Process for green synthesis of high-purity stearyl glycyrrhetinate based on enzyme catalysis

Abstract

The invention relates to the technical field of organic synthesis and biochemical engineering, in particular to a process for green synthesis of high-purity stearyl glycyrrhetinic acid ester based on enzyme catalysis, which comprises the following steps: S1, dissolving glycyrrhizic acid or salt thereof in a buffer solution, adding immobilized beta-glucosidase, and treating after reaction to obtain a glycyrrhetinic acid intermediate; and S2, adding the glycyrrhetinic acid intermediate obtained in the step S1, stearyl alcohol and a catalyst into an organic solvent, reacting, purifying, and carrying out supercritical CO2 drying to obtain stearyl alcohol glycyrrhetinic acid ester. The high-purity stearyl glycyrrhetinic acid ester is prepared by preparing the trinity structured composite catalyst with stable MOF as a skeleton, the functionalized ionic liquid as an interface layer and the high-activity lipase as a catalytic core, the synthesis process is green and environmentally friendly, the reaction conditions are mild, and the catalyst can be reused.

Description

Technical Field

[0001] This invention relates to the fields of organic synthesis and biochemical technology, specifically to a process for the green synthesis of high-purity stearyl glycyrrhizic acid ester based on enzyme catalysis. Background Technology

[0002] Licorice, a traditional medicinal plant, has had its skincare value fully validated by modern science for its active ingredients. Among them, glycyrrhizic acid, with its significant anti-inflammatory and soothing effects, has become one of the core directions for the development of functional cosmetic raw materials. However, natural glycyrrhizic acid suffers from drawbacks such as poor lipid solubility, low skin penetration, and poor formulation compatibility, limiting its application efficacy in mainstream cosmetic formulations such as creams and sunscreens. To address this issue, optimizing its physicochemical properties through chemical modification has become a hot research topic in the industry. Stearyl glycyrrhizic acid ester is a derivative obtained by the esterification reaction of glycyrrhizic acid and stearyl alcohol. With the performance improvement brought about by the introduction of a lipophilic higher alkanol structure, it is gradually becoming a preferred active ingredient in the cosmetic field.

[0003] Existing research confirms that stearyl glycyrrhizic acid ester possesses multiple skincare benefits. Its anti-inflammatory activity is superior to that of natural glycyrrhizic acid. It can reduce the expression of inflammatory factors such as IL-6 and TNF-α by inhibiting the NF-κB signaling pathway, while competitively inhibiting tyrosinase activity, achieving a synergistic effect of soothing, anti-allergy, and whitening / removing blemishes. Furthermore, this ingredient has excellent UV absorption capabilities, enhancing the photoprotective effect of sunscreens. It also promotes the synthesis of skin filaggrin and hyaluronic acid, aiding in barrier repair and moisturizing stability. Suitable for various skincare scenarios, including sensitive skin and acne-prone skin, its market demand continues to rise. To meet this market demand, researchers in the field have developed different processes for synthesizing stearyl glycyrrhizic acid ester.

[0004] Patent CN103833820A discloses a method for synthesizing 3-succinic acid-30-stearyl glycyrrhetinic acid ester. This method uses glycyrrhetinic acid as a starting material, which undergoes nucleophilic substitution with octadecyl bromide, followed by condensation with succinic anhydride to obtain 3-succinic acid-30-stearyl glycyrrhetinic acid ester. This invention improves upon the intermediate synthesis method, eliminating the use of condensing agents in the reaction, reducing reaction waste, simplifying process steps, and significantly increasing reaction yield. It has advantages such as simple operation, suitability for industrial production, and high product purity. However, this invention directly uses glycyrrhetinic acid as a reactant, resulting in higher costs.

[0005] Patent CN111171106A discloses a method for preparing 24-hydroxystearyl glycyrrhetinic acid ester, which relates to the fields of food, cosmetics, pharmaceuticals, and health products. This invention uses glycyrrhizin as a raw material, and the extraction process is simple and highly operable. It has a high yield of effective substances and makes full use of resources. Compared with 24-hydroxy-glycyrrhetinic acid, the prepared 24-hydroxy-stearyl glycyrrhetinic acid ester has significantly changed its solubility in oils due to the introduction of lipophilic higher alkanols. At the same time, it has a better anti-inflammatory effect than 24-hydroxy-glycyrrhetinic acid, making it suitable for widespread promotion and application. However, this invention requires column chromatography purification, which is complex to operate.

[0006] Therefore, there is an urgent need in the market to develop a low-cost and simple process for synthesizing stearyl glycyrrhetinic acid ester. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention aims to provide a green synthesis process for stearyl glycyrrhetinic acid ester. This process is simple, easy to operate, has mild reaction conditions, allows for the reuse of catalysts, and produces a product with a purity of not less than 99.5%.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a process for the green synthesis of high-purity stearyl glycyrrhizic acid ester based on enzyme catalysis, comprising the following steps: S1. Glycyrrhizic acid or its salt is dissolved in a buffer solution, immobilized β-glucosidase is added, and the reaction is carried out at 40-60℃ and 180-250rpm for 4-6 hours. After post-treatment, glycyrrhizic acid intermediate is obtained. S2. Add the glycyrrhetinic acid intermediate obtained in step S1, stearyl alcohol, and catalyst to an organic solvent, react at 50-70℃ for 17-20h, purify, and dry with supercritical CO2 to obtain stearyl glycyrrhetinic acid ester.

[0009] In some embodiments, the method for preparing the immobilized β-glucosidase includes the following steps: β-glucosidase was added to a citrate-disodium hydrogen phosphate buffer solution, then resin was added and shaken at 30-40℃ for 60-70 min. The mixture was then filtered and dried to obtain immobilized β-glucosidase.

[0010] In some embodiments, the mass ratio of the β-glucosidase to the resin is 1:(38-42).

[0011] In some embodiments, the resin is an anion exchange resin.

[0012] This invention first uses immobilized β-glucosidase to specifically hydrolyze glycyrrhizic acid glycosidic bonds to generate glycyrrhetinic acid, avoiding side reactions and structural damage that may result from chemical hydrolysis. This ensures the high purity and bioactivity of the intermediate. Furthermore, the mild reaction conditions prevent high temperatures from damaging the glycyrrhizic acid triterpenoid core and enzyme catalyst. In addition, the entire process does not require the use of strong acids, strong bases, or halogenated reagents, resulting in minimal emissions of waste. The organic solvents used are mostly low in toxicity, and the enzyme catalyst itself is a biodegradable material, which aligns with the concept of green chemistry. Finally, the purified product is dried using supercritical CO2, which effectively prevents the degradation of heat-sensitive products and avoids organic solvent residues, meeting the high standards required for pharmaceutical and high-end cosmetic raw materials.

[0013] In some embodiments, the glycyrrhizic acid salt is a monoammonium glycyrrhizate.

[0014] In some embodiments, the buffer solution in step S1 is a 0.05-0.2 mol / L acetate-sodium acetate buffer or citrate-disodium hydrogen phosphate buffer.

[0015] In some embodiments, the amount of immobilized β-glucosidase added in step S1 is 1%-5% of the mass of glycyrrhizic acid or its salt.

