Sodium alginate-gelatin hydrogel coated multi-enzyme system and application thereof

Abstract

The invention provides a sodium alginate-gelatin hydrogel coated multi-enzyme system and application, and belongs to the technical field of bioengineering. A P protein (a phosphopyridoxal dependent glycine decarboxylase), a T protein (aminomethyltransferase) and an H protein (a lipoic acid-containing protein) are wrapped in a sodium alginate-gelatin hydrogel, so that the stability and activity of enzymes can be remarkably improved, the multi-enzyme cascade reaction efficiency is improved, and the yield of glycine converted from carbon dioxide is further improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a multi-enzyme system encapsulated in sodium alginate-gelatin hydrogel and its application in improving the yield of converting carbon dioxide into glycine. Background Art

[0002] The reversed Glycine Cleavage Reaction (rGCR) is an important pathway for the biosynthesis of glycine. This reaction involves the coordinated action of multiple enzymes, including Protein P (a pyridoxal phosphate-dependent glycine decarboxylase), Protein T (aminomethyltransferase), Protein H (a lipoic acid-containing protein), and Protein L (lipoamide dehydrogenase), etc. In the traditional reaction system, this multi-enzyme system directly converts carbon dioxide and methanol into glycine, but the reaction depends on NAD(P)H and ATP, and has a low thermodynamic driving force, resulting in low reaction efficiency and limited glycine production. The research group of Zeng Anping at Westlake University has developed a method that combines chemical and bioengineering methods, relying on the engineering modification of dithiothreitol (DTT) and Protein H, without relying on NAD(P)H, ATP, and Protein L to produce glycine, which improves the thermodynamic driving force and greatly improves the production efficiency.

[0003] In recent years, hydrogels, as materials with high biocompatibility, have been widely used in the immobilization and protection of enzymes. Sodium alginate-gelatin hydrogels have good mechanical properties and biocompatibility, and can effectively improve the stability and activity of enzymes, thereby improving the efficiency of multi-enzyme cascade reactions. Summary of the Invention

[0004] The present invention aims to provide a multi-enzyme system encapsulated in sodium alginate-gelatin hydrogel and its application to improve the efficiency of the multi-enzyme cascade reaction of the reversed glycine cleavage reaction and the local concentration of enzymes, thereby improving the yield of converting carbon dioxide into glycine.

[0005] To achieve the above object, the first aspect of the present invention provides a multi-enzyme system encapsulated in sodium alginate-gelatin hydrogel, which is prepared by the following steps:

[0006] 1. Expression and purification of enzymes: Protein P (pyridoxal phosphate-dependent glycine decarboxylase), Protein T (aminomethyltransferase), and Protein H (lipoic acid-containing protein) are three of the constituent proteins of the glycine cleavage system (GCS).

[0007] 2. Preparation of hydrogel: Mix P protein, T protein, H protein and buffer to obtain an enzyme mixture, such that the final concentration ratio of P protein, T protein and H protein in the enzyme mixture is 5:3:40. Add sodium alginate and stir well to obtain a mixture, such that the concentration of sodium alginate in the mixture is 1-3% w / v. Dissolve CaCl2 in buffer to make the CaCl2 concentration 70-100 mM to obtain a curing solution. Drop the mixture into the curing solution to form hydrogel microspheres. Wash the hydrogel microspheres with deionized water and buffer in sequence.

[0008] As a preferred embodiment, the curing solution further includes gelatin with a concentration of 1-2% w / v.

[0009] Furthermore, the final concentration of P protein in the enzyme mixture is 5-30 μM.

[0010] As a preferred embodiment, when dropping the mixture into the curing solution, continuously stir the curing solution during dropping.

[0011] Furthermore, the diameter of the hydrogel microspheres is 1-5 mm.

[0012] Furthermore, the buffer is Tris-HCl buffer with a pH of 7.3-7.5.

[0013] As a second aspect, a method for improving the efficiency of the reverse glycine cleavage reaction is provided, including the following steps:

[0014] Suspend the above multi-enzyme system in a reaction system containing DTT, HCHO, THF, PLP, NH4Cl, NaHCO3 to carry out the reverse glycine cleavage reaction; carry out the reaction under the conditions of 37 °C and pH 7.3-7.5, and the reaction time is 24-48 hours.

[0015] As a preferred embodiment, the concentrations of DTT, HCHO, THF, PLP, NH4Cl, NaHCO3 in the reaction system are respectively: 20 mM, 10 mM, 0.5 mM, 25 μM, 50 mM and 50 mM.

[0016] Evaluate the efficiency of the multi-enzyme system by monitoring the amount of glycine generated during the reaction.

[0017] The beneficial effects of the present invention are as follows:

[0018] (1) Improve the stability and activity of the enzyme: Sodium alginate-gelatin hydrogel can provide a stable microenvironment for the enzyme, protect the enzyme from the influence of the external environment, and thus improve the activity and stability of the enzyme;

[0019] (2) Improving the efficiency of multi-enzyme cascade reactions: Hydrogel microspheres can effectively immobilize multiple enzymes, increase the local concentration of enzymes, promote the synergy between multiple enzymes, and improve the reaction efficiency;

[0020] (3) Increasing the glycine yield: By optimizing the multi-enzyme system, the glycine yield is significantly increased. Description of the Drawings

[0021] Figure 1 Images of sodium alginate-gelatin (left) and sodium alginate (right) hydrogel microspheres;

[0022] Figure 2 Standard curve graph of glycine for high-throughput mass spectrometry detection;

[0023] Figure 3 Comparison graph of glycine production amounts with enzymes encapsulated in different types of hydrogels;

[0024] Figure 4 Comparison graph of glycine production amounts in hydrogels encapsulating enzyme systems with different concentrations. Detailed Embodiments

[0025] The embodiments, features, and aspects of the present invention will be described in detail below with reference to the drawings, but this does not limit the present invention. Any embodiment extended based on the embodiments of the present invention, without creative efforts, for all other embodiments obtained by those of ordinary skill in the art, falls within the scope of protection of the present invention.

[0026] Example 1:

[0027] 1. Preparation of enzymes: Each protein solution (P protein, T protein, and H protein dissolved in Tris-HCl buffer with a pH of 7.5) was expressed and purified through an Escherichia coli overexpression system. Then, the protein solutions were mixed to form an enzyme mixture such that the final concentration of P protein in the enzyme mixture was 5 μM, the final concentration of T protein was 3 μM, and the final concentration of H protein was 40 μM.

[0028] 2. Preparation of hydrogels: Sodium alginate was dissolved in the enzyme mixture prepared in step 1, and stirred thoroughly to form a uniform sodium alginate solution with a sodium alginate concentration of 2% (w / v). CaCl2 was dissolved in Tris-HCl (pH 7.5) buffer to a final concentration of 75 mM to obtain a solidifying solution. The mixture was dropped into the stirring CaCl2-containing solidifying solution using a 1 mL syringe, and left standing until hydrogel microspheres with a diameter of 1 - 2 mm were formed, as shown on the right in Figure 1 . The hydrogel microspheres (i.e., the multi-enzyme system) were washed successively with deionized water and Tris-HCl buffer to remove the excess solidifying agent.

[0029] 3. Application of multi-enzyme system: Suspend the prepared hydrogel microspheres (2 pieces) in a reaction system containing DTT (20 mM), HCHO (10 mM), THF (0.5 mM), PLP (25 μM), NH4Cl (50 mM), NaHCO3 (50 mM) and Tris-HCl buffer, and carry out the reverse glycine cleavage reaction, with the total volumes being 100 μL and 200 μL respectively. Set the reaction conditions: temperature is 37 °C, pH is 7.5, and the reaction time is 48 hours (sampling at different time intervals).

[0030] 4. Detect the production amount of glycine during the reaction by high-throughput mass spectrometry (RapidFire).

[0031] Example 2:

[0032] 1. Preparation of enzymes: Express and purify each protein solution (P protein, T protein, and H protein are dissolved in Tris-HCl buffer with a pH of 7.5) through the Escherichia coli overexpression system respectively. Then mix the protein solutions to make an enzyme mixture, so that the final concentration ratio of P protein, T protein, and H protein is 5:3:40. In this example, three enzyme mixtures with different concentrations are prepared, containing 5 μM, 20 μM, and 30 μM of P protein respectively, and the corresponding T protein and H protein are added in a fixed ratio. The three groups are denoted as P5, P20, and P30 respectively.

[0033] 2. Preparation of hydrogel: Dissolve sodium alginate in the enzyme mixture prepared in step 1, and stir well to dissolve to form a uniform sodium alginate solution, so that the concentration of sodium alginate is 2% (w / v). Dissolve CaCl2 and gelatin in Tris-HCl (pH 7.5) buffer to make their final concentrations 75 mM and 1% (w / v) respectively to obtain a solidifying solution. Drop the mixed solution into the solidifying solution containing CaCl2 and gelatin and under stirring, and let it stand until hydrogel microspheres with a diameter of 1 - 2 mm are formed, as shown on the left in Figure 1 Wash the hydrogel microspheres with deionized water and Tris-HCl buffer in sequence to remove the excess solidifying agent.

