Preparation method of immobilized nitrilase for brivaracetam synthesis
Immobilized nitrile hydrolase was prepared by supporting carboxy-terminated polylactic acid-hydroxyglycolic acid copolymer and hydroxyapatite on the surface of macroporous resin, and combining adsorption and cross-linking methods, the problems of unstable nitrile hydrolase activity and low catalytic efficiency were solved, and the efficient immobilization and stability of enzymes in bovacetam synthesis were achieved.
Patent Information
- Application Number
- CN202411409433.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2025-03-14
AI Technical Summary
The existing nitrile hydrolase has problems such as unstable activity, low catalytic efficiency and difficulty in recycling in bovacetam synthesis, which has affected the industrial green synthesis of bovacetam.
Immobilized nitrile hydrolase enzyme is prepared by loading carboxy-terminated polylactic acid-glycolic acid copolymer and hydroxyapatite on the surface of macroporous resin, and combining adsorption and cross-linking methods to reduce the steric hindrance effect and improve the immobilization effect and stability of the enzyme.
The immobilized nitrile hydrolase has achieved high activity and good stability preparation, which improves the catalytic efficiency and product yield of bovacetam synthesis, and extends the enzyme usage cycle.
Smart Images
Figure BDA0005077588560000101
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of drug synthesis, and in particular to a method for preparing immobilized nitrilase for brivaracetam synthesis. Background Art
[0002] Brivaracetam is a new generation of anti-epileptic drugs with broad market prospects. Its efficient and green synthesis method has become one of the research hotspots of pharmaceutical engineering. Among them, (R)-3-aminomethylhexanoic acid is a key chiral intermediate for the synthesis of Brivaracetam. It can be synthesized by 3-cyanohexanenitrile using nitrilase catalytic hydrolysis to (R)-3-cyanohexanoic acid, and then by (R)-3-cyanohexanoic acid hydrogenation reduction to (R)-3-aminomethylhexanoic acid. A new enzymatic synthesis process of Brivaracetam has been constructed, which is of great significance for the realization of industrial green synthesis of Brivaracetam.
[0003] The active site of nitrilase contains three residues of Glu-Lys-Cys, which can degrade inorganic cyanide. The biocatalytic process mediated by it has the advantages of mild reaction conditions, high catalytic efficiency, strict selectivity and environmental friendliness. However, due to the sensitivity of free enzyme to substrate, low catalytic efficiency to substrate, low stability of free enzyme, and difficulty in recycling, nitrilase can be immobilized to improve the catalytic stability of nitrilase, improve catalytic efficiency and product yield, and extend the service life. Enzyme immobilization technology is divided into adsorption, embedding, cross-linking, coupling and other methods according to the fixation method. The adsorption method is a physical adsorption method with simple operation and mild conditions, but the binding force is not strong and it is easy to fall off; the embedding method has a low degree of damage to the biological activity of the enzyme, but it is not suitable for macromolecular substrates; the carrier obtained by the cross-linking method and the coupling method has a higher binding firmness with the enzyme and better stability, but it is easy to affect the activity and conformation of the enzyme. Therefore, it is particularly important to select a suitable carrier and immobilization method for immobilizing nitrilase. Summary of the invention
[0004] In order to solve the above technical problems, the present invention provides a method for preparing immobilized nitrilase for the synthesis of brivaracetam, by modifying the macroporous resin to reduce the steric hindrance effect of directly immobilizing the enzyme on the resin micropore wall, combining the adsorption method with the cross-linking method to prepare the immobilized nitrilase, which can ensure the firmness of the combination between the carrier and the enzyme and obtain an immobilized nitrilase with higher activity.
[0005] The purpose of the present invention is achieved through the following technical solutions: The present invention provides a method for preparing an immobilized nitrilase for the synthesis of brivaracetam, comprising the following steps: (1) dissolving a carboxyl-terminated poly(lactic acid-glycolic acid) copolymer in dichloromethane, adding a n-hexane solution containing hydroxyapatite and stirring to obtain a suspension; (2) adding a macroporous weakly basic anion exchange resin to the suspension and then sonicating, and then adding an aqueous solution of polyvinyl alcohol and sonicating again; (3) heating to volatilize the organic solvent, filtering, adding the obtained product to a lysine solution, then adding a dicyclohexylcarbodiimide ethanol solution, stirring for reaction, and filtering to obtain a carrier; (4) adding the carrier into a buffer solution containing glutaraldehyde, and then adding nitrilase to carry out an immobilized enzyme reaction to obtain immobilized nitrilase.
[0006] Macroporous resin is a commonly used immobilized enzyme carrier, but the enzyme is fixed on the surface of the macroporous resin and in the macroporous structure by cross-linking, which greatly affects the conformation and activity of the enzyme. The present invention combines the adsorption method with the cross-linking method to prepare the immobilized nitrilase, which can ensure the firmness of the combination between the carrier and the enzyme and obtain an immobilized enzyme with higher activity.
[0007] First, carboxyl-terminated polylactic acid-glycolic acid copolymer and hydroxyapatite are adsorbed on the surface of a macroporous resin, which is a macroporous weakly alkaline anion exchange resin. The free amine on the surface of the macroporous resin interacts electrostatically with the carboxyl group in the carboxyl-terminated polylactic acid-glycolic acid copolymer, and the calcium ions in the hydroxyapatite coordinate with the carboxyl group and the amino group, so that a polylactic acid film containing hydroxyapatite is formed on the surface of the macroporous resin. The introduction of polylactic acid with biological activity will also promote the adsorption effect on the enzyme. Then, a polyvinyl alcohol aqueous solution is added to help the deposition of the carboxyl-terminated polylactic acid-glycolic acid copolymer on the surface of the resin particles, and form a structure with a certain shape of protrusions, while improving the load stability of the polylactic acid through the interface effect. Then, the added n-hexane is a poor solvent for polylactic acid. During the heating and volatilization of the organic solvent, several protrusion structures on the surface of the resin will become porous, thereby increasing the adsorption specific surface area for the enzyme, and the hydroxyapatite exposed through the porous structure will have a good adsorption effect on the enzyme. The added lysine will dehydrate with the free amine in the macroporous resin under the catalysis of N, N-dicyclohexylcarbodiimide to generate an amide bond, and the lysine side chain will be grafted on the resin surface, so that the nitrilase can be better fixed by the cross-linking method, reducing the loss of enzyme activity. Therefore, the present invention can first adsorb the nitrilase on the carrier by the adsorption method through the prepared carrier, and compared with the resin adsorption on the conventional flat surface, the steric hindrance effect generated by the direct immobilization of the enzyme on the resin micropore wall can be reduced, and the spatial range of the immobilized nitrilase catalysis can be expanded, and then the nitrilase is cross-linked and fixed by glutaraldehyde, while the enzyme loading rate is increased, the structural stability of the enzyme fixed by the adsorption method is improved, and then the activity and stability of the obtained immobilized nitrilase are both high.
[0008] Preferably, in step (1), the n-hexane solution containing hydroxyapatite is prepared by dispersing hydroxyapatite powder in n-hexane, and the amount of hydroxyapatite added relative to the concentration of n-hexane is 0.05-0.08 g / mL; the particle size of the hydroxyapatite is 50-100 nm.
[0009] Preferably, in step (1), the concentration of the carboxyl-terminated polylactic acid-glycolic acid copolymer added is 40-60 g / L relative to the mixed volume of dichloromethane and n-hexane; and the mixed volume ratio of dichloromethane and n-hexane is 8-9:1.
[0010] The mixing volume ratio of dichloromethane and n-hexane, the concentration of polylactic acid, the concentration of hydroxyapatite in n-hexane, etc. will all affect the pore size and porosity of the porous structure, and the formed pore size is 10-25 μm.
[0011] Preferably, in step (2), the macroporous weakly basic anion exchange resin is a macroporous weakly basic acrylic anion exchange resin; and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer is 10-12:1.
[0012] Preferably, in step (2), the volume ratio of the suspension to the aqueous solution of polyvinyl alcohol is 1:10; and the concentration of the aqueous solution of polyvinyl alcohol is 2-4 g / L.
[0013] Preferably, in step (2), the ultrasonication time is 5-10 min; and the second ultrasonication time is 1-2 h.
[0014] Preferably, in step (3), the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 3-6 mL: 0.4-0.5 mL; the concentration of the lysine solution is 0.05-0.1 g / mL; the concentration of the dicyclohexylcarbodiimide ethanol solution is 0.1-0.2 g / mL.
