An enzyme and cofactor co-immobilized millimeter ball catalyst and its preparation and application

Abstract

The invention belongs to the field of catalytic material preparation, and specifically relates to a millimeter ball catalyst with enzyme and cofactor co-immobilized, and its preparation and application. The millimeter ball particle size is 1.0-5.0 mm, the enzyme content is 7.9-72.1 mg / g, the cofactor content is 9.9-39.5 mg / g, and the mechanical strength is 3-12 N. The preparation process is to add solid nanoparticles, biological enzymes, and cofactors to an aqueous solution of amines and stir them evenly to form a mixed solution, and then continuously drip the mixed solution on a layer of hydrophobic nano silicon oxide powder spread on a flat surface, rolling to form droplet millimeter balls with silicon oxide shells; collect the droplet millimeter balls and slowly dry them, and finally obtain the millimeter balls with enzyme and cofactor co-immobilized. The millimeter balls with enzyme and cofactor co-immobilized prepared by the method can be directly loaded into a fixed bed reactor for continuous flow enzyme catalysis to prepare chiral amines, and the continuous reaction lasts for more than 1500 hours, and the reaction conversion rate remains basically unchanged, and the enzyme activity remains basically unchanged, and the cofactor is also basically not lost.

Description

Technical Field

[0001] The invention belongs to the field of catalytic material preparation, and in particular relates to a millimeter ball catalyst with enzyme and cofactor co-immobilized thereon, and the preparation and application thereof. Background Art

[0002] Due to the advantages of high enzyme activity, high chiral selectivity and mild reaction conditions, enzyme-catalyzed synthesis has become one of the most promising synthetic methods for preparing chiral amine drug intermediates. At present, enzyme immobilization is mainly carried out by physical adsorption and covalent bonding. The disadvantage of physical adsorption is that weak binding force can easily lead to enzyme loss, while chemical bonding has strong binding force but will reduce enzyme activity and stability. Cofactors are important components for enzymes to maintain catalytic activity. Transaminases need to bind cofactors to synergistically catalyze the preparation of chiral amines. Traditionally, cofactors are fixed mainly by entering the fixed bed from the continuous phase or by adsorption of solid materials. This method is prone to cofactor loss and loss, and it is difficult to achieve effective recycling of cofactors. At present, the carriers for immobilizing enzymes and cofactors are mainly submicron particles with small particle size and low mechanical strength. Directly filling these materials into fixed bed reactors is prone to high bed pressure drop, bed blockage and other problems, making it difficult to achieve continuous reactions. Summary of the invention

[0003] The present invention aims to solve the problems in the prior art of reduced activity of immobilized transaminase, loss of immobilized transaminase during circulation, inability to efficiently regenerate cofactors, and difficulty in continuous and industrial production of catalysts of immobilized transaminase and cofactors. The present invention provides a method for preparing a millimeter-sphere catalyst in which enzyme and cofactor are co-immobilized. The catalyst can be directly loaded into a fixed bed reactor for continuous flow enzyme catalysis to prepare chiral amines.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] On one hand, the present invention provides an enzyme and cofactor co-immobilized millimeter ball catalyst, wherein the enzyme and cofactor co-immobilized millimeter ball particle size is 1.0-5.0 mm, the enzyme content is 7.9-72.1 mg / g, the cofactor content is 9.9-39.5 mg / g, and the mechanical strength is 3-12N.

[0006] Another aspect of the present invention provides a method for preparing a millimeter-sphere catalyst in which an enzyme and a cofactor are co-immobilized, comprising the following steps:

[0007] Step 1, adding solid nanoparticles, biological enzymes, cofactors and amines into deionized water, and magnetically stirring to obtain a mixed solution;

[0008] Step 2, adding the above mixed solution into a syringe, and continuously dropping the mixed solution onto a layer of hydrophobic silicon oxide powder spread on a flat surface through a syringe pump, and the droplets roll under the action of a shaker to form droplet millimeter balls with a silicon oxide hydrophobic shell layer;

[0009] Step 3, placing the above-mentioned droplet millimeter ball into a constant temperature shaker, and slowly drying it during rolling to obtain the millimeter ball catalyst with enzyme and cofactor co-immobilized.

[0010] The enzyme and cofactor co-immