TG enzyme solution (50 / L dissolved in PBS buffer) with a pH of 7.8, add 4 g of gelatin soaked in purified water, control the temperature at 15°C, and then add 1 mL of the supernatant enzyme solution obtained in Example 1 (T195 supernatant enzyme solution). Adsorb for 4 hours with stirring at 100 rpm and filter to obtain 3.83 g of gelatin-immobilized enzyme.
[0030] Example 4
[0031] Effect of immobilization mode on enzyme purification of TG enzyme catalysis
[0032] This example is to compare the differences in the hydrolysis of alanine methyl ester (L-Ala-OMe) between gelatin immobilization and amino immobilization and non-immobilization. The specific operation is as follows:
[0033] A 10 g / L L-Ala-OMe solution was prepared using a 0.2 mol / L boric acid aqueous solution and added to four 50 ml centrifuge tubes. The four centrifuge tubes were numbered as follows: no enzyme, crude enzyme solution, amino-immobilized enzyme obtained in Example 2, and gelatin-immobilized enzyme obtained in Example 3 (the same amount of enzyme protein was added to each of the four centrifuge tubes). The total volume of each centrifuge tube was 20 ml. The reaction was then carried out on a shaker at 200 rpm, with the reaction temperature at 15°C and the pH at 8.0. After 3 h of reaction, samples were taken to determine the concentration change.
[0034] The results showed that the residual L-Ala-OMe levels in the four centrifuge tubes containing no enzyme, crude enzyme solution, amino-immobilized enzyme obtained in Example 2, and gelatin-immobilized enzyme obtained in Example 3 were 8.07 g / L, 4.18 g / L, 2.96 g / L, and 6.49 g / L, respectively. These analytical data indicate that the systems using crude enzyme solution and amino-immobilized amino-immobilized enzyme in the centrifuge tubes exhibited higher L-Ala-OMe-degrading enzyme activity. Furthermore, gelatin-immobilized enzyme immobilized with the aforementioned α-amino acid ester acyltransferase (gene sequence shown in SEQ ID NO. 1, amino acid sequence shown in SEQ ID NO. 2) of the present invention and gelatin effectively reduced the degradation of L-Ala-OMe.
[0035] Example 5
[0036] Synthesis of Alanylglutamine catalyzed by immobilized QQQQQ-AET enzyme (gelatin-immobilized enzyme):
[0037] 0.5 g of the gelatin-immobilized enzyme of the present invention was added to 20 mL of a reaction solution containing 100 mM L-Gln, 100 mM L-Ala-OMe, and 200 mM boric acid. The enzyme activity was measured at 25°C for 10 min on a water bath shaker at 200 rpm. The enzyme catalyzed the production of 1.99 g / L alanyl-glutathione, with an enzyme activity of 36.76 U / g.
[0038] Example 6
[0039] Reusability of immobilized QQQQQ-AET enzyme (gelatin-immobilized enzyme)
[0040] 1.46 g L-Gln and 0.618 g boric acid were weighed and placed in a beaker. 10 mL of purified water was added, and the temperature was lowered to 15°C. Stirring was started, and 2 M sodium hydroxide was used to adjust the pH of the reaction system to about 8.0. 3.83 g of the gelatin-immobilized enzyme obtained in Example 3 was added. 2.09 g L-Ala-OMe was weighed and dissolved in 5 ml of purified water, and the pH was adjusted to 6.60. The mixture was slowly pumped into the beaker using a peristaltic pump for 1 h. At the same time, 2 M sodium hydroxide was added dropwise to maintain the pH at 8.0. The reaction was completed after 2.5 h.
[0041] After the reaction, the gelatin-immobilized enzyme was washed with purified water and used for the next reaction. Four batches were reacted in total. The concentration of glutathione generated was detected by HPLC. The specific data of the four batches are as follows:
[0042] Table 1:
[0043]
[0044] The conditions for HPLC liquid phase detection are:
[0045] The detection wavelength was 215 nm, the mobile phase was acetonitrile: 50 mM potassium dihydrogen phosphate = 65:35, the column temperature was 30 °C, the flow rate was 1 ml / min, the injection volume was 10 μl, the column model was ShimNex HE NH2, 5 μm, and the column length was 4.6 × 250 mm.
[0046] The results in Table 1 above show that the composite has good reusability.
[0047] Example 7
[0048] Gelatin-immobilized enzyme immobilized with gelatin
[0049] To a 50 mL reaction flask, 10 mL of TG enzyme solution (50 g / L dissolved in PBS buffer) at pH 8.0 was added, along with 4 g of gelatin soaked in purified water. The temperature was controlled at 20°C, and 1 mL of the supernatant enzyme solution (T195 supernatant enzyme solution) obtained by the method in Example 1 was added. The mixture was adsorbed for 4 hours with stirring at 100 rpm. The mixture was filtered to obtain 3.82 g of gelatin-immobilized enzyme.
[0050] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.
[0051] Although the present invention has been described in detail and certain specific embodiments have been cited, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.
Claims
1. A method for immobilizing an α-amino acid ester acyltransferase immobilized enzyme, characterized in that: The method comprises the following steps: Under the catalytic action of TG enzyme, an α-amino acid ester acyltransferase containing multiple glutamine residues is subjected to an enzyme-protein cross-linking immobilization reaction with gelatin to obtain an immobilized α-amino acid ester acyltransferase immobilized enzyme.
2. The method for immobilizing the immobilized α-amino acid ester acyltransferase according to claim 1, characterized in that: The α-amino acid ester acyltransferase has five linked glutamine residues at the N-terminus or the C-terminus.
3. The method for immobilizing the immobilized α-amino acid ester acyltransferase according to claim 1, characterized in that: The gene sequence of the α-amino acid ester acyltransferase is shown in SEQ ID NO.1, and the amino acid sequence of the α-amino acid ester acyltransferase is shown in SEQ ID NO.
2.
4. The method for immobilizing an immobilized α-amino acid ester acyltransferase according to claim 1, 2 or 3, characterized in that: The temperature of the enzyme protein cross-linking immobilization reaction is 15°C-20°C.
5. The method for immobilizing the immobilized α-amino acid ester acyltransferase according to claim 1, 2 or 3, characterized in that: The enzyme protein cross-linking immobilization reaction is carried out in a PBS buffer solution with a pH value of 7.5-8.
5.
6. An application of an immobilized α-amino acid ester acyltransferase, characterized in that: Alanine dipeptide was synthesized from glutamine and alanine methyl ester under the catalysis of immobilized α-amino acid ester acyltransferase.