Process for the enzymatic synthesis of santalol

Abstract

The application relates to the technical field of organic synthesis, in particular to a process for biologically catalyzing and synthesizing squalyl alcohol, which comprises the following steps: S1, solidification treatment: fixing lipase on a solidification material to obtain solidified lipase; S2, enzyme catalysis reaction: adding glycerol, oleyl acetate, the solidified lipase and molecular sieves into an organic solvent, and performing oscillation reaction at 30 DEG C to 45 DEG C, then performing solid-liquid separation, and obtaining an intermediate SN1 through concentration of the obtained filtrate; S3, saponification and post-treatment: mixing the intermediate SN1 with an alkali solution to perform saponification reaction, then performing neutralization, extraction and drying after the reaction is completed, and obtaining an organic phase containing squalyl alcohol; and S4, purification: performing column chromatography purification on the obtained organic phase, and obtaining squalyl alcohol after concentration of an eluent. The application realizes 1-position specific catalysis by using the regional selectivity of the enzyme, the product has high purity, the reaction efficiency and the recycling property of the catalyst are remarkably improved, the total yield is more than 85%, the whole process has mild conditions, is green and environment-friendly, is simple to operate, and is easy to industrialize.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, specifically to a bio-enzyme-catalyzed synthesis process for salinol. Background Technology

[0002] Saloyl alcohol (glycerol 1-oleyl ether) is a compound with important biological activity and application value. Currently, its synthesis process mainly faces two types of technical bottlenecks:

[0003] 1. Inherent drawbacks of traditional chemical synthesis methods (such as Williamson etherification and direct dehydration etherification):

[0004] Environmental and safety issues: Strong acids, strong bases, or highly toxic raw materials (such as haloalkanes and boron trifluoride ether) are often used, the reaction conditions are harsh, and a large amount of inorganic salts are produced as byproducts, which is not environmentally friendly.

[0005] Selectivity issues: Poor regioselectivity, easy generation of polyether byproducts such as 2-substituted ethers and diethers, resulting in low purity of the final product and difficulty in separation and purification.

[0006] Product quality issues: The reaction often requires high temperatures, which can easily lead to the oxidation and isomerization of unsaturated double bonds in oil-based raw materials, resulting in a darker product color that is difficult to meet the high standards required by pharmaceutical, daily chemical and other fields.

[0007] 2. Bottlenecks in the development of existing bio-enzyme catalysis methods:

[0008] Catalytic efficiency and cost issues: Free enzymes have poor stability and are difficult to recover and reuse, resulting in high production costs; in addition, the accumulation of water in the reaction system can inhibit the equilibrium of the acylation reaction, resulting in unsatisfactory conversion rate.

[0009] Issues with process maturity: There is a lack of a mature, stable, and scalable industrial process that covers enzyme immobilization, reaction system optimization, and efficient product separation.

[0010] Therefore, developing a highly selective, mild, efficient, green, economical, and industrially scalable bio-enzyme-catalyzed synthesis process for salinol has become an urgent technical problem to be solved in this field. Summary of the Invention

[0011] The present invention aims to overcome the defects existing in the above-mentioned background technology and provide a bio-enzymatic catalytic synthesis process for salinol that is mild, highly selective, environmentally friendly and has recyclable catalyst.

[0012] This invention is achieved through the following technical solution:

[0013] A process for the bio-enzymatic synthesis of salinol is provided, comprising the following steps:

[0014] S1. Curing treatment: The lipase is fixed onto the curing material to obtain cured lipase;

[0015] S2, Enzyme-catalyzed reaction: Glycerol, oleyl acetate, the solidified lipase obtained in S1 and molecular sieve are added to an organic solvent and shaken at 30℃~45℃ for 24~36h. After the reaction is completed, solid and liquid are separated and the filtrate is concentrated to obtain intermediate SN1.

[0016] S3, Saponification and Post-treatment: The intermediate SN1 obtained from S2 is mixed with an alkaline solution for saponification. After the reaction is completed, it is neutralized, extracted and dried to obtain an organic phase containing saponin.

[0017] S4. Purification: The organic phase obtained in S3 was purified by column chromatography, and the eluent was concentrated to obtain salool.

[0018] To address the lack of a complete set of processes, this invention integrates and optimizes four key steps to form a complete process chain. It employs immobilized lipase for catalysis under mild conditions (30-45°C), utilizing the enzyme's inherent regioselectivity to specifically catalyze the reaction of oleyl alcohol acetate with the 1-hydroxyl group of glycerol, thus avoiding the generation of isomeric byproducts at the source. The immobilized lipase is prepared by fixing it onto a solidification material such as diatomaceous earth, making it easy to separate and directly recycle. Adding molecular sieves to the reaction system immediately removes the byproduct water, shifting the reaction equilibrium to the right and significantly improving the conversion rate.

[0019] Furthermore, in S1, the lipase is selected from one of Pseudomonas lipase, Candida lipase, Candida antarcticis lipase, or Rhizopus miltiorrhiza lipase.

[0020] Lipases possess precise substrate recognition and position selectivity. In this invention, lipases specifically recognize the primary hydroxyl group (1-position hydroxyl group) of glycerol and catalyze its acylation reaction with oleyl acetate, while hardly acting on the secondary hydroxyl group (2-position hydroxyl group) of glycerol. This eliminates the generation of byproducts such as 2-position substituted isomers and disubstituted ethers from the source of the reaction, resulting in stable purity of the final product and significantly simplifying the subsequent separation and purification burden.

[0021] Furthermore, in S1, the curing material is selected from one of diatomaceous earth, acrylic resin, and silicone.

[0022] By immobilizing the enzyme on porous solidification materials such as diatomaceous earth, the contact interface between the enzyme and the reactants is increased, the mass transfer resistance is reduced, and the catalytic efficiency is improved, enabling efficient conversion to be completed within 24 to 36 hours at lower enzyme dosage and temperature (30~45℃).

[0023] Further, in S1, the curing process is achieved by adsorption, including: dissolving lipase in phosphate buffer solution with pH=7 and a concentration of 0.05~0.1 mol / L to prepare an enzyme solution, mixing it with pretreated curing material at a ratio of 1g curing material: 10~20mL enzyme solution, adsorbing at 4~25℃ and 100~150rpm for 4~6h, and then obtaining the cured lipase by filtration, washing and drying.

[0024] Furthermore, in S2, the molecular sieve is selected from one of 3A, 4A, 5A, and 13X molecular sieves.

[0025] This invention adds molecular sieves as a desiccant, which can remove water molecules in the reaction system instantly and selectively. The removal of water disrupts the reaction equilibrium and drives the reaction to continuously move towards the formation of the target intermediate SN1, thereby significantly improving the conversion rate.

[0026] Furthermore, in S2, the organic solvent is selected from one of anhydrous tert-butanol, dichloromethane, carbon tetrachloride, cyclohexane, and petroleum ether.

[0027] Furthermore, in S2, the molar ratio of glycerol to oleyl acetate is 1:1.2~1.5.

[0028] Furthermore, in S2, the amount of molecular sieve added is 0.4 to 0.6 times the mass of oleyl acetate; the amount of organic solvent added is 1.5 to 2 times the mass of oleyl acetate; and the amount of immobilized lipase added is 0.1 to 0.2 times the mass of oleyl acetate.

[0029] Furthermore, in S3, the alkaline solution is a 30% sodium hydroxide solution, and its addition amount is 1.5 to 2 times the mass of intermediate SN1; the saponification reaction is stirred at room temperature for 2 to 6 hours.

[0030] Preferably, in S4, column chromatography uses a silica gel column, and the concentration is achieved by rotary evaporation.

[0031] The beneficial effects of this invention are:

[0032] I. High regional selectivity and high product purity

[0033] Utilizing the biocatalytic specificity of immobilized lipase, the acylation reaction between oleyl acetate and the 1-hydroxyl group of glycerol molecules is precisely catalyzed, effectively inhibiting the formation of byproducts such as 2-substituted isomers and diethers from the source. After purification, the purity of the final product, saloyl alcohol, is consistently above 98.0%, significantly superior to the purity of products obtained by traditional chemical methods, and better meets the stringent requirements for raw materials in the pharmaceutical and high-end daily chemical industries.

[0034] II. High reaction efficiency and yield

[0035] By optimizing the reaction system, particularly by adding molecular sieves to immediately remove the byproduct water, the reversible reaction equilibrium is shifted towards the forward direction, significantly improving the conversion rate. Compared to the traditional chemical synthesis route mentioned in the background section, the yield is significantly increased.

[0036] III. Green and environmentally friendly, with mild conditions

[0037] The entire synthesis process utilizes bio-enzyme catalysis, completely avoiding the use of toxic and corrosive catalysts such as strong acids, strong bases, or boron trifluoride ether required by traditional chemical methods, thus reducing waste generation at the source. The reaction is carried out at a low temperature of 30℃~45℃, effectively preventing the oxidation and isomerization of unsaturated double bonds in the oil-based raw materials at high temperatures, ensuring the stability of product quality and its light color.

[0038] IV. The catalyst is recyclable and economical.

[0039] By immobilizing lipase on a solidifying material such as diatomaceous earth, the resulting solidified lipase can be recovered from the reaction system through a simple filtration process and directly used in the next batch of reaction. This greatly reduces the cost of using expensive biocatalysts and solves the economic bottleneck of the free enzyme method, which is difficult to industrialize.

[0040] V. The process is safe, easy to operate, and readily industrialized.

[0041] The entire process is carried out at normal pressure and medium to low temperatures. The raw materials and solvents used are all conventional reagents, requiring no special high-pressure or high-risk equipment. It can be completed in a conventional reactor, ensuring high safety and strong operability. The process route is clear, the steps are standardized, and it has good reproducibility and scale-up potential.

[0042] This invention achieves green and recyclable catalysts through "immobilized enzymes", ensures product purity through "enzyme regioselectivity", and drives efficient reaction through "molecular sieve dehydration". The three work synergistically to achieve significant technological progress with high yield, high purity, green and environmentally friendly, economically feasible and easy to industrialize, and comprehensively solves the various technical problems raised in the background art. Attached Figure Description

[0043] Figure 1 This is a reaction flow diagram of the present invention.

[0044] Figure 2 This is a gas chromatogram of the target product after purification by the process in Example 1 of this invention.

[0045] Figure 3 This is a gas chromatogram of the target product after purification by the process in Example 6 of this invention.

[0046] Figure 4 This is a gas chromatogram of the target product after purification by the process in Example 11 of this invention.

[0047] Figure 5 This is a gas chromatogram of the target product after purification by the process in Example 12 of this invention.

[0048] Figure 6 This is a gas chromatogram of the target product after purification by the process in Example 16 of this invention.

[0049] Figure 7 This is a gas chromatogram of the target product purified by conventional process in Example 20 of this invention. Detailed Implementation

[0050] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to describe the solution.

[0051] Example 1:

[0052] A process for the bio-enzymatic synthesis of sarool includes the following steps:

[0053] S1. Solidification Treatment: Pseudomonas lipase was dissolved in a 0.05 mol / L potassium dihydrogen phosphate solution (pH=7) to prepare an enzyme solution of 5 mg / mL for later use. Diatomaceous earth was washed twice with anhydrous ethanol, vacuum dried, and mixed with enzyme solution at a ratio of 1 g: 10 mL. The mixture was shaken at 25°C and 100 rpm for 4 hours, filtered, and the filter cake was washed twice with buffer solution and vacuum dried to obtain solidified lipase.

[0054] S2. Enzyme-catalyzed reaction: Glycerol and oleyl acetate were added to the reaction flask at a molar ratio of 1:1.2, followed by the addition of 4A molecular sieve (0.4 times the mass of oleyl acetate), immobilized lipase (0.1 times the mass of oleyl acetate), and anhydrous tert-butanol (1.5 times the mass of oleyl acetate). The reaction was carried out at 30°C with shaking for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated to obtain intermediate SN1. The immobilized lipase was recovered and recycled.

[0055] S3, Saponification and Post-treatment: Add intermediate SN1 to 30% sodium hydroxide solution (mass of intermediate SN1), stir at room temperature for 2 hours, then add 30% hydrochloric acid solution to adjust pH to 7, and then extract the organic phase with ethyl acetate. The organic phase is dried with anhydrous sodium sulfate. When the moisture content is less than 0.5%, proceed to the next step.

[0056] S4. Purification: The organic phase from reaction S3 was subjected to silica gel column chromatography. The liquid that passed through the column was then rotary evaporated to obtain the final product. The product purity was 99.0%, and the yield was 88%.

[0057] like Figure 2The image shows the gas chromatogram of the product obtained in Example 1 (optimal process conditions). A very sharp and tall main peak (peak area 99.029%) was observed at a retention time of approximately 13.263 minutes, while only a very small impurity peak (peak area 0.971%) was observed at 13.384 minutes.

[0058] This demonstrates that, under optimal process parameters, the method of this invention can efficiently synthesize the target product with extremely high purity (up to 99.0%) and extremely low by-product content, proving the significant advantages of this process in terms of product selectivity and purity.

[0059] Example 2:

[0060] The difference from Example 1 is that in S1, the buffer solution was replaced with 0.05 mol / L sodium dihydrogen phosphate; all other process conditions were exactly the same as in Example 1. The yield of the target product was tested to be 85.0%, and the purity was 98.0%.

[0061] Therefore, it can be seen that the buffer solutions prepared with potassium dihydrogen phosphate or sodium dihydrogen phosphate have a basically consistent effect on the overall reaction, with no significant changes.

[0062] Example 3:

[0063] The only difference from Example 1 is that the buffer solution in S1 was prepared as 0.15 mol / L potassium dihydrogen phosphate; all other process conditions were exactly the same as in Example 1. The yield of the target product was measured to be 83.0%, and the purity was 96.0%.

[0064] Example 4:

[0065] The only difference from Example 1 is that the buffer solution in S1 was prepared as 0.04 mol / L potassium dihydrogen phosphate; all other process conditions were exactly the same as in Example 1. The yield of the target product was measured to be 84.0%, and the purity was 95.5%.

[0066] Buffer concentrations above 0.1 mol / L affect enzyme activity, leading to a decrease in overall reaction yield. When the buffer concentration is below 0.05 mol / L, the amount of enzyme adhering to the solidification material decreases, resulting in a decrease in overall reaction conversion rate and final product purity.

[0067] Example 5:

[0068] The only difference from Example 1 is that the ratio of solidifying material to enzyme solution in S1 is changed to 1g:25mL. All other process conditions are exactly the same as in Example 1. Testing showed that the yield of the target product was 85.0% and the purity was 98.1%.

[0069] Example 6:

[0070] The only difference from Example 1 is that the ratio of solidifying material to enzyme solution in S1 is changed to 1g:5mL. All other process conditions are exactly the same as in Example 1. Testing showed that the yield of the target product was 77.0% and the purity was 84.8%.

[0071] Therefore, when the ratio of solidified material to enzyme solution exceeds 1g:20mL, the enzyme reaction effect is consistent with that when the ratio is 1g:20mL, with no significant change. When the ratio of solidified material to enzyme solution is less than 1g:10mL, the enzyme reaction effect decreases, the overall conversion rate decreases, impurities increase, and both the content and yield are significantly reduced.

[0072] like Figure 3 As shown, this embodiment altered the ratio of "curing material: enzyme solution" (1g:5mL, lower than the optimal ratio). Its chromatogram shows that the main peak area at retention time 13.247 minutes accounted for 96.542%, while a distinct impurity peak (peak area 3.458%) appeared at 13.556 minutes.

[0073] and Figure 2 In comparison, the purity of the main peak decreased, while the area of ​​the impurity peaks increased significantly. This indicates that the ratio of the solidified material to the enzyme solution has a direct impact on catalytic efficiency and product purity; an excessively low ratio leads to an increase in byproducts, thus verifying the necessity of the parameter ranges in the examples.

[0074] Example 7:

[0075] The only difference from Example 1 is that the reaction time in S1 is 3 hours. All other process conditions are exactly the same as in Example 1. The yield of the target product was 79.0%, and the purity was 95.4%.

[0076] Example 8:

[0077] The only difference from Example 1 is that the reaction time in S1 is 7 hours. All other process conditions are exactly the same as in Example 1. Testing showed that the yield of the target product was 85.4%, and the purity of the product was 98.3%.

[0078] During the process of solidifying lipase, when the reaction time is less than 4 hours, the amount of solidified lipase is small, resulting in poor reaction effect in the subsequent enzyme catalysis process, and a decrease in product content and yield. When the reaction time exceeds 6 hours, the amount of solidified lipase is consistent with that at 6 hours, and there is no significant change in product yield and content.

[0079] Example 9:

[0080] Screening of biological enzymes: The same process as in Example 1 was used, the only difference being that the biological enzymes used were different from those in S1. Different biological enzymes (Pseudomonas lipase, Candida lipase, Candida antarcticis lipase, and Rhizopus oryzae lipase) were selected for testing and verification. The specific test results are shown in the table below:

[0081] Table 1: Comparison of results of various enzyme catalysis experiments 1 Pseudomonas lipase ≥85% ≥98% 2 Candida lipase 80%~86% ≥96% 3 Antarctic Candida fat 70%~80% ≥91% 4 Rhizopus micranthum lipase 65%~75% ≥93%

[0082] Example 10:

[0083] Screening of curing materials: The same process as in Example 1 was used, the only difference being the material used to cure the lipase in S1. Different curing materials (diatomaceous earth, acrylic resin, and silica gel) were selected and tested. The specific test results are shown in the table below:

[0084] Table 2: Comparison of test results for various curing materials 1 diatomite The catalytic efficiency reached 86.0% after 5 cycles of testing. 8 2 acrylic resin The catalytic efficiency reached 79.2% after 5 cycles of testing. 5 3 silicone The catalytic efficiency reached 75.1% after 5 cycles of testing. 4

[0085] Based on the experimental results, diatomaceous earth is the best material for solidifying lipase.

[0086] The recyclability of solidified lipase significantly reduces production costs. As shown in Example 10, the solidified lipase prepared using diatomaceous earth as the solidification material maintained a high catalytic efficiency of 86.0% after being reused 5 times, and could be recycled up to 8 times. This demonstrates that the solidification process of the present invention not only achieves efficient catalyst recovery but also ensures its activity stability during multiple uses, laying a solid foundation for continuous industrial production.

[0087] Example 11:

[0088] The only difference from Example 1 is that the molar ratio of glycerol to oleyl acetate in S2 is 1:1.6. All other process conditions are exactly the same as in Example 1. The yield of the target product was 87.4%, and the purity was 98.4%.

[0089] This example adjusts the substrate molar ratio (glycerol: oleyl acetate = 1:1.6). Figure 4 In the chromatogram shown, the main peak (retention time 13.242 min) accounts for 98.473% of the area, while the impurity peak (13.339 min) accounts for 1.527%. Although the product purity remains high, the proportion of the main peak is slightly lower than that in Example 1 (99.029%). This indicates that even a slight imbalance in the substrate ratio can affect the selectivity of the reaction, further demonstrating the value of optimizing process parameters.

[0090] Example 12:

[0091] The only difference from Example 1 is that the molar ratio of glycerol to oleyl acetate in S2 is 1:1.1. All other process conditions are exactly the same as in Example 1. The yield of the target product was 77.4%, and the purity was 86.5%.

[0092] When the molar ratio of glycerol to oleyl acetate is less than 1:1.1, the reaction conversion rate is low and the yield of the target product is reduced; when the molar ratio of glycerol to oleyl acetate is greater than 1:1.5, the reaction conversion rate is consistent with that when the molar ratio is 1:1.5, and the yield is close.

[0093] This example alters the substrate molar ratio (glycerol: oleyl acetate = 1:1.1, deviating from the optimal ratio). For example... Figure 5 As shown, a main peak appears at a retention time of 13.354 minutes, but its peak area accounts for only 86.577%. Meanwhile, significant impurity peaks appear at multiple locations, including 10.160 minutes and 11.865 minutes, indicating a high total impurity content. The purity of the main peak decreases sharply, and the impurities are numerous and abundant. This demonstrates that when the substrate ratio deviates significantly from the optimal range, it leads to severe side reactions and a sharp decline in product purity, highlighting the importance of strictly controlling the substrate ratio in the process of this invention.

[0094] Example 13:

[0095] The only difference from Example 1 is that the amount of molecular sieve added in S2 is 0.3 times the mass of oleyl ethylene ester. All other process conditions are exactly the same as in Example 1. Testing showed that the yield of the target product was 81.5%, and the purity of the product was 95.3%.

[0096] Example 14:

[0097] The only difference from Example 1 is that the amount of molecular sieve added in S2 is 0.7 times the mass of oleyl ethylene ester. All other process conditions are exactly the same as in Example 1. Testing showed that the yield of the target product was 85.6%, and the purity of the product was 94.8%.

[0098] When the amount of molecular sieve added is less than 0.3 times, the moisture in the reaction process will affect the reaction conversion rate, which will lead to a decrease in yield. When the amount added exceeds 0.6 times, the effect is basically the same as that at 0.6 times, with no significant change.

[0099] Example 15:

[0100] Molecular sieve screening: The same process as in Example 1 was used, with the only difference being the molecular sieve in S2. Different molecular sieves (3A, 4A, 5A, 13X) were selected for testing, and the specific test results are shown in the table below:

[0101] Table 3: Experimental results of different molecular sieves 1 Molecular sieve 3A 86.7% 0.44% 2 Molecular sieve 4A 88.3% 0.31% 3 Molecular sieve 5A 85.8% 0.55% 4 Molecular sieve 13X 85.3% 0.57%

[0102] Based on the comparison of experimental results, the best molecular sieve for dehydration is molecular sieve 4A.

[0103] Example 16:

[0104] The only difference from Example 1 is that the amount of solidified lipase added in S2 is 0.05 times the mass of oleyl acetate. All other process conditions are exactly the same as in Example 1. The yield of the target product was 66.3%, and the purity was 79.1%.

[0105] This embodiment significantly reduces the amount of curing enzyme added (only 0.05 times the mass of oleyl acetate). Figure 6 The chromatogram shows that the main peak area ratio at 13.363 minutes decreased to 79.157%, while a large number of impurity peaks appeared at multiple positions such as 10.160 minutes and 11.865 minutes, with the impurity peak area ratio at 11.865 minutes reaching as high as 5.265%.

[0106] This indicates that a severe deficiency in enzyme dosage led to low catalytic efficiency and serious side reactions. The product purity decreased significantly, while the impurity content increased substantially, further validating the significant impact of process parameters (such as enzyme dosage) on the technical effectiveness of this invention.

[0107] Example 17:

[0108] The only difference from Example 1 is that the amount of solidified lipase added in S2 is 0.25 times the mass of oleyl acetate. All other process conditions are exactly the same as in Example 1. Testing showed that the yield of the target product was 87.3%, and the purity of the product was 98.2%.

[0109] When the amount of solidified lipase added is less than 0.1 times, the overall conversion rate and yield of the reaction decrease. When the amount of solidified lipase added is greater than 0.2 times, the conversion rate is basically the same as that at 0.2 times, and the yield and purity do not change significantly.

[0110] Example 18:

[0111] The only difference from Example 1 is that the mass of the 30% sodium hydroxide solution added in S3 is twice that of the intermediate. All other process conditions are exactly the same as in Example 1. Testing showed that the yield of the target product was 77.3%, and the purity of the product was 94.4%.

[0112] Example 19:

[0113] The only difference from Example 1 is that the mass of the 30% sodium hydroxide solution added in S3 is 2.5 times that of the intermediate. All other process conditions are exactly the same as in Example 1. Testing showed that the yield of the target product was 85.1%, and the purity of the product was 98.1%.

[0114] Example 20:

[0115] According to the traditional process, oleyl alcohol is first added to dichloromethane, followed by boron trifluoride ethyl ether (the molar ratio of boron trifluoride ethyl ether to oleyl alcohol is 0.1:1). Epichlorohydrin (the molar ratio of oleyl alcohol to epichlorohydrin is 1:1.2) is then slowly added dropwise at a temperature below 20°C. The mixture is stirred at room temperature for 6-8 hours, and the raw material content is checked to be less than 1.0%. The mixture is washed twice with water, and 30% sodium hydroxide is added to adjust the pH to around 8. The temperature is then raised to 45°C for hydrolysis for 2-3 hours. The pH is then adjusted to around 6 with hydrochloric acid, followed by extraction with ethyl acetate. The product is obtained by column chromatography and rotary evaporation, with a yield of 70% and a purity of 96.5%.

[0116] To more clearly compare the effects of various process parameters on the yield and purity of the target product in this invention, the yield and purity of the target products obtained in Examples 1-8, 11-14 and 16-20 of this invention are listed again in Table 4 below.

[0117] Table 4. Yield and purity of the target product under different process conditions <![CDATA[ Example 1 ]]> <![CDATA[ 88% ]]> <![CDATA[ 99.0% ]]> Example 2 85% 98.0% Example 3 83% 96.0% Example 4 84% 95.5% Example 5 85% 98.1% Example 6 77% 84.8% Example 7 79% 95.4% Example 8 85.4% 98.3% Example 11 87.4% 98.4% Example 12 77.4% 86.5% Example 13 81.5% 95.3% Example 14 85.6% 94.8% Example 16 66.3% 79.1% Example 17 87.3% 98.2% Example 18 77.3% 94.4% Example 19 85.1% 98.1% <![CDATA[ Example 20 ]]> <![CDATA[ 70% ]]> <![CDATA[ 96.5% ]]>

[0118] As can be seen from the results in Table 4, the process of the present invention can successfully prepare the target product under certain conditions. However, only under the process conditions and parameters of Example 1, the total yield and purity of the prepared saline alcohol are the highest. In particular, the product yield is as high as 88%, which is 18 percentage points higher than the total yield of 70% of the product prepared by the traditional method in Example 20.

[0119] Figure 7 The product chromatogram of the conventional chemical synthesis process (Example 20) is shown. The main peak is at a retention time of 13.208 min (accounting for 84.857%), but there are a large number of obvious impurity peaks at multiple positions such as 11.517 min, 11.861 min, 12.022 min, and 13.249 min. In particular, the impurity peak at 11.861 min accounts for 5.231%, and the impurity peak at 13.249 min accounts for 3.013%.

[0120] Compared with traditional processes, the best implementation of this invention (e.g.) Figure 2This invention exhibits overwhelming advantages in terms of peak purity and impurity control. Traditional processes generate a wide variety and high concentration of impurities, which not only increases the difficulty of separation and purification but also leads to a decrease in yield and quality. This clearly demonstrates the remarkable effectiveness of this invention in overcoming the shortcomings of existing technologies.

[0121] Furthermore, screening experiments determined that:

[0122] Optimal biological enzyme: Pseudomonas lipase (yield ≥85%, purity ≥98%).

[0123] Optimal curing material: diatomaceous earth (catalytic efficiency still reaches 86.0% after 5 cycles, and can be cycled up to 8 times).

[0124] The optimal molecular sieve is 4A molecular sieve (yield 88.3%, and water content of the system after reaction is only 0.31%).

[0125] contrast Figure 2 (Optimal conditions of this invention) and Figure 7 (Traditional process) It can be clearly seen that the chromatographic peaks of the product obtained by the present invention are sharper, and the impurity peaks ( Figure 7 The peak area with a retention time of approximately 13.356 minutes (accounting for 3.458%) is significantly less than that of the traditional process, which directly demonstrates the advantage of this invention in terms of product purity.

[0126] In summary, this invention provides a mild, highly selective, environmentally friendly, and industrially suitable enzymatic synthesis process for salinol, effectively overcoming the shortcomings of existing technologies.

[0127] Of course, the above description is not limited to the examples above. Technical features not described in this invention can be implemented by or using existing technology, and will not be repeated here. The above embodiments and drawings are only used to illustrate the technical solutions of this invention and are not intended to limit this invention. This invention has been described in detail with reference to preferred embodiments. Those skilled in the art should understand that any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention do not depart from the spirit of this invention and should also fall within the scope of protection of the claims of this invention.

Claims

1. A process for the bio-enzymatic synthesis of salinol, characterized in that: Includes the following steps: S1. Curing treatment: The lipase is fixed onto the curing material to obtain cured lipase; S2, Enzyme-catalyzed reaction: Glycerol, oleyl acetate, the solidified lipase obtained in S1 and molecular sieve are added to an organic solvent and shaken at 30℃~45℃ for 24~36h. After the reaction is completed, solid and liquid are separated and the filtrate is concentrated to obtain intermediate SN1. S3, Saponification and Post-treatment: The intermediate SN1 obtained from S2 is mixed with an alkaline solution for saponification. After the reaction is completed, it is neutralized, extracted and dried to obtain an organic phase containing saponin. S4. Purification: The organic phase obtained in S3 was purified by column chromatography, and the eluent was concentrated to obtain salool.

2. The bio-enzyme-catalyzed synthesis process of salinol according to claim 1, characterized in that: In S1, the lipase is selected from one of the following: Pseudomonas lipase, Candida lipase, Candida antarcticis lipase, or Rhizopus miltiorrhiza lipase.

3. The bio-enzyme-catalyzed synthesis process of salinol according to claim 1, characterized in that: In S1, the curing material is selected from one of diatomaceous earth, acrylic resin, and silicone.

4. The bio-enzyme-catalyzed synthesis process of salinol according to claim 1, characterized in that: In S1, the curing process is achieved by adsorption, which includes: dissolving lipase in phosphate buffer solution with pH=7 and concentration of 0.05~0.1 mol / L to prepare an enzyme solution, mixing it with pretreated curing material at a ratio of 1g curing material: 10~20mL enzyme solution, adsorbing at 4~25℃ and 100~150rpm for 4~6h, and then filtering, washing and drying to obtain the cured lipase.

5. The bio-enzyme-catalyzed synthesis process of salinol according to claim 1, characterized in that: In S2, the molecular sieve is selected from one of the following: 3A, 4A, 5A, and 13X molecular sieves.

6. The bio-enzyme-catalyzed synthesis process of salinol according to claim 1, characterized in that: In S2, the organic solvent is selected from one of anhydrous tert-butanol, dichloromethane, carbon tetrachloride, cyclohexane, and petroleum ether.

7. The bio-enzyme-catalyzed synthesis process of salinol according to claim 1, characterized in that: In S2, the molar ratio of glycerol to oleyl acetate is 1:1.2~1.

5.

8. The bio-enzyme-catalyzed synthesis process of salinol according to claim 1, characterized in that: In S2, the amount of molecular sieve added is 0.4 to 0.6 times the mass of oleyl acetate; the amount of organic solvent added is 1.5 to 2 times the mass of oleyl acetate; and the amount of immobilized lipase added is 0.1 to 0.2 times the mass of oleyl acetate.

9. The bio-enzyme-catalyzed synthesis process of salinol according to claim 1, characterized in that: In S3, the alkaline solution is a 30% sodium hydroxide solution, and its addition amount is 1.5 to 2 times the mass of intermediate SN1; the saponification reaction is stirred at room temperature for 2 to 6 hours.

10. The bio-enzyme-catalyzed synthesis process of salinol according to claim 1, characterized in that: In S4, column chromatography uses a silica gel column, and the concentration is achieved by rotary evaporation.

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