[0016] In some embodiments, the method for preparing the catalyst includes the following steps: (1) Add 4-dimethylaminobenzoic acid and 1-hydroxyethyl-3-methylimidazolium chloride to toluene, add concentrated sulfuric acid while stirring, react at 70-80℃ for 5-6h, wash and rotary evaporate to obtain the compound; add potassium acetate and ethanol to the obtained compound, stir at 40-60℃ for 36-48h, filter and rotary evaporate to obtain the product; (2) Zirconium tetrachloride, 2-aminoterephthalic acid, and acetic acid were added to DMF, ultrasonically dispersed, and then reacted at 119-125℃ for 12-14h. After washing and drying, UiO-66-NH2 was obtained. (3) Add the product obtained in step (1) and the UiO-66-NH2 from step (2) into methanol and stir at 35-45℃ for 24-30h to obtain modified UiO-66-NH2; (4) Add lipase to phosphate buffer, disperse by ultrasonication, and then add it to the modified UiO-66-NH2 obtained in step (3). Stir at 30-40℃ for 3-4 hours, filter, wash and dry to obtain the catalyst.

[0017] This invention uses 1-hydroxyethyl-3-methylimidazolium chloride, 4-dimethylaminobenzoic acid, and potassium acetate as raw materials to generate an ionic liquid product with lipophilicity, reactive sites, and hydrogen bonding. This product is then grafted onto UiO-66-NH2 to obtain modified UiO-66-NH2. Finally, lipase is loaded onto the modified UiO-66-NH2 using a physical adsorption-crosslinking method to obtain a catalyst. This improves the purity of stearyl glycyrrhetinic acid ester and reduces the formation of byproducts. The possible reason is that the ionic liquid introduces pores into UiO-66-NH2, making it more... It facilitates the enrichment and mass transfer of substrates stearyl alcohol and glycyrrhetinic acid, and is conducive to their binding with the enzyme's catalytic active site, thereby increasing the reaction rate. In addition, the lipase immobilized in modified UiO-66-NH2 can efficiently and selectively catalyze the formation of an ester bond between the C3 hydroxyl group of glycyrrhetinic acid and stearyl alcohol, generating the target product stearyl glycyrrhetinic acid ester. Furthermore, the final catalyst resembles a "nanoreactor," which can simultaneously enrich the aqueous phase (the micro-aqueous environment required by the enzyme) and the organic substrate phase (glycyrrhetinic acid and stearyl alcohol) at its interface, creating an optimal local reaction microenvironment for the enzyme and overcoming the problem of mass transfer limitations in traditional heterogeneous biocatalysis.

[0018] In some embodiments, the molar ratio of 4-dimethylaminobenzoic acid to 1-hydroxyethyl-3-methylimidazolium chloride and potassium acetate is (1-1.3):1:(1-1.2).

[0019] In some embodiments, the ratio of zirconium tetrachloride to 2-aminoterephthalic acid and acetic acid is 1 mmol: (1-1.1) mmol: (4-5.5) ml.

[0020] In some embodiments, the mass ratio of the product in step (3) to UiO-66-NH2 is (0.1-0.3):1.

[0021] In some embodiments, the mass ratio of the lipase to the modified UiO-66-NH2 in step (4) is (2-2.5):1.

[0022] In some embodiments, the amount of catalyst added in step S2 is 10%-30% of the mass of the glycyrrhetinic acid intermediate.

[0023] In some embodiments, the molar ratio of stearyl alcohol to glycyrrhetinic acid intermediate in step S2 is 1:(1-1.5).

[0024] In some embodiments, the organic solvent in step S2 is any one or more of tert-amyl alcohol, isooctane, and methyl tert-butyl ether. In some embodiments, the purification step in step S2 is as follows: after filtering to remove the catalyst, the product is concentrated and dried under reduced pressure, and the dried product is dissolved in methanol or ethanol at 50-75°C and stirred for 2-3 hours, followed by programmed cooling crystallization.

[0025] In some embodiments, the stearyl glycyrrhetinic acid ester has a purity of not less than 99.5%, an APHA color number of less than 20, and a solvent residue of less than 10 ppm.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a green synthesis process for stearyl glycyrrhetinic acid ester with a clear process flow, mild reaction conditions, reusable catalyst and high product purity. Through two innovative systems, namely "two-step enzyme catalytic synthesis" and "combined high-efficiency purification", the purity of the product is not less than 99.5%, the APHA color number is less than 20 and the solvent residue is less than 10 ppm.

[0027] (2) This invention first uses immobilized β-glucosidase to specifically hydrolyze the glycosidic bond of glycyrrhizic acid, efficiently converting glycyrrhizic acid into glycyrrhetinic acid intermediate. Then, through catalyst catalysis, the ester bond between the C3 hydroxyl group of glycyrrhetinic acid and stearyl alcohol is formed to generate the target product stearyl glycyrrhetinic acid ester. The reaction conditions are mild, and there is no need to use strong acids, strong bases or halogenated reagents, resulting in less waste discharge. Most of the organic solvents used are low in toxicity, which is in line with the concept of green chemistry development.

[0028] (3) The present invention loads lipase onto modified UiO-66-NH2 to obtain a catalyst, which is more conducive to the enrichment and mass transfer of substrate stearyl alcohol and glycyrrhetinic acid, and is conducive to combining with the enzyme catalytic active site to improve the reaction rate. In addition, it can simultaneously enrich the aqueous phase (the micro-aqueous environment required by the enzyme) and the organic phase substrate (glycyrrhetinic acid and stearyl alcohol) at its interface, creating the optimal local reaction microenvironment for the enzyme and overcoming the problem of mass transfer limitation in traditional heterogeneous biocatalysis. Detailed Implementation

[0029] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0030] In the following examples and comparative examples, the compounds and related reagents used were all commercially available. The β-glucosidase was Novozymes Cellic Ctec 2, and the resin was Lanxiao LX1000EP resin. The lipase was Candida antarcticis lipase B with a purity of 5000 LU / g, purchased from Shanghai Yingxin Laboratory Equipment Co., Ltd. The immobilized lipase Novozym 435 with a purity of 10000 U / g was purchased from Shanghai Puzhen Biotechnology Co., Ltd.

[0031] Unless otherwise specified, the post-processing steps such as "washing", "extraction", "depressurized concentration", "drying", "rotary evaporation", and "filtration" used below are routine operations for those skilled in the art, and can be selected according to actual operation.

[0032] Preparation Example 1 The preparation method of catalyst-1 includes the following steps: (1) 0.12 mol of 4-dimethylaminobenzoic acid and 0.1 mol of 1-hydroxyethyl-3-methylimidazolium chloride were added to 200 ml of toluene. While stirring, 0.03 mol of 95 wt% concentrated sulfuric acid was added. The mixture was reacted at 75 °C for 5.5 h. The mixture was washed with deionized water until neutral and then rotary evaporated to obtain the compound. 0.11 mol of potassium acetate and 100 ml of anhydrous ethanol were added to the obtained compound. The mixture was stirred at 50 °C for 42 h. The mixture was filtered and then rotary evaporated to obtain the product. (2) 0.69 mmol zirconium tetrachloride, 0.72 mmol 2-aminoterephthalic acid, and 3.6 ml acetic acid were added to 45 ml DMF and dispersed by sonication at 60 W for 20 min at room temperature. Then the mixture was reacted at 120 °C for 13 h. After centrifugation, the mixture was washed three times with DMF and anhydrous ethanol and dried to obtain UiO-66-NH2. (3) Add 0.02g of the product obtained in step (1) and 0.1g of UiO-66-NH2 from step (2) to anhydrous methanol, stir at 40°C for 28h, filter, and dry to obtain modified UiO-66-NH2; (4) Add 220 mg of lipase to 20 ml of phosphate buffer (pH=6.5), disperse by sonication at 40 W for 10 min, then add 100 mg of the modified UiO-66-NH2 obtained in step (3), stir at 35 °C for 3.5 h, filter, wash with deionized water, freeze dry to obtain catalyst-1.

[0033] Preparation Example 2 The preparation method of catalyst-2 is the same as that of preparation example 1, except that the amount of product added in step (3) is 0.04g.

[0034] Preparation Example 3 The preparation method of catalyst-3 is the same as that of preparation example 1, except that the amount of lipase added in step (4) is 260 mg.

[0035] Preparation Example 4 The preparation method of catalyst-4 includes the following steps: (1) 0.69 mmol zirconium tetrachloride, 0.69 mmol 2-aminoterephthalic acid, and 3.6 ml acetic acid were added to 45 ml DMF and dispersed by sonication at 60 W for 20 min at room temperature. Then the mixture was reacted at 120 °C for 13 h. After centrifugation, the mixture was washed three times with DMF and anhydrous ethanol and dried to obtain UiO-66-NH2. (2) Add 220 mg of lipase to 20 ml of phosphate buffer (pH=6.5), disperse by sonication at 40 W for 10 min, then add 100 mg of UiO-66-NH2 obtained in step (1), stir at 35 °C for 3.5 h, filter, wash with deionized water, freeze dry to obtain catalyst-4.

[0036] Preparation Example 5 A method for preparing immobilized β-glucosidase includes the following steps: 1 g of β-glucosidase was added to 100 ml of citrate-disodium hydrogen phosphate buffer solution, and then 40 g of LX1000EP resin was added. The mixture was shaken at 150 rpm for 65 min at 38 °C, filtered, and freeze-dried to obtain immobilized β-glucosidase.

[0037] Example 1 A process for the green synthesis of high-purity stearyl glycyrrhizic acid ester based on enzyme catalysis includes the following steps: S1. Dissolve 5g of glycyrrhizic acid monoammonium salt in 100ml of 0.2mol / L acetate-sodium acetate buffer solution (pH=5), add 0.1g of immobilized β-glucosidase, and react at 50℃ and 200rpm for 4-6h. Then cool the reaction solution to 25℃ and adjust the pH to 2.5 with 1mol / L HCl solution, resulting in the precipitation of a large amount of white precipitate. Extract twice with 100mL of ethyl acetate, combine the organic phases, concentrate and dry under reduced pressure at 40℃ to obtain glycyrrhizic acid intermediate; S2. Add 3.8g of glycyrrhetinic acid intermediate obtained in step S1, 5.6g of stearyl alcohol, and 0.76g of catalyst-1 to 100ml of tert-amyl alcohol. React at 60℃ for 18h. After filtering to remove the catalyst, concentrate and dry under reduced pressure. Dissolve the dried product in anhydrous methanol at 60℃ and stir for 2.5h. The mass of anhydrous methanol is 15 times the mass of the dried product. Then, reduce the temperature to 2℃ at 0.8℃ / min. Finally, dry under supercritical CO2 (40℃, 10 MPa) for 4 hours to obtain stearyl glycyrrhetinic acid ester.

[0038] Example 2 A process for the green synthesis of high-purity stearyl glycyrrhizic acid ester based on enzyme catalysis includes the following steps: S1. Dissolve 5g of glycyrrhizic acid monoammonium salt in 100ml of 0.2mol / L acetate-sodium acetate buffer solution (pH=5), add 0.05g of immobilized β-glucosidase, and react at 40℃ and 180rpm for 6h. Then cool the reaction solution to 25℃ and adjust the pH to 2.5 with 1mol / L HCl, resulting in the precipitation of a large amount of white precipitate. Extract twice with 100mL of ethyl acetate, combine the organic phases, concentrate and dry under reduced pressure at 40℃ to obtain glycyrrhizic acid intermediate; S2. Add 3.8g of glycyrrhetinic acid intermediate obtained in step S1, 5.6g of stearyl alcohol, and 0.38g of catalyst-1 to 100ml of tert-amyl alcohol. React at 50℃ for 20h. After filtering to remove the catalyst, concentrate and dry under reduced pressure. Dissolve the dried product in anhydrous methanol at 50℃ and stir for 3h. The amount of anhydrous methanol is 8 times that of the dried product. Then, reduce the temperature to 2℃ at 0.8℃ / min. Finally, dry under supercritical CO2 (40℃, 10 MPa) for 4h to obtain stearyl glycyrrhetinic acid ester.

[0039] Example 3 A process for the green synthesis of high-purity stearyl glycyrrhizic acid ester based on enzyme catalysis includes the following steps: S1. Dissolve 5g of glycyrrhizic acid monoammonium salt in 100ml of 0.2mol / L acetate-sodium acetate buffer solution (pH=5), add 0.25g of immobilized β-glucosidase, and react at 70℃ and 250rpm for 4h. Then cool the reaction solution to 25℃ and adjust the pH to 2.5 with 1mol / L HCl, resulting in the precipitation of a large amount of white precipitate. Extract twice with 100mL of ethyl acetate, combine the organic phases, concentrate and dry under reduced pressure at 40℃ to obtain glycyrrhizic acid intermediate; S2. Add 3.8g of glycyrrhetinic acid intermediate obtained in step S1, 5.6g of stearyl alcohol, and 1.14g of catalyst-1 to 100ml of tert-amyl alcohol. React at 70℃ for 17h. After filtering to remove the catalyst, concentrate and dry under reduced pressure. Dissolve the dried product in anhydrous methanol at 75℃ and stir for 2h. The amount of anhydrous methanol is 20 times that of the dried product. Then, reduce the temperature to 2℃ at 0.8℃ / min. Finally, dry under supercritical CO2 (40℃, 10MPa) for 4h to obtain stearyl glycyrrhetinic acid ester.

[0040] Example 4 A process for the green synthesis of high-purity stearyl glycyrrhetinic acid ester based on enzyme catalysis is described. The specific implementation method is the same as in Example 1, except that catalyst-1 is replaced by catalyst-2 in an equal amount.

[0041] Example 5 A process for the green synthesis of high-purity stearyl glycyrrhetinic acid ester based on enzyme catalysis is described. The specific implementation method is the same as in Example 1, except that catalyst-1 is replaced by catalyst-3 in an equal amount.

[0042] Example 6 A process for the green synthesis of high-purity stearyl glycyrrhetinic acid ester based on enzyme catalysis is described. The specific implementation method is the same as in Example 1, except that catalyst-1 is replaced by catalyst-4 in an equal amount.

[0043] Example 7 A process for the green synthesis of high-purity stearyl glycyrrhetinic acid ester based on enzyme catalysis is described. The specific implementation method is the same as in Example 1, except that the amount of catalyst-1 added is 1.52g.

[0044] Example 8 A process for the green synthesis of high-purity stearyl glycyrrhizic acid ester based on enzyme catalysis is described. The specific implementation method is the same as in Example 1, except that catalyst-1 is replaced by an equal amount of immobilized lipase Novozym 435.

[0045] Example 9 A process for the green synthesis of high-purity stearyl glycyrrhetinic acid ester based on enzyme catalysis is described. The specific implementation method is the same as in Example 1, except that catalyst-1 is catalyst-1 that has been reused 7 times.

[0046] Comparative Example 1 A process for the green synthesis of high-purity stearyl glycyrrhizic acid ester based on enzyme catalysis includes the following steps: S1. Dissolve 5g of glycyrrhizic acid monoammonium salt in 100ml of 0.2mol / L acetate-sodium acetate buffer solution with pH=5, add 0.1g of immobilized β-glucosidase, and react at 50℃ and 200rpm for 4-6h. Then add 120ml of tert-amyl alcohol, and remove water by distillation to obtain a tert-amyl alcohol solution containing glycyrrhizic acid. S2. Add 5.6g stearyl alcohol and 0.76g catalyst-1 to the tert-amyl alcohol solution containing glycyrrhetinic acid obtained in step S1. React at 60℃ for 18h. After filtering to remove the catalyst, concentrate and dry under reduced pressure. Dissolve the dried product in anhydrous methanol at 60℃ and stir for 2.5h. The mass of anhydrous methanol is 15 times the mass of the dried product. Then, reduce the temperature to 2℃ at 0.8℃ / min. Finally, dry with supercritical CO2 (40℃, 10 MPa) for 4 hours to obtain stearyl glycyrrhetinic acid ester.

[0047] Comparative Example 2 A process for the green synthesis of high-purity stearyl glycyrrhizic acid ester based on enzyme catalysis includes the following steps: 5g of glycyrrhizic acid monoammonium salt, 5.6g of stearyl alcohol, 0.1g of immobilized β-glucosidase, and 0.76g of catalyst-1 were added to 100ml of tert-amyl alcohol and reacted at 60℃ and 200rpm for 24 hours. The conversion rate was monitored by HPLC. After filtering to remove the catalyst, the product was concentrated and dried under reduced pressure. The dried product was dissolved in anhydrous methanol at 60℃ and stirred for 2.5h. The mass of anhydrous methanol was 15 times the mass of the dried product. The temperature was then reduced to 2℃ at 0.8℃ / min. Finally, the product was dried under supercritical CO2 (40℃, 10 MPa) for 4 hours to obtain stearyl glycyrrhizic acid ester.

[0048] Performance testing 1. The stearyl glycyrrhetinic acid esters obtained in each example were weighed, and their purity was determined by HPLC.

[0049] The test results are shown in Table 1: Table 1

[0050] As shown in Table 1, the synthesis processes in Examples 1-3 yielded high amounts of high-purity products. A comparison of the data from Example 4 and Example 1 reveals that the altered ratio of the product to UiO-66-NH2 may have blocked the pores of UiO-66-NH2, hindering substrate diffusion and leading to a decrease in reaction rate and yield. Furthermore, the chemical catalysis of the ionic liquid caused side reactions, resulting in decreased product purity. A comparison of the data from Example 5 and Example 1 shows that the altered ratio of lipase to modified UiO-66-NH2 caused enzyme molecules to form multilayers or aggregates on the carrier surface, obscuring the enzyme and preventing it from contacting the substrate, resulting in wasted activity. Moreover, an excessively thick enzyme layer itself becomes a dense diffusion barrier. This can hinder the entry of substrates into the carrier and into the product. Furthermore, overloaded enzymes are not firmly bound and are prone to detaching during the reaction, catalyzing the formation of different stereoisomers, thus reducing product purity and yield. A comparison between Example 6 and Example 1 shows that directly using lipase-loaded UiO-66-NH2 may affect the enrichment and mass transfer of the substrates stearyl alcohol and glycyrrhetinic acid, leading to a decrease in product yield and purity. A comparison between Example 7 and Example 1 shows that changing the amount of catalyst added can lead to excessively long contact time between the substrate and the active site, resulting in side reactions such as over-esterification or isomerization, thus reducing product purity. A comparison between Example 8 and Example 1 shows that directly using commercially available immobilized lipase Novozym... 435, the substrate may have limited binding affinity to the enzyme's catalytic active site, leading to a decrease in product yield; a comparison of Example 9 and Example 1 shows that the catalyst prepared by this invention has good cyclicity; a comparison of Comparative Example 1 and Example 1 shows that the change in the post-processing step S1 resulted in a decrease in both product yield and purity; a comparison of Comparative Example 2 and Example 1 shows that the one-pot preparation method may have problems with incompatible reaction conditions and potential inter-enzyme interference, resulting in a decrease in both product yield and purity.

[0051] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A process for the green synthesis of high-purity stearyl glycyrrhizic acid ester based on enzyme catalysis, characterized in that, Includes the following steps: S1. Glycyrrhizic acid or its salt is dissolved in a buffer solution, immobilized β-glucosidase is added, and the reaction is carried out at 40-60℃ and 180-250rpm for 4-6 hours. After post-treatment, glycyrrhizic acid intermediate is obtained. S2. Add the glycyrrhetinic acid intermediate obtained in step S1, stearyl alcohol, and catalyst to an organic solvent, react at 50-70℃ for 17-20h, purify, and dry with supercritical CO2 to obtain stearyl glycyrrhetinic acid ester.

2. The process for the enzyme-catalyzed green synthesis of high-purity stearyl glycyrrhizic acid ester according to claim 1, characterized in that, The buffer solution in step S1 is a 0.05-0.2 mol / L acetate-sodium acetate buffer or citrate-disodium hydrogen phosphate buffer.

3. The process for the enzyme-catalyzed green synthesis of high-purity stearyl glycyrrhizic acid ester according to claim 1, characterized in that, The amount of immobilized β-glucosidase added in step S1 is 1%-5% of the mass of glycyrrhizic acid or its salt.

4. The process for the enzyme-catalyzed green synthesis of high-purity stearyl glycyrrhizic acid ester according to claim 1, characterized in that, The method for preparing the catalyst includes the following steps: (1) Add 4-dimethylaminobenzoic acid and 1-hydroxyethyl-3-methylimidazolium chloride to toluene, add concentrated sulfuric acid while stirring, react at 70-80℃ for 5-6h, wash and rotary evaporate to obtain the compound; add potassium acetate and ethanol to the obtained compound, stir at 40-60℃ for 36-48h, filter and rotary evaporate to obtain the product; (2) Zirconium tetrachloride, 2-aminoterephthalic acid, and acetic acid were added to DMF, ultrasonically dispersed, and then reacted at 120-130℃ for 12-14h. After washing and drying, UiO-66-NH2 was obtained. (3) Add the product obtained in step (1) and the UiO-66-NH2 from step (2) into methanol and stir at 35-45℃ for 24-30h to obtain modified UiO-66-NH2; (4) Add lipase to phosphate buffer, disperse by ultrasonication, and then add it to the modified UiO-66-NH2 obtained in step (3). Stir at 30-40℃ for 3-4 hours, filter, wash and dry to obtain the catalyst.

5. The process for the enzyme-catalyzed green synthesis of high-purity stearyl glycyrrhizic acid ester according to claim 4, characterized in that, The mass ratio of the product to UiO-66-NH2 in step (3) is (0.1-0.3):

1.

6. The process for the enzyme-catalyzed green synthesis of high-purity stearyl glycyrrhizic acid ester according to claim 4, characterized in that, The mass ratio of the lipase to the modified UiO-66-NH2 in step (4) is (2-2.5):

1.

7. The process for the enzyme-catalyzed green synthesis of high-purity stearyl glycyrrhizic acid ester according to claim 1, characterized in that, The amount of catalyst added in step S2 is 10%-30% of the mass of glycyrrhetinic acid intermediate.

8. The process for the enzyme-catalyzed green synthesis of high-purity stearyl glycyrrhizic acid ester according to claim 1, characterized in that, The molar ratio of stearyl alcohol to glycyrrhetinic acid intermediate in step S2 is 1:(1-1.5).

9. The process for the enzyme-catalyzed green synthesis of high-purity stearyl glycyrrhizic acid ester according to claim 1, characterized in that, The purification steps described in step S2 are as follows: after filtering to remove the catalyst, the product is concentrated and dried under reduced pressure. The dried product is dissolved in methanol or ethanol at 50-75℃ and stirred for 2-3 hours, followed by programmed cooling crystallization.

10. The process for the enzyme-catalyzed green synthesis of high-purity stearyl glycyrrhizic acid ester according to claim 1, characterized in that, The purity of the stearyl glycyrrhetinic acid ester is not less than 99.5%, the APHA color number is less than 20, and the solvent residue is less than 10 ppm.

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