[0034] 3. Application of multi-enzyme system: Suspend the prepared hydrogel microspheres (2 pieces) in a reaction system containing DTT (20 mM), HCHO (10 mM), THF (0.5 mM), PLP (25 μM), NH4Cl (50 mM), NaHCO3 (50 mM) and Tris-HCl buffer solution for the reverse glycine cleavage reaction, with the total volumes being 100 μL and 200 μL respectively. Reaction conditions: temperature 37 °C, pH 7.5, reaction time 48 hours. Samples are taken at different time intervals. In this example, samples are taken at 1 hour, 2 hours, 3 hours, and 24 hours of the reaction respectively.

[0035] 4. Wash the hydrogel microspheres (2 pieces) that have completed the reaction in the first round with Tris-HCl buffer solution to remove the remaining reaction solution. Then reuse them and add them to a new reaction system containing DTT (20 mM), HCHO (10 mM), THF (0.5 mM), PLP (25 μM), NH4Cl (50 mM), and NaHCO3 (50 mM) for the reverse glycine cleavage reaction, with the total volume being 100 μL respectively. Reaction conditions: temperature 37 °C, pH 7.5, reaction time 3 hours. Samples are taken at different time intervals. In this example, samples are taken at 1 hour, 2 hours, and 3 hours of the reaction (starting from the time when the hydrogel microspheres are placed in the reaction system in this round), and are denoted as reuse2-1, reuse2-2, and reuse2-3 respectively.

[0036] 5. Detect the amount of glycine generated during the reaction by high-throughput mass spectrometry (RapidFire).

[0037] As Figure 2 shown is the standard curve graph of glycine detected by high-throughput mass spectrometry, Figure 3 showing the statistical graph of the change in the amount of glycine generated over time within 48 hours. Alg (sodium alginate) is the result of Example 1, and Alg+Gelatin (sodium alginate + gelatin) is the result of Group P5 in Example 2. It can be seen that adding gelatin can increase the glycine yield. Figure 4 showing the statistical graph of the change in the glycine yield over time obtained by reacting with different concentrations of P protein, T protein, and H protein in Example 2. It can be seen that when the final concentrations of P protein, T protein, and H protein in the enzyme mixture are 20 μM, 12 μM, and 160 μM respectively, the glycine yield is higher.

[0038] It should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Without making creative efforts, any modifications, equivalent replacements, improvements, etc. should be included within the protection scope of the present invention.

Claims

1. A multi-enzyme system encapsulated by sodium alginate-gelatin hydrogel, characterized in that: Prepared by the following steps: Mixing the P protein, T protein, H protein and buffer to obtain an enzyme mixture, wherein the final concentration ratio of the P protein, T protein and H protein in the enzyme mixture is 5:3:40, adding sodium alginate, and stirring well to obtain a mixture, wherein the concentration of sodium alginate in the mixture is 1-3% w / v; CaCl2 is dissolved in a buffer solution to make the CaCl2 concentration be 70-100 mM to obtain a solidifying solution, and the mixed solution is dropped into the solidifying solution to form hydrogel microspheres; the hydrogel microspheres are washed with deionized water and a buffer solution in turn.

2. The multi-enzyme system according to claim 1, characterized in that: The solidifying liquid also includes gelatin at a concentration of 1-2% w / v.

3. The multi-enzyme system according to claim 1, characterized in that: The final concentration of P protein in the enzyme mixture is 5-30 μM.

4. The multi-enzyme system according to claim 1, characterized in that: The mixed liquid is dripped into the solidifying liquid, and the solidifying liquid is continuously stirred during the dripping.

5. The multi-enzyme system according to claim 1, characterized in that: The diameter of the hydrogel microspheres is 1-5 mm.

6. The multi-enzyme system according to claim 1, characterized in that: The buffer is Tris-HCl buffer with a pH of 7.3-7.

5.

7. A method for improving the efficiency of reverse glycine cleavage reaction, characterized in that: The following steps are involved: Suspending the multi-enzyme system according to any one of claims 1 to 6 in a reaction system containing DTT, HCHO, THF, PLP, NH4Cl, and NaHCO3; The reaction is carried out at 37°C and pH 7.3-7.5 for 24-48 hours.

8. The method according to claim 7, characterized in that The concentrations of DTT, HCHO, THF, PLP, NH4Cl and NaHCO3 in the reaction system are 20 mM, 10 mM, 0.5 mM, 25 μM, 50 mM and 50 mM, respectively.

Images