Immobilized enzyme catalyst for preparing chiral alcohol and chiral ester as well as preparation method and application of immobilized enzyme catalyst

Abstract

The invention belongs to the technical field of catalytic materials, and particularly relates to an immobilized enzyme catalyst for preparing chiral alcohol and chiral ester as well as a preparation method and application of the immobilized enzyme catalyst. The preparation method comprises the following steps: 1) adding anionic polyelectrolyte and cationic polyelectrolyte into water, and mixing to form an aggregate; adding an enzyme solution into the aggregate, and uniformly mixing to obtain an enzyme-containing aggregate; (2) adding a porous material into the enzyme-containing aggregate in the step (1), and uniformly stirring to obtain a mixture; and (3) adding a thickening agent into the mixture in the step (2), heating, stirring, carrying out extrusion forming, and drying, so as to obtain the product. The immobilized enzyme catalyst can be directly filled into a fixed bed reactor to carry out an enzyme catalysis reaction so as to obtain chiral alcohol and chiral alcohol ester with high added values, a high-concentration substrate continuously flows to react for 10-30 days, and the enzyme catalysis activity and selectivity are basically kept unchanged.

Description

Technical Field

[0001] This invention belongs to the field of catalytic materials technology, specifically relating to an immobilized enzyme catalyst for preparing chiral alcohols and chiral esters, its preparation method, and its application. Background Technology

[0002] Immobilized enzymes possess advantages such as high catalytic activity, selectivity, stability, and ease of recovery and reuse, making them highly promising for industrial biocatalysis in the production of high-value-added chiral drug intermediates, alcohols. Immobilizing enzymes in macromolecular scaffolds or micro / nano spaces using zeolites, mesoporous silica, nanoparticles, metal-organic frameworks (MOFs), and covalent organic frameworks (COFs) can improve catalytic efficiency to some extent. However, the complex immobilization process and alterations to the support structure often unpredictably cause changes in the enzyme protein structure, leading to reduced enzyme activity or even inactivation, failing to provide an ideal microenvironment for the enzyme. While enzyme-loaded catalysts prepared using surfactant-stabilized emulsions, Pickering emulsions made from mesoporous silica, or simple sol-gel methods provide an ideal microenvironment, these emulsions suffer from inherent drawbacks: low stability or uncontrollable silica gel formation often leads to emulsion rupture or irregular morphology. Furthermore, filling them into fixed-bed reactors can easily increase fluid resistance, cause equipment blockage, and result in enzyme loss or inactivation during the reaction.

[0003] It is evident that existing technologies for immobilizing enzyme catalytic materials suffer from several technical challenges, including complex immobilization processes, low immobilization efficiency, inability to provide an ideal microenvironment for the enzyme, low and unstable strength, and difficulty in recycling. Therefore, developing novel methods that simplify the immobilization process, ensure strong enzyme-carrier bonding, and preserve high enzyme activity and selectivity remains a significant challenge. Summary of the Invention

[0004] One of the objectives of this invention is to provide a method for preparing immobilized enzyme catalysts for chiral alcohols and chiral esters. This method is simple, uses mild conditions, is low in cost, is easy to scale up, and can preserve the integrity and activity of the enzyme structure to the greatest extent.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing immobilized enzyme catalysts for chiral alcohols and chiral esters includes the following steps:

[0007] 1) Add anionic polyelectrolytes and cationic polyelectrolytes to water and mix to form aggregates; add enzyme solution to the aggregates and mix well to obtain enzyme-containing aggregates;

[0008] 2) Add porous material to the enzyme-containing aggregates from step 1), stir until homogeneous, and obtain a mixture;

[0009] 3) Add a thickener to the mixture in step 2), heat and stir, then extrude and dry to obtain the final product.

[0010] Further, the ratio of the anionic polyelectrolyte, cationic polyelectrolyte, enzyme solution, and porous material is (0.074-0.34)g:(0.1-0.7)g:(100-600)μL:(0.5-2)g; and the concentration of the enzyme in the enzyme solution is 6-10mg / mL.

[0011] Further, the anionic polyelectrolyte in step 1) is selected from one of sodium polyacrylate, adenine nucleoside triphosphate, polyacrylic acid, CM-glucan, and carboxymethylated amyl starch; the cationic polyelectrolyte is polydiallyldimethylammonium chloride or quaternary ammonium amyl starch.

[0012] Further, the enzyme in the enzyme solution described in step 1) is selected from one of the following: Candida antarcticis lipase A, Candida antarcticis lipase B, Penicillium salmonella lipase, Candida pumilus lipase, and Burkholderia cepacia lipase.

[0013] Furthermore, the porous material mentioned in step 2) is selected from one of mesoporous silica, molecular sieve, and activated carbon.

[0014] Further, the stirring speed in step 2) is 600-1200 rpm, and the time is 30-120 min.

[0015] Further, in step 3), the amount of thickener added is 10-30% of the total mass of the mixture, and the thickener is selected from one of guar gum powder, guar gum, and starch.

[0016] Further, in step 3), the heating temperature is 30-50℃, the stirring speed is 1000-2000rpm, and the stirring time is 2-6h; the drying temperature is 30-40℃, and the time is 12-24h.

[0017] A second objective of this invention is to provide an immobilized enzyme catalyst for preparing chiral alcohols and chiral esters, which exhibits excellent enzyme activity and stability.

[0018] To achieve the above objectives, the present invention adopts the following technical solution:

[0019] An immobilized enzyme catalyst for preparing chiral alcohols and chiral esters is prepared using the method described above.

[0020] The third objective of this invention is to provide an application of an immobilized enzyme catalyst for the preparation of chiral alcohols and chiral esters, which has broad application prospects.

[0021] To achieve the above objectives, the present invention adopts the following technical solution:

[0022] The above-mentioned immobilized enzyme catalysts for the preparation of chiral alcohols and chiral esters are used in the catalytic synthesis of chiral alcohols and chiral ester compounds.

[0023] Furthermore, the alcohol compound and vinyl acetate are passed into a fixed-bed reactor containing the immobilized enzyme catalyst, and a transesterification chiral resolution reaction is carried out at 45°C to obtain chiral alcohols and chiral esters.

[0024] Further, the alcohol compound is any one of 1-[3,5-bis(trifluoromethyl)phenyl]ethanol, 6,10-dimethylundec-5,9-dien-2-ol, 2-(1-hydroxyethyl)pyridine, and 1-(5-fluoro-2-iodophenyl)ethanol.

[0025] Compared with the prior art, the beneficial effects of this invention are as follows:

[0026] 1. This invention provides a method for preparing immobilized enzyme catalysts for chiral alcohols and chiral esters. This method is simple, has mild conditions, is low in cost, and is easy to scale up. It can also preserve the integrity and activity of the enzyme structure to the maximum extent during the preparation process.

[0027] 2. This invention prepares an immobilized enzyme catalyst using anionic polyelectrolytes, cationic polyelectrolytes, enzyme solutions, porous materials, and thickeners as raw materials. The anionic and cationic polyelectrolytes exhibit excellent electrostatic interactions and non-conjugated interactions with the enzyme, such as hydrogen bonds and van der Waals forces. These non-covalent interactions stably confine the enzyme molecules within the aggregates formed by the anionic and cationic polyelectrolytes, providing an ideal microenvironment for the enzyme. This crowded environment effectively improves enzyme conformation, significantly enhancing enzyme activity. The porous material possesses a good pore structure and a large specific surface area, which is more conducive to rapid mass transfer of the reaction substrate, effectively ensuring the distribution of the reaction substrate and the diffusion of products, achieving high efficiency and high quality. Furthermore, the porous material also exhibits good chemical stability and biocompatibility, helping to retain enzyme activity. The thickener has good biocompatibility, is compatible with both the enzyme and the porous material, does not affect enzyme activity or the performance of the porous material, and can enhance the thermal stability and mechanical strength of the material.

[0028] 3. The immobilized enzyme catalyst prepared by this invention can be directly loaded into a fixed-bed reactor to carry out enzyme catalytic reactions to obtain high-value chiral alcohols and chiral alcohol esters. Moreover, the catalytic activity and selectivity of the enzyme remain basically unchanged even after 10-30 days of continuous flow reaction with high-concentration substrate.

[0029] 4. The immobilized enzyme catalyst prepared by this invention has strong mechanical properties and is easy to recycle and reuse, which helps to reduce production costs and environmental impact. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the structure of the enzyme catalyst immobilized in Example 1;

[0031] Figure 2 This is a graph showing the changes in the excess of 1-[3,5-bis(trifluoromethyl)phenyl]ethanol enantiomeric and excess of 1-[3,5-bis(trifluoromethyl)phenyl]ethanol acetate enantiomeric as a function of flow time during the transesterification chiral resolution reaction of the immobilized enzyme catalyst prepared in Example 1.

[0032] Figure 3 The graph shows the changes in the excess of 6,10-dimethylundec-5,9-dien-2-ol enantiomeric and 6,10-dimethylundec-5,9-dien-2-ol acetate enantiomeric as a function of flow time during the transesterification chiral resolution reaction of the immobilized enzyme catalyst prepared in Example 2.

[0033] Figure 4 This is a graph showing the changes in the excess of 2-(1-hydroxyethyl)pyridine enantiomer and the excess of 2-(1-hydroxyethyl)pyridine acetate enantiomer as a function of flow time during the transesterification chiral resolution reaction of the immobilized enzyme catalyst prepared in Example 3.

[0034] Figure 5 This is a graph showing the changes in the excess of 1-(5-fluoro-2-iodophenyl)ethanol enantiomeric and 1-(5-fluoro-2-iodophenyl)ethanol acetate enantiomeric as a function of flow time during the transesterification chiral resolution reaction of the immobilized enzyme catalyst prepared in Example 4. Detailed Implementation

[0035] The technical solution of the present invention will be further explained and described below with reference to specific embodiments, application examples and accompanying drawings.

[0036] Unless otherwise specified, the raw materials and preparation methods used in the following examples and application cases are conventional materials and techniques in the art.

[0037] Example

[0038] Example 1

[0039] A method for preparing immobilized enzyme catalysts for chiral alcohols and chiral esters, the specific process of which is as follows:

[0040] 1) Dissolve 0.052g sodium polyacrylate and 0.7g polydiallyldimethylammonium chloride in 3.5mL of deionized water, mix to form aggregates, then add 500μL of Candida antarcticis lipase B solution with an enzyme content of 8mg / mL, shake to mix evenly, and obtain enzyme-containing aggregates;

[0041] 2) Add 1.5g of mesoporous silica to the enzyme-containing aggregates in step 1), and magnetically stir at 1000rpm for 60min to obtain a mixture;

[0042] 3) Add 0.8g of guar gum powder to the mixture in step 2), heat and stir at 30℃ and 1800rpm for 6h until it becomes viscous, granulate it and dry it at 30℃ for 24h to obtain the immobilized enzyme catalyst.

[0043] An immobilized enzyme catalyst for preparing chiral alcohols and chiral esters was prepared using the method described above, and its structural schematic diagram is shown below. Figure 1 shown.

[0044] Example 2

[0045] A method for preparing immobilized enzyme catalysts for chiral alcohols and chiral esters, the specific process of which is as follows:

[0046] 1) Dissolve 0.074 g adenine nucleoside triphosphate and 0.1 g polydiallyl dimethyl ammonium chloride in 1.5 mL of deionized water, mix to form aggregates, then add 100 μL of Candida antarcticis lipase A solution with an enzyme content of 6 mg / mL, shake to mix evenly, and obtain enzyme-containing aggregates;

[0047] 2) Add 0.5g of activated carbon to the enzyme-containing aggregates in step 1), and magnetically stir at 600rpm for 120min to obtain a mixture;

[0048] 3) Add 0.5g of guar gum to the mixture in step 2), stir at 40℃ and 1500rpm for 3h until it becomes viscous, granulate it and dry it at 30℃ for 12h to obtain the immobilized enzyme catalyst.

[0049] An immobilized enzyme catalyst for preparing chiral alcohols and chiral esters was prepared using the method described above.

[0050] Example 3

[0051] A method for preparing immobilized enzyme catalysts for chiral alcohols and chiral esters, the specific process of which is as follows:

[0052] 1) Dissolve 0.22g of carboxymethylated amylose and 0.3g of quaternary amylose in 2mL of deionized water, mix well to form aggregates, then add 600μL of Burkholderia cepacia lipase solution with an enzyme content of 10mg / mL, shake to mix well, and obtain enzyme-containing aggregates;

[0053] 2) Add 0.8 g of molecular sieve to the enzyme-containing aggregates in step 1) and stir at 1200 rpm for 30 min to obtain a mixture;

[0054] 3) Add 0.6g of starch to the mixture in step 2), stir at 50℃ and 1000rpm for 2h until it becomes viscous, extrude it into strips and dry at 40℃ for 24h to obtain the immobilized enzyme catalyst.

[0055] An immobilized enzyme catalyst for preparing chiral alcohols and chiral esters was prepared using the method described above.

[0056] Example 4

[0057] An immobilized enzyme catalyst for the preparation of chiral alcohols and chiral esters, the specific process of which is as follows:

[0058] 1) Dissolve 0.34g CM-glucan and 0.6g polydiallyldimethylammonium chloride in 4mL of deionized water, mix to form aggregates, then add 300μL of Candida pleuropsii lipase solution with an enzyme content of 8mg / mL, shake to mix evenly, and obtain enzyme-containing aggregates;

[0059] 2) Add 2g of activated carbon to the enzyme-containing aggregates in step 1) and stir at 800rpm for 40min to obtain a mixture;

[0060] 3) Add 1.2g of guar gum powder to the mixture in step 2), stir at 50℃ and 2000rpm for 4h until it becomes viscous, extrude it into strips and dry at 35℃ for 18h to obtain the immobilized enzyme catalyst.

[0061] An immobilized enzyme catalyst for preparing chiral alcohols and chiral esters was prepared using the method described above.

[0062] Application examples

[0063] Application Example 1

[0064] The immobilized enzyme catalyst prepared in Example 1 was directly packed into a fixed-bed reactor with an inner diameter of 2.0 cm, and the reactor temperature was set to 45 °C. A mixed solution of 1.2 mol / L 1-[3,5-bis(trifluoromethyl)phenyl]ethanol and 4.8 mol / L vinyl acetate in octane was introduced by a constant flow pump at a flow rate of 3 mL / h to carry out the transesterification chiral resolution reaction. The curves showing the changes in the excess of 1-[3,5-bis(trifluoromethyl)phenyl]ethanol enantiomeric and 1-[3,5-bis(trifluoromethyl)phenyl]ethanol acetate enantiomeric as a function of flow time are shown in the figure. Figure 2 shown.

[0065] Figure 2 This is a graph showing the changes in the excess of 1-[3,5-bis(trifluoromethyl)phenyl]ethanol enantiomeric and 1-[3,5-bis(trifluoromethyl)phenyl]ethanol acetate enantiomeric as a function of flow time during the transesterification chiral resolution reaction of the immobilized enzyme catalyst prepared in Example 1. Observation Figure 2It can be seen that after 30 days of continuous operation, the enzyme catalytic activity and selectivity remained essentially unchanged. The ee value of the chiral alcohols generated during the reaction remained above 95%, and the ee value of the chiral esters remained above 99%.

[0066] Application Example 2

[0067] The immobilized enzyme catalyst prepared in Example 2 was directly packed into a fixed-bed reactor with an inner diameter of 2.0 cm, and the reactor temperature was set to 45 °C. A mixed solution of 1.6 mol / L 6,10-dimethylundec-5,9-dien-2-ol and 6.4 mol / L vinyl acetate in octane was introduced by a constant flow pump at a flow rate of 3 mL / h to carry out a transesterification chiral resolution reaction. The curves showing the changes in the excess of 6,10-dimethylundec-5,9-dien-2-ol enantiomer and the excess of 6,10-dimethylundec-5,9-dien-2-ol acetate enantiomer with flow time are shown in the figure. Figure 3 shown.

[0068] Figure 3 This is a graph showing the changes in the excess of 6,10-dimethylundec-5,9-dien-2-ol enantiomeric and 6,10-dimethylundec-5,9-dien-2-ol acetate enantiomeric as a function of flow time during the transesterification chiral resolution reaction of the immobilized enzyme catalyst prepared in Example 2. (Observation) Figure 3 It can be seen that after 30 days of continuous operation, the enzyme catalytic activity and selectivity remained essentially unchanged. The ee values ​​of the chiral alcohols and chiral esters generated during the reaction were both maintained above 99%.

[0069] Application Example 3

[0070] The immobilized enzyme catalyst prepared in Example 3 was directly packed into a fixed-bed reactor with an inner diameter of 2.0 cm, and the reactor temperature was set to 45 °C. A mixed solution of 1.6 mol / L 2-(1-hydroxyethyl)pyridine and 6.4 mol / L vinyl acetate in octane was introduced at a flow rate of 3 mL / h using a constant flow pump to carry out a transesterification chiral resolution reaction. The curves showing the changes in the excess of 2-(1-hydroxyethyl)pyridine enantiomer and the excess of 2-(1-hydroxyethyl)pyridine acetate enantiomer as a function of flow time are shown in the figure. Figure 4 shown.

[0071] Figure 4 This is a graph showing the changes in the excess of 2-(1-hydroxyethyl)pyridine enantiomer and the excess of 2-(1-hydroxyethyl)pyridine acetate enantiomer as a function of flow time during the transesterification chiral resolution reaction of the immobilized enzyme catalyst prepared in Example 3. (Observation) Figure 4 It can be seen that after 30 days of continuous operation, the enzyme catalytic activity and selectivity remained essentially unchanged. The ee value of the chiral alcohols generated during the reaction remained above 97%, and the ee value of the chiral esters remained above 99%.

[0072] Application Example 4

[0073] The immobilized enzyme catalyst prepared in Example 4 was directly packed into a fixed-bed reactor with an inner diameter of 2.0 cm, and the reactor temperature was set to 45 °C. A mixed solution of 1.2 mol / L 1-(5-fluoro-2-iodophenyl)ethanol and 4.8 mol / L vinyl acetate in octane was introduced by a constant flow pump at a flow rate of 3 mL / h to carry out a transesterification chiral resolution reaction. The curves showing the changes in the excess of 1-(5-fluoro-2-iodophenyl)ethanol enantiomer and the excess of 1-(5-fluoro-2-iodophenyl)ethanol acetate enantiomer with flow time are shown in the figure. Figure 5 shown.

[0074] Figure 5 This is a graph showing the changes in the excess of 1-(5-fluoro-2-iodophenyl)ethanol enantiomeric and 1-(5-fluoro-2-iodophenyl)ethanol acetate enantiomeric as a function of flow time during the transesterification chiral resolution reaction of the immobilized enzyme catalyst prepared in Example 4. (Observation) Figure 5 It can be seen that after 30 days of continuous operation, the enzyme catalytic activity and selectivity remained essentially unchanged. The ee values ​​of the chiral alcohols and chiral esters generated during the reaction were both maintained above 99%.

[0075] The above are merely preferred embodiments of the present invention and are not limited to the examples described above. Those skilled in the art will recognize that various modifications and variations can be made based on the principles of the present invention. Any modifications or improvements made should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an immobilized enzyme catalyst for preparing chiral alcohols and chiral esters, characterized in that: The following steps are involved: 1) adding anionic polyelectrolyte and cationic polyelectrolyte into water and mixing to form aggregates; adding enzyme solution into the aggregates and mixing evenly to obtain enzyme-containing aggregates; 2) adding the porous material to the enzyme-containing aggregates of step 1), stirring evenly to obtain a mixture; 3) adding a thickener to the mixture of step 2), heating and stirring, extruding and drying to obtain a product.

2. The method for preparing the immobilized enzyme catalyst for preparing chiral alcohols and chiral esters according to claim 1, characterized in that: The usage ratio of the anionic polyelectrolyte, the cationic polyelectrolyte, the enzyme solution and the porous material is (0.074-0.34) g: (0.1-0.7) g: (100-600) μL: (0.5-2) g; The concentration of the enzyme in the enzyme solution is 6-10 mg / mL.

3. The method for preparing the immobilized enzyme catalyst for preparing chiral alcohols and chiral esters according to claim 1, characterized in that: In step 1), the anionic polyelectrolyte is selected from sodium polyacrylate, adenosine triphosphate, polyacrylic acid, CM-dextran, and carboxymethylated amylose; and the cationic polyelectrolyte is polydiallyldimethylammonium chloride or quaternary ammonium amylose.

4. The method for preparing the immobilized enzyme catalyst for preparing chiral alcohols and chiral esters according to claim 1, characterized in that: The enzyme in the enzyme solution in step 1) is selected from one of Candida antarctica lipase A, Candida antarctica lipase B, Penicillium salmonellae lipase, Candida plicatilis lipase and Burkholderia cepacia lipase.

5. The method for preparing an immobilized enzyme catalyst for preparing chiral alcohols and chiral esters according to claim 1, characterized in that: The porous material in step 2) is selected from mesoporous silica, molecular sieve, and activated carbon.

6. The method for preparing the immobilized enzyme catalyst for preparing chiral alcohols and chiral esters according to claim 1, characterized in that: The stirring speed in step 2) is 600-1200 rpm and the time is 30-120 min.

7. The method for preparing the immobilized enzyme catalyst for preparing chiral alcohols and chiral esters according to claim 1, characterized in that: In step 3), the amount of the thickener added is 10-30% of the total mass of the mixture, and the thickener is selected from one of sesbania gum powder, guar gum and starch.

8. The method for preparing the immobilized enzyme catalyst for preparing chiral alcohols and chiral esters according to claim 1, characterized in that: In step 3), the heating temperature is 30-50° C., the stirring speed is 1000-2000 rpm, and the stirring time is 2-6 hours; the drying temperature is 30-40° C., and the drying time is 12-24 hours.

9. An immobilized enzyme catalyst for preparing chiral alcohols and chiral esters, characterized in that: The preparation method is described in any one of claims 1 to 8.

10. The use of an immobilized enzyme catalyst for preparing chiral alcohols and chiral esters as claimed in claim 9, characterized in that: Application in the catalytic synthesis of chiral alcohols and chiral esters.

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