Process for preparing L-2-aminobutyric acid by immobilized enzyme method

Abstract

The invention provides a process for preparing L-2-aminobutyric acid by an immobilized enzyme method. The preparation method comprises the following steps: firstly, enabling graphene oxide, hexadecyl trimethyl ammonium bromide and tetraethoxysilane to react, growing fibrous mesoporous silica in situ, forming a compound with a mesoporous structure and a through large-aperture channel, providing binding sites for enzyme and promoting diffusion of a substrate / product; secondly, through ternary polymerization of a double-bond-containing graphene oxide-silicon dioxide compound, double-bond-containing chitosan and double-bond-containing cyclodextrin, a stable carrier network is constructed, and the enzyme immobilization efficiency is enhanced by virtue of the biocompatibility of chitosan and cyclodextrin and active groups; and finally, embedding the carrier and the enzyme by using sodium alginate-calcium chloride gel, protecting the enzyme activity conformation and constructing a stable microenvironment. The method solves the problems that the existing enzyme is poor in stability and cannot be reused, maintains high catalytic activity and enantiomer purity while reducing the enzyme cost, and has industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of L-2-aminobutyric acid preparation, and more specifically to a process for preparing L-2-aminobutyric acid using an immobilized enzyme method. Background Technology

[0002] L-2-Aminobutyric acid (L-2-aminobutyric acid) is an important chiral compound with wide applications in pharmaceuticals, biochemicals, and other fields. Its preparation methods mainly include chemical synthesis, microbial fermentation, and enzymatic catalysis. Among these, enzymatic catalysis, with its advantages of mild reaction conditions, high stereoselectivity, and environmental friendliness, has gradually become the preferred route for preparing high-purity L-2-aminobutyric acid.

[0003] Traditional enzymatic preparation of L-2-aminobutyric acid (LABA) typically involves the hydrolysis of N-acetyl-DL-2-aminobutyric acid by free enzymes, utilizing the specific catalytic function of the enzyme to generate the product. However, this method is difficult to separate and recover the free enzyme after the reaction, leading to significant consumption of the enzyme protein and requiring frequent replenishment of fresh enzyme preparations. This not only increases raw material costs but also limits the large-scale application of the process due to the poor reusability of the enzyme. To address this issue, immobilized enzyme technology has been introduced into this preparation system, enabling enzyme reuse by immobilizing the enzyme on a carrier.

[0004] However, current immobilized enzyme technology faces a series of challenging problems in the preparation of L-2-aminobutyric acid (GABA). The immobilization process often requires complex and costly chemical reagents and processes, increasing upfront costs and potentially damaging enzyme activity, resulting in a significant reduction in the activity of the immobilized enzyme. Furthermore, the enzyme-carrier system constructed using traditional immobilization methods suffers from poor stability; during repeated recycling, the enzyme easily detaches from the carrier, making it difficult to maintain high and stable catalytic efficiency and hindering the full realization of the advantages of reusable immobilized enzymes. In addition, the structural characteristics of the immobilization carrier limit the full potential of enzyme activity, severely hindering the catalytic reaction process and slowing the overall reaction rate, thus greatly restricting the widespread application of immobilized enzyme technology in the large-scale industrial production of L-2-aminobutyric acid.

[0005] In conclusion, a new technical solution is urgently needed to address the problems existing in the current technology. Summary of the Invention

[0006] Based on this, the present invention provides a process for preparing L-2-aminobutyric acid (GABA) using an immobilized enzyme method. Through multiple modifications to the carrier material, the performance of the immobilized enzyme is significantly improved. First, the present invention utilizes the reaction of graphene oxide, hexadecyltrimethylammonium bromide, and tetraethyl orthosilicate to grow fibrous mesoporous silica in situ, forming a complex with both mesoporous structure and interconnected macroporous channels, providing binding sites for the enzyme and promoting substrate / product diffusion. Second, a stable carrier network is constructed through the ternary copolymerization of a graphene oxide-silica complex containing double bonds, chitosan containing double bonds, and cyclodextrin containing double bonds. The biocompatibility and active groups of chitosan and cyclodextrin enhance the enzyme immobilization efficiency. Finally, sodium alginate-calcium chloride gel is used to encapsulate the carrier and enzyme, protecting the enzyme's active conformation and constructing a stable microenvironment. This multiple modification strategy solves the problems of poor enzyme stability and inability to be reused in existing enzymes, maintaining high catalytic activity and enantiomeric purity while reducing enzyme costs, and has promising prospects for industrial application.

[0007] One object of the present invention is to provide a process for preparing L-2-aminobutyric acid by immobilized enzyme, wherein the process for preparing L-2-aminobutyric acid by immobilized enzyme includes the following steps: S1. Graphene oxide, surfactant and catalyst are added to water and dispersed evenly to obtain solution A; n-pentanol is added to cyclohexane and dispersed evenly to obtain solution B; solution A and solution B are mixed, tetraethyl orthosilicate is added, and the mixture is heated to react. After the reaction is completed, the mixture is purified and dried to obtain graphene oxide-silica composite. S2. The graphene oxide-silica composite is mixed with KH570 and heated and stirred to obtain a graphene oxide-silica composite containing double bonds. S3. Chitosan is blended with ethylacryloyl chloride and reacted at low temperature to obtain chitosan containing double bonds; S4. Blend cyclodextrin with ethylacryloyl chloride, heat and stir to react, and obtain cyclodextrin containing double bonds; S5. The graphene oxide-silica composite containing double bonds, chitosan containing double bonds and cyclodextrin containing double bonds are blended together, an initiator is added, and the mixture is heated and stirred to obtain an immobilized carrier. S6. The L-aminoacylase is mixed with the immobilized carrier, and then mixed with sodium alginate solution. Then, it is added to calcium chloride solution to obtain immobilized L-aminoacylase. S7. Add N-acetyl-DL-2-aminobutyric acid to a buffer solution, add the immobilized L-aminoacylase, and heat to react to obtain L-2-aminobutyric acid.

[0008] Furthermore, L-aminoacylase is an important hydrolytic enzyme in organisms that can selectively catalyze the metabolism of amino acids. It can asymmetrically hydrolyze N-acetyl-DL-2-aminobutyric acid to generate optically active L-2-aminobutyric acid.

[0009] Furthermore, in step S1, the heating temperature is 110-130℃.

[0010] Furthermore, in step S2, the heating temperature is 70-90℃.

[0011] Furthermore, in step S3, the low temperature is an ice-water bath.

[0012] Furthermore, in step S4, the heating temperature is 60-80°C.

[0013] Furthermore, in step S5, the heating temperature is 60-80℃.

[0014] Furthermore, in step S7, the heating temperature is 30-40°C.

[0015] Further, in step S2, the mass ratio of the graphene oxide-silica composite to KH570 is 1:(1-5).

[0016] Further, in step S3, the mass ratio of chitosan to ethylacryloyl chloride is 20:(1-5).

[0017] Further, in step S4, the mass ratio of the cyclodextrin to ethylacryloyl chloride is 10:(1-3).

[0018] Further, in step S5, the mass ratio of the graphene oxide-silica composite containing double bonds, the chitosan containing double bonds, and the cyclodextrin containing double bonds is 1:(3-5):(1-3).

[0019] The present invention has the following beneficial effects: This invention provides a process for preparing L-2-aminobutyric acid (GABA) using an immobilized enzyme. Through multiple modifications to the carrier material, the performance of the immobilized enzyme is significantly improved. Firstly, this invention involves the in-situ growth of fibrous mesoporous silica on the surface of graphene oxide via a reaction of graphene oxide, hexadecyltrimethylammonium bromide, and tetraethyl orthosilicate, forming a graphene oxide-silica composite. This material not only possesses a mesoporous structure constructed from an internal template but also forms large-aperture channels communicating with the outside through the gaps between the folded layers. This provides stable and abundant binding sites for the enzyme and facilitates efficient diffusion of substrates and products. Secondly, by copolymerizing a graphene oxide-silica complex containing double bonds, chitosan containing double bonds, and cyclodextrin containing double bonds to form an immobilized support, chitosan and cyclodextrin are grafted onto the graphene oxide-silica complex, integrating them into a single unit. This process not only enhances the structural stability of the support itself, thereby improving the conformational stability of the enzyme molecule, but also, the biocompatibility and active groups such as amino and hydroxyl groups of chitosan and cyclodextrin can enhance the enzyme immobilization efficiency through hydrogen and ionic bonds. On the other hand, encapsulating the immobilized support and enzyme using a gel network formed by sodium alginate and calcium chloride not only protects the active conformation of the enzyme but also significantly improves the cycling stability of the immobilized enzyme by constructing a stable microenvironment. This multi-modification approach solves the problems of poor enzyme stability and inability to be reused in existing technologies, reduces the cost of enzyme use, and maintains high catalytic activity and enantiomeric purity, showing promising prospects for industrial application. Detailed Implementation

[0020] To more clearly illustrate the technical solution of the present invention, the following embodiments are provided. Unless otherwise stated, the raw materials, reactions, and post-processing methods appearing in the embodiments are all commercially available raw materials and technical methods well known to those skilled in the art.

[0021] The terms "preferred," "more preferably," and "more suitable" used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0022] It should be understood that, except in any operational instance or otherwise indicated, the amounts or all figures representing ingredients used, for example, in the specification and claims, should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​varying according to the desired performance to be obtained according to the invention.

[0023] The present invention uses the following raw materials: Chitosan: Brand name S52721, purchased from Shanghai Yuanye.

[0024] α-Cyclodextrin: Brand name S11009, purchased from Shanghai Yuanye.

[0025] Sodium alginate: brand name S100128, purchased from Aladdin Reagent.

[0026] L-aminoacylase: ≥30000 (u / g), purchased from Shanghai Qiude Biochemical Co., Ltd.

[0027] All water used in this invention is deionized water.

[0028] In this invention, "parts" refers to parts by mass. Example 1

[0029] A process for preparing L-2-aminobutyric acid using an immobilized enzyme method, the process comprising the following steps: S1. 20 mg of graphene oxide was ultrasonically dispersed in 80 ml of water, 1.32 g of hexadecyltrimethylammonium bromide and 0.8 g of urea were added, and the mixture was stirred and ultrasonically mixed evenly to obtain solution A. 2.66 ml of n-pentanol and 80 ml of cyclohexane were mixed and stirred evenly to obtain solution B. Solution A and solution B were mixed and stirred evenly, and 3.2 ml of TEOS was added. The mixture was stirred for 1 h and then reacted at 125 °C for 5 h. After centrifugation, washing and drying, the product was washed three times with hydrochloric acid ethanol solution (8 ml of 12 M hydrochloric acid was added to 88 ml of anhydrous ethanol and stirred evenly), water, and anhydrous ethanol, respectively, and dried to obtain the graphene oxide-silica composite. S2. Using an ethanol-water mixture (ethanol:water = 9:1, v / v) as a solvent, the graphene oxide-silica composite was added and stirred for 1 h. The pH was adjusted to 4 with hydrochloric acid solution (concentration of 0.1 wt%), and then KH570 (graphene oxide-silica composite:KH570 = 1:4, m / m) was added. The mixture was stirred and reacted at 70℃ for 12 h. After washing and drying, the graphene oxide-silica composite containing double bonds was obtained. S3. Dissolve 40 g of chitosan in 20 ml of methanesulfonic acid to form a chitosan solution; dissolve 2 g of ethylacrylyl chloride in 16 ml of methanesulfonic acid to form an ethylacrylyl chloride solution; mix the chitosan solution and the ethylacrylyl chloride solution under ice-water bath conditions, react overnight, then add anhydrous diethyl ether to precipitate, and dry in a vacuum drying oven for two days to obtain chitosan containing double bonds; S4. Using DMSO as solvent, 40 g of α-cyclodextrin was mixed with 4 g of ethylacryloyl chloride and reacted at 70 °C for 12 h. After cooling, methanol was added for dilution, and acetone was added for precipitation. The mixture was filtered, washed, and dried to obtain cyclodextrin containing double bonds. S5. Using sodium hydroxide aqueous solution (16 wt%) as solvent, the graphene oxide-silica complex containing double bonds and the cyclodextrin containing double bonds were mixed evenly, and then a chitosan solution containing double bonds (20 g of chitosan containing double bonds dissolved in 20 ml of 0.1 M hydrochloric acid) was added. Sodium persulfate was added (graphene oxide-silica complex containing double bonds: chitosan containing double bonds: cyclodextrin containing double bonds: sodium persulfate = 1:4:2:0.07, m / m / m / m). The reaction was carried out at 70℃ for 10 h. After filtration, washing and drying, the immobilized carrier was obtained. S6. 0.02 g of L-aminoacylase and 0.18 g of the immobilized carrier were mixed and added to 2 ml of physiological saline. The mixture was stirred and dispersed evenly. Then, 10 ml of sodium alginate aqueous solution (3 wt%) was added and stirred and mixed evenly. The mixture was squeezed into CaCl2 aqueous solution (0.1 M) using a No. 6 needle to form immobilized microspheres. The immobilized microspheres were soaked in 0.1 M CaCl2 aqueous solution for 0.5 h, then transferred to 0.01 M CaCl2 aqueous solution and hardened in a 4°C refrigerator for 2 h. After washing with physiological saline, immobilized L-aminoacylase was obtained. S7. Add 14.5 mg of N-acetyl-DL-2-aminobutyric acid to 10 ml of Tris-HCl buffer (50 mM, pH 7.5), add the immobilized L-aminoacylase (100 U), and react in a constant temperature shaker at 30°C (180 rpm) for 4 h to obtain L-2-aminobutyric acid. Example 2

[0030] A process for preparing L-2-aminobutyric acid by immobilized enzyme method. The difference between this embodiment and Example 1 is that in step S7, the amount of immobilized L-aminoacylase added is 150 U, the reaction time is 3.5 h, and the remaining steps and dosages are the same as in Example 1. Example 3

[0031] A process for preparing L-2-aminobutyric acid by immobilized enzyme method. The difference between this embodiment and Example 1 is that in step S7, the amount of immobilized L-aminoacylase added is 75 U, the reaction time is 5 h, and the remaining steps and dosages are the same as in Example 1.

[0032] Comparative Example 1 A process for preparing L-2-aminobutyric acid by immobilized enzyme method. The difference between this comparative example and Example 1 is that step S1 is omitted. In step S2, the graphene oxide-silica composite is replaced with silica by an equal mass. The remaining steps and amounts are the same as in Example 1.

[0033] Comparative Example 2 A process for preparing L-2-aminobutyric acid by immobilized enzyme method. The difference between this comparative example and Example 1 is that step S4 is omitted. In step S5, the mass of the double-bonded cyclodextrin is replaced with double-bonded chitosan. The remaining steps and amounts are the same as in Example 1.

[0034] Test case The performance of the immobilized enzyme method for preparing L-2-aminobutyric acid in Examples 1-3 and Comparative Examples 1-2 was tested.

[0035] Test method: After immobilized L-aminoacylase catalyzes N-acetyl-DL-2-aminobutyric acid (NABA), the immobilized enzyme is recovered by filtration and thoroughly washed with Tris-HCl buffer (50 mM, pH 7.5), then stored at 4°C for reuse. Following the above storage conditions and referring to step S7 of Example, NABA is subjected to multiple catalytic reactions using immobilized L-aminoacylase, and the conversion rate and enantiomeric excess percentage (ee%) of L-2-aminobutyric acid prepared in each reaction are determined.

[0036] L-2-Aminobutyric acid (ABA) detection method: Chromatographic column: Crownpak CR(+), 5 μm, 4.0 × 150 mm; Mobile phase: distilled water, pH adjusted to 1.30 with perchloric acid; Detection wavelength: 210 nm; Flow rate: 0.6 mL / min; Injection volume: 20 μL; Column temperature: 20℃; Instrument: Shimadzu LC-15C liquid chromatograph. The retention time of ABA was 3.5 min, and the retention time of D-2-aminobutyric acid was 2.7 min.

[0037] The test results are shown in Table 1.

[0038]

[0039] The test results above show that when L-2-aminobutyric acid is prepared using this invention, the substrate conversion rate can reach 44.21% on the first use, with an ee% exceeding 99%; on the fourth use, the substrate conversion rate can reach 32.89%, with an ee% exceeding 99%; even after multiple uses, it still has a high substrate conversion rate and optical purity, indicating good application prospects.

[0040] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0041] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A process for preparing L-2-aminobutyric acid using an immobilized enzyme method, characterized in that, The process for preparing L-2-aminobutyric acid by immobilized enzyme method includes the following steps: S1. Graphene oxide, surfactant and catalyst are added to water and dispersed evenly to obtain solution A; n-pentanol is added to cyclohexane and dispersed evenly to obtain solution B; solution A and solution B are mixed, tetraethyl orthosilicate is added, and the mixture is heated to react. After the reaction is completed, the mixture is purified and dried to obtain graphene oxide-silica composite. S2. The graphene oxide-silica composite is mixed with KH570 and heated and stirred to obtain a graphene oxide-silica composite containing double bonds. S3. Chitosan is blended with ethylacryloyl chloride and reacted at low temperature to obtain chitosan containing double bonds; S4. Blend cyclodextrin with ethylacryloyl chloride, heat and stir to react, and obtain cyclodextrin containing double bonds; S5. The graphene oxide-silica composite containing double bonds, chitosan containing double bonds and cyclodextrin containing double bonds are blended together, an initiator is added, and the mixture is heated and stirred to obtain an immobilized carrier. S6. The L-aminoacylase is mixed with the immobilized carrier, and then mixed with sodium alginate solution. Then, it is added to calcium chloride solution to obtain immobilized L-aminoacylase. S7. N-acetyl-DL-2-aminobutyric acid is added to a buffer solution, followed by the immobilized L-aminoacylase. The mixture is heated to react and yield L-2-aminobutyric acid.

2. The process for preparing L-2-aminobutyric acid by immobilized enzyme method according to claim 1, characterized in that, In step S1, the heating temperature is 110-130℃.

3. The process for preparing L-2-aminobutyric acid by immobilized enzyme method according to claim 1, characterized in that, In step S2, the heating temperature is 70-90℃.

4. The process for preparing L-2-aminobutyric acid by immobilized enzyme method according to claim 1, characterized in that, In step S4, the heating temperature is 60-80℃.

5. The process for preparing L-2-aminobutyric acid by immobilized enzyme method according to claim 1, characterized in that, In step S5, the heating temperature is 60-80℃.

6. The process for preparing L-2-aminobutyric acid by immobilized enzyme method according to claim 1, characterized in that, In step S7, the heating temperature is 30-40℃.

7. The process for preparing L-2-aminobutyric acid by immobilized enzyme method according to claim 1, characterized in that, In step S2, the mass ratio of the graphene oxide-silica composite to KH570 is 1:(1-5).

8. The process for preparing L-2-aminobutyric acid by immobilized enzyme method according to claim 1, characterized in that, In step S3, the mass ratio of chitosan to ethylacryloyl chloride is 20:(1-5).

9. The process for preparing L-2-aminobutyric acid by immobilized enzyme method according to claim 1, characterized in that, In step S4, the mass ratio of the cyclodextrin to ethylacryloyl chloride is 10:(1-3).

10. The process for preparing L-2-aminobutyric acid by immobilized enzyme method according to claim 1, characterized in that, In step S5, the mass ratio of the graphene oxide-silica composite containing double bonds, the chitosan containing double bonds, and the cyclodextrin containing double bonds is 1:(3-5):(1-3).

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