[0015] Preferably, in step (3), the heating to volatilize the organic solvent is performed by heating to 40-50° C. and stirring for 3-5 hours at a stirring speed of 1000-1500 rpm; and the stirring reaction is performed by stirring at room temperature for 1-2 hours.
[0016] Preferably, in step (4), the mass volume ratio of the carrier, buffer solution and glutaraldehyde solution is 1 g:10 mL:1-5 mL; and the mass concentration of glutaraldehyde in the glutaraldehyde solution is 10-15%.
[0017] Preferably, in step (4), the amount of the nitrilase used is 16-20 mg / g resin in terms of protein content; and the immobilized enzyme reaction is stirred at 30-40° C. for 1-3 h.
[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Carboxyl-terminated poly(lactic acid-glycolic acid) copolymer and hydroxyapatite are loaded on the surface of a macroporous resin to form a porous surface protrusion structure, thereby increasing the specific surface area and the adsorption active groups, improving the immobilization effect of nitrilase, reducing the steric hindrance effect of the enzyme directly immobilized on the resin micropore wall, and expanding the spatial range of catalysis of immobilized nitrilase; (2) By adding lysine grafting, the nitrilase can be better fixed by cross-linking method, reducing the loss of enzyme activity; and by first adsorbing and then cross-linking the nitrilase to fix it, while increasing the enzyme loading rate, the structural stability of the enzyme fixed by adsorption is improved, and the activity and stability of the immobilized nitrilase are higher. DETAILED DESCRIPTION
[0019] The technical solution of the present invention is described below with specific embodiments, but the protection scope of the present invention is not limited thereto.
[0020] 1. Immobilized nitrilase (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) is dissolved in dichloromethane, and a n-hexane solution containing hydroxyapatite (average particle size 50-100 nm) is added and stirred. The concentration of the carboxyl-terminated polylactic acid-glycolic acid copolymer added is 40-60 g / L relative to the mixed volume of dichloromethane and n-hexane, and the mixed volume ratio of dichloromethane and n-hexane is 8-9:1 to obtain a suspension.
[0021] (2) After adding a macroporous weakly basic anion exchange resin to the suspension, ultrasonically treat for 5-10 minutes, wherein the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer is 10-12:1; then, add a 2-4 g / L aqueous solution of polyvinyl alcohol, wherein the volume ratio of the suspension to the aqueous solution of polyvinyl alcohol is 1:10, and ultrasonically treat again for 1-2 hours.
[0022] (3) The reactant obtained in step (2) is heated to 40-50° C. and stirred for 3-5 hours at a stirring speed of 1000-1500 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.05-0.1 g / mL, stirred at room temperature for 1-2 hours, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.1-0.2 g / mL is added, and the reaction is stirred at room temperature for 1-2 hours, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 3-6 mL: 0.4-0.5 mL, and after the reaction is completed, the carrier is filtered to obtain the carrier.
[0023] (4) The carrier is added to a PBS buffer solution, and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content is added, and the amount of nitrilase used is 16-20 mg / g resin in terms of protein content, and an adsorption reaction is carried out in a water bath shaker at 30-40° C. and 100-150 rpm for 1-2 hours; then a PBS buffer solution containing 10-15% glutaraldehyde is added, and an immobilized enzyme reaction is carried out in a water bath shaker at 30-40° C. and 100-150 rpm for 1-2 hours, and then the mixture is washed with deionized water for 3-5 times, and dried on a filter paper at room temperature to obtain an immobilized nitrilase, which is stored in a refrigerator at 4° C. for later use.
[0024] In a specific embodiment of the present invention, in step (1), the n-hexane solution containing hydroxyapatite is prepared by ultrasonically dispersing hydroxyapatite powder in n-hexane, and the concentration of hydroxyapatite added relative to the volume of n-hexane is 0.05-0.08 g / mL.
[0025] In a specific embodiment of the present invention, in step (2), the macroporous weakly basic anion exchange resin is a macroporous weakly basic acrylic anion exchange resin D314 / D315, containing free amine and having a particle size of 0.80-1.25 mm.
[0026] In a specific embodiment of the present invention, in step (4), the mass volume ratio of the carrier, the PBS buffer and the PBS buffer containing glutaraldehyde is 1 g:10 mL:1-5 mL, and the pH of the PBS buffer is 6-8.
[0027] In a specific embodiment of the present invention, in step (4), the nitrilase is commercially available.
[0028] 2. Application in the synthesis of (R)-3-cyanohexanoic acid In 10mL 100mM, pH=7.4 PBS buffer, add 1g immobilized nitrilase and 0.3g 3-cyanohexanenitrile, and react for 24h at 30℃ and 700r / min in a water bath. Take 200μL of sample, add 30μL 6M HCl to terminate the reaction and extract with ethyl acetate. Take 200μL of the upper organic phase and dry it with anhydrous sodium sulfate, add 7.5μL methanol and 3.75μL diazomethane, and use gas chromatography to determine the conversion rate of the substrate and the enantiomeric excess (ee) of the product (R)-3-cyanohexanoic acid. According to the above steps, the immobilized nitrilase was reused 5 times in the synthesis of (R)-3-cyanohexanoic acid, and the conversion rate of the substrate was determined by gas chromatography.
[0029] Activity assay reaction system: Add 1g immobilized nitrilase and 0.3g 3-cyanohexanenitrile to 10mL 100mM, pH=7.4 PBS buffer, and react for 15min at 30℃ and 700r / min in a water bath. Take 200μL of sample, add 30μL 6M HCl to terminate the reaction and extract with ethyl acetate. Take 200μL of the upper organic phase, dry it with anhydrous sodium sulfate, add 7.5μL methanol and 3.75μL diazomethane, detect the product concentration by liquid chromatography, and calculate the enzyme activity.
[0030] Pure enzyme activity determination reaction system: Take 1 mL of pure enzyme solution with a protein concentration of 1 mg / mL, add 0.03 g of 3-cyanohexanenitrile, and react for 15 minutes in a water bath at 30°C and 700 r / min. The post-treatment and detection methods are the same as above, and the free enzyme activity is calculated.
[0031] Protein adsorption rate = (total protein amount in the initial enzyme solution - total protein amount in the residual solution) / total protein amount in the initial enzyme solution.
[0032] Definition of enzyme activity: Under the conditions of PBS buffer (100 mM, pH = 7.4) at 30°C, the amount of enzyme required to generate 1 μmol (R)-3-cyanohexanoic acid per minute is one activity unit (1U).
[0033] Enzyme activity recovery rate = enzyme activity of immobilized enzyme / total free enzyme activity used for immobilization * 100%.
[0034] Example 1 The macroporous weakly basic anion exchange resin was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content, and an adsorption reaction was carried out for 2 hours at 30° C. and 130 rpm in a water bath shaker; then a PBS buffer (100 mM, pH = 7.4) containing 10% glutaraldehyde was added, and the mass volume ratio of the carrier, the PBS buffer and the PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL, and an immobilized enzyme reaction was carried out for 1.5 hours at 30° C. and 130 rpm in a water bath shaker, and then the mixture was washed with deionized water 4 times, and dried on a filter paper at room temperature to obtain an immobilized nitrilase, which was stored in a refrigerator at 4° C. for later use.
[0035] Example 2 (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and a n-hexane solution containing hydroxyapatite (average particle size 100 nm) was added and stirred. The concentration of the added hydroxyapatite relative to the volume of n-hexane was 0.06 g / mL, and the concentration of the added carboxyl-terminated polylactic acid-glycolic acid copolymer relative to the mixed volume of dichloromethane and n-hexane was 50 g / L. The mixed volume ratio of dichloromethane to n-hexane was 9:1, and a suspension was obtained.
[0036] (2) After adding a macroporous weakly basic anion exchange resin to the suspension, ultrasonic treatment was performed for 10 minutes, and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer was 10:1; then, a 3 g / L aqueous solution of polyvinyl alcohol was added, and the volume ratio of the suspension to the aqueous solution of polyvinyl alcohol was 1:10, and ultrasonic treatment was performed again for 1 hour.
[0037] (3) The reactant obtained in step (2) is heated to 45° C. and stirred for 4 h at a stirring speed of 1000 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.08 g / mL, stirred at room temperature for 1 h, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.1 g / mL is added, and the reaction is stirred at room temperature for 1 h, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 5 mL: 0.4 mL. After the reaction is completed, the carrier is filtered to obtain the carrier.
[0038] (4) The carrier was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content. The adsorption reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 2 h. Then, a PBS buffer (100 mM, pH = 7.4) containing 10% glutaraldehyde was added, and the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL. The immobilized enzyme reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 1.5 h, and then the mixture was washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a refrigerator at 4° C. for future use.
[0039] Example 3 (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and a n-hexane solution containing hydroxyapatite (average particle size 50 nm) was added and stirred. The amount of hydroxyapatite added relative to the concentration of n-hexane was 0.08 g / mL, the amount of carboxyl-terminated polylactic acid-glycolic acid copolymer added relative to the concentration of the mixed volume of dichloromethane and n-hexane was 45 g / L, and the mixed volume ratio of dichloromethane and n-hexane was 9:1, to obtain a suspension.
[0040] (2) After adding a macroporous weakly basic anion exchange resin to the suspension, ultrasonic treatment was performed for 10 minutes, and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer was 12:1; then, a 2 g / L aqueous solution of polyvinyl alcohol was added, and the volume ratio of the suspension to the aqueous solution of polyvinyl alcohol was 1:10, and ultrasonic treatment was performed again for 2 hours.
[0041] (3) The reactant obtained in step (2) is heated to 45° C. and stirred for 4 hours at a stirring speed of 1500 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.1 g / mL, stirred at room temperature for 2 hours, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.2 g / mL is added, and stirred at room temperature for 2 hours, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 5 mL: 0.5 mL. After the reaction is completed, the carrier is filtered to obtain the carrier.
[0042] (4) The carrier was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content. The adsorption reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 2 h. Then, a PBS buffer (100 mM, pH = 7.4) containing 10% glutaraldehyde was added, and the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL. The immobilized enzyme reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 1.5 h, and then the mixture was washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a refrigerator at 4° C. for future use.
[0043] Example 4 (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and a n-hexane solution containing hydroxyapatite (average particle size 100 nm) was added and stirred. The amount of hydroxyapatite added relative to the concentration of n-hexane was 0.08 g / mL, the amount of carboxyl-terminated polylactic acid-glycolic acid copolymer added relative to the concentration of the mixed volume of dichloromethane and n-hexane was 55 g / L, and the mixed volume ratio of dichloromethane and n-hexane was 8:1, to obtain a suspension.
[0044] (2) After adding macroporous weakly basic anion exchange resin to the suspension, ultrasonic treatment was performed for 10 minutes, and the mass ratio of macroporous weakly basic anion exchange resin to carboxyl-terminated polylactic acid-glycolic acid copolymer was 10:1; then, 3 g / L polyvinyl alcohol aqueous solution was added, and the volume ratio of the suspension to the polyvinyl alcohol aqueous solution was 1:10, and ultrasonic treatment was performed again for 2 hours.
[0045] (3) The reactant obtained in step (2) is heated to 40° C. and stirred for 5 h at a stirring speed of 1000 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.08 g / mL, stirred at room temperature for 1 h, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.2 g / mL is added, and the reaction is stirred at room temperature for 1-2 h, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g:6 mL:0.5 mL. After the reaction is completed, the carrier is filtered to obtain the carrier.
[0046] (4) The carrier was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content. The adsorption reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 2 h. Then, a PBS buffer (100 mM, pH = 7.4) containing 10% glutaraldehyde was added, and the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL. The immobilized enzyme reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 1.5 h, and then the mixture was washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a refrigerator at 4° C. for future use.
[0047] Comparative Example 1 The difference from Example 2 is that hydroxyapatite is not added.
[0048] (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and an n-hexane solution was added and stirred. The concentration of the carboxyl-terminated polylactic acid-glycolic acid copolymer added was 50 g / L relative to the mixed volume of dichloromethane and n-hexane, and the mixed volume ratio of dichloromethane and n-hexane was 9:1 to obtain a suspension.
[0049] (2) After adding a macroporous weakly basic anion exchange resin to the suspension, ultrasonic treatment was performed for 10 minutes, and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer was 10:1; then, a 3 g / L aqueous solution of polyvinyl alcohol was added, and the volume ratio of the suspension to the aqueous solution of polyvinyl alcohol was 1:10, and ultrasonic treatment was performed again for 1 hour.
[0050] (3) The reactant obtained in step (2) is heated to 45° C. and stirred for 4 h at a stirring speed of 1000 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.08 g / mL, stirred at room temperature for 1 h, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.1 g / mL is added, and the reaction is stirred at room temperature for 1 h, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 5 mL: 0.4 mL. After the reaction is completed, the carrier is filtered to obtain the carrier.
[0051] (4) The carrier was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content. The adsorption reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 2 h. Then, a PBS buffer (100 mM, pH = 7.4) containing 10% glutaraldehyde was added, and the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL. The immobilized enzyme reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 1.5 h, and then the mixture was washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a refrigerator at 4° C. for future use.
[0052] Comparative Example 2 The difference from Example 2 is that the concentration of hydroxyapatite in n-hexane is too high.
[0053] (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and a n-hexane solution containing hydroxyapatite (average particle size 100 nm) was added and stirred. The concentration of the added hydroxyapatite relative to the volume of n-hexane was 0.12 g / mL, and the concentration of the added carboxyl-terminated polylactic acid-glycolic acid copolymer relative to the mixed volume of dichloromethane and n-hexane was 50 g / L. The mixed volume ratio of dichloromethane to n-hexane was 9:1, and a suspension was obtained.
[0054] (2) After adding a macroporous weakly basic anion exchange resin to the suspension, ultrasonic treatment was performed for 10 minutes, and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer was 10:1; then, a 3 g / L aqueous solution of polyvinyl alcohol was added, and the volume ratio of the suspension to the aqueous solution of polyvinyl alcohol was 1:10, and ultrasonic treatment was performed again for 1 hour.
[0055] (3) The reactant obtained in step (2) is heated to 45° C. and stirred for 4 h at a stirring speed of 1000 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.08 g / mL, stirred at room temperature for 1 h, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.1 g / mL is added, and the reaction is stirred at room temperature for 1 h, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 5 mL: 0.4 mL. After the reaction is completed, the carrier is filtered to obtain the carrier.
[0056] (4) The carrier was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content. The adsorption reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 2 h. Then, a PBS buffer (100 mM, pH = 7.4) containing 10% glutaraldehyde was added, and the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL. The immobilized enzyme reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 1.5 h, and then the mixture was washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a refrigerator at 4° C. for future use.
[0057] Comparative Example 3 The difference from Example 2 is that no polyvinyl alcohol is added.
[0058] (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and a n-hexane solution containing hydroxyapatite (average particle size 100 nm) was added and stirred. The concentration of the added hydroxyapatite relative to the volume of n-hexane was 0.06 g / mL, and the concentration of the added carboxyl-terminated polylactic acid-glycolic acid copolymer relative to the mixed volume of dichloromethane and n-hexane was 50 g / L. The mixed volume ratio of dichloromethane to n-hexane was 9:1, and a suspension was obtained.
[0059] (2) After adding a macroporous weakly basic anion exchange resin to the suspension, ultrasonic treatment was performed for 10 minutes, and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer was 10:1.
[0060] (3) The reactant obtained in step (2) is heated to 45° C. and stirred for 2 h at a stirring speed of 1000 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.08 g / mL, stirred at room temperature for 1 h, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.1 g / mL is added, and the reaction is stirred at room temperature for 1 h, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 5 mL: 0.4 mL. After the reaction is completed, the carrier is filtered to obtain the carrier.
[0061] (4) The carrier was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content. The adsorption reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 2 h. Then, a PBS buffer (100 mM, pH = 7.4) containing 10% glutaraldehyde was added, and the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL. The immobilized enzyme reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 1.5 h, and then the mixture was washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a refrigerator at 4° C. for future use.
[0062] Comparative Example 4 The difference from Example 2 is that no lysine is added.
[0063] (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and a n-hexane solution containing hydroxyapatite (average particle size 100 nm) was added and stirred. The concentration of the added hydroxyapatite relative to the volume of n-hexane was 0.06 g / mL, and the concentration of the added carboxyl-terminated polylactic acid-glycolic acid copolymer relative to the mixed volume of dichloromethane and n-hexane was 50 g / L. The mixed volume ratio of dichloromethane to n-hexane was 9:1, and a suspension was obtained.
[0064] (2) After adding a macroporous weakly basic anion exchange resin to the suspension, ultrasonic treatment was performed for 10 minutes, and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer was 10:1; then, a 3 g / L aqueous solution of polyvinyl alcohol was added, and the volume ratio of the suspension to the aqueous solution of polyvinyl alcohol was 1:10, and ultrasonic treatment was performed again for 1 hour.
[0065] (3) The reactant obtained in step (2) was heated to 45° C. and stirred for 4 h at a stirring speed of 1000 rpm to evaporate the organic solvent, and then filtered to obtain the carrier.
[0066] (4) The carrier was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content. The adsorption reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 2 h. Then, a PBS buffer (100 mM, pH = 7.4) containing 10% glutaraldehyde was added, and the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL. The immobilized enzyme reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 1.5 h, and then the mixture was washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a refrigerator at 4° C. for future use.
[0067] Comparative Example 5 The difference from Example 2 is that the concentration of glutaraldehyde is too high.
[0068] (1) A carboxyl-terminated polylactic acid-glycolic acid copolymer (average molecular weight 50 kDa) was dissolved in dichloromethane, and a n-hexane solution containing hydroxyapatite (average particle size 100 nm) was added and stirred. The concentration of the added hydroxyapatite relative to the volume of n-hexane was 0.06 g / mL, and the concentration of the added carboxyl-terminated polylactic acid-glycolic acid copolymer relative to the mixed volume of dichloromethane and n-hexane was 50 g / L. The mixed volume ratio of dichloromethane to n-hexane was 9:1, and a suspension was obtained.
[0069] (2) After adding a macroporous weakly basic anion exchange resin to the suspension, ultrasonic treatment was performed for 10 minutes, and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer was 10:1; then, a 3 g / L aqueous solution of polyvinyl alcohol was added, and the volume ratio of the suspension to the aqueous solution of polyvinyl alcohol was 1:10, and ultrasonic treatment was performed again for 1 hour.
[0070] (3) The reactant obtained in step (2) is heated to 45° C. and stirred for 4 h at a stirring speed of 1000 rpm to evaporate the organic solvent, and then filtered; the obtained product is added to a lysine solution with a concentration of 0.08 g / mL, stirred at room temperature for 1 h, and then a dicyclohexylcarbodiimide ethanol solution with a concentration of 0.1 g / mL is added, and the reaction is stirred at room temperature for 1 h, wherein the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 5 mL: 0.4 mL. After the reaction is completed, the carrier is filtered to obtain the carrier.
[0071] (4) The carrier was added to a PBS buffer (100 mM, pH = 7.4), and then a pure enzyme solution containing 1 mg / mL nitrilase in terms of protein content was added, and the amount of nitrilase used was 18 mg / g resin in terms of protein content, and the adsorption reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 2 h; then a PBS buffer (100 mM, pH = 7.4) containing 20% glutaraldehyde was added, and the mass volume ratio of the carrier, PBS buffer and PBS buffer containing glutaraldehyde was 1 g: 10 mL: 5 mL, and the immobilized enzyme reaction was carried out in a water bath shaker at 30° C. and 130 rpm for 1.5 h, and then washed with deionized water 4 times, placed on filter paper and dried at room temperature to obtain immobilized nitrilase, which was stored in a refrigerator at 4° C. for future use.
[0072] Table 1 As shown in Table 1, the comparison between Example 1 and Example 2-4 shows that the present invention can not only ensure the firmness of the combination between the carrier and the enzyme, but also obtain an immobilized nitrilase with higher activity by modifying the macroporous resin. The carrier prepared in Comparative Example 1 does not contain hydroxyapatite, which will cause the immobilization effect of nitrilase to deteriorate and the structural stability of immobilized nitrilase (surface protrusion structure of macroporous resin) to deteriorate, thereby causing a lower substrate conversion rate. The concentration of hydroxyapatite in n-hexane in Comparative Example 2 is relatively large, which will cause the dispersibility of hydroxyapatite to be affected, and will also affect the problems such as pore size distribution and structural stability, thereby causing a lower substrate conversion rate and enzyme activity. In Comparative Example 3, the carrier is prepared without adding polyvinyl alcohol, which will seriously affect the structural stability of immobilized nitrilase (surface protrusion structure of macroporous resin), thereby causing a low substrate conversion rate and a deterioration of enzyme activity. In Comparative Example 4, the carrier prepared does not add lysine, which will cause the immobilization effect of nitrilase to deteriorate, thereby causing a lower substrate conversion rate and enzyme activity. In Comparative Example 5, the concentration of glutaraldehyde added is too high, which is not conducive to improving the protein adsorption rate, and affects the enzyme activity, while resulting in a low substrate conversion rate.
[0073] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the specification of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.
Claims
1. A method for preparing immobilized nitrilase for the synthesis of brivaracetam, characterized in that: The steps include: (1) dissolving a carboxyl-terminated poly(lactic acid-glycolic acid) copolymer in dichloromethane, adding a n-hexane solution containing hydroxyapatite and stirring to obtain a suspension; (2) adding a macroporous weakly basic anion exchange resin to the suspension and then sonicating, and then adding an aqueous solution of polyvinyl alcohol and sonicating again; (3) heating to volatilize the organic solvent, filtering, adding the obtained product to a lysine solution, then adding a dicyclohexylcarbodiimide ethanol solution, stirring to react, and filtering to obtain a carrier; (4) The carrier is added to a buffer solution, and then nitrilase is added for adsorption; and then glutaraldehyde solution is added for immobilized enzyme reaction to obtain immobilized nitrilase.
2. The method for preparing immobilized nitrilase for brivaracetam synthesis according to claim 1, characterized in that: In step (1), the n-hexane solution containing hydroxyapatite is prepared by dispersing hydroxyapatite powder in n-hexane, and the amount of hydroxyapatite added relative to the concentration of n-hexane is 0.05-0.08 g / mL; the particle size of the hydroxyapatite is 50-100 nm.
3. The method for preparing the immobilized nitrilase for brivaracetam synthesis according to claim 1 or 2, characterized in that: In step (1), the concentration of the carboxyl-terminated polylactic acid-glycolic acid copolymer added relative to the mixed volume of dichloromethane and n-hexane is 40-60 g / L; the mixed volume ratio of dichloromethane and n-hexane is 8-9:
1.
4. The method for preparing immobilized nitrilase for brivaracetam synthesis according to claim 1, characterized in that: In step (2), the macroporous weakly basic anion exchange resin is a macroporous weakly basic acrylic acid anion exchange resin; and the mass ratio of the macroporous weakly basic anion exchange resin to the carboxyl-terminated polylactic acid-glycolic acid copolymer is 10-12:
1.
5. The method for preparing immobilized nitrilase for brivaracetam synthesis according to claim 1, characterized in that: In step (2), the volume ratio of the suspension to the aqueous solution of polyvinyl alcohol is 1:10; the concentration of the aqueous solution of polyvinyl alcohol is 2-4 g / L.
6. The method for preparing immobilized nitrilase for brivaracetam synthesis according to claim 1, 4 or 5, characterized in that: In step (2), the ultrasonic treatment time is 5-10 minutes; and the second ultrasonic treatment time is 1-2 hours.
7. The method for preparing immobilized nitrilase for brivaracetam synthesis according to claim 1, characterized in that: In step (3), the mass volume ratio of the obtained product to the lysine solution and the dicyclohexylcarbodiimide ethanol solution is 1 g: 3-6 mL: 0.4-0.5 mL; the concentration of the lysine solution is 0.05-0.1 g / mL; the concentration of the dicyclohexylcarbodiimide ethanol solution is 0.1-0.2 g / mL.
8. The method for preparing immobilized nitrilase for brivaracetam synthesis according to claim 1 or 7, characterized in that: In step (3), the heating to volatilize the organic solvent is performed by heating to 40-50° C. and stirring for 3-5 hours at a stirring speed of 1000-1500 rpm; and the stirring reaction is performed by stirring at room temperature for 1-2 hours.
9. The method for preparing immobilized nitrilase for brivaracetam synthesis according to claim 1, characterized in that: In step (4), the mass volume ratio of the carrier, the buffer solution and the glutaraldehyde solution is 1 g:10 mL:1-5 mL; and the mass concentration of glutaraldehyde in the glutaraldehyde solution is 10-15%.
10. The method for preparing immobilized nitrilase for brivaracetam synthesis according to claim 1 or 9, characterized in that: In step (4), the amount of the nitrilase used is 16-20 mg / g resin in terms of protein content; and the immobilized enzyme reaction is stirred at 30-40° C. for 1-3 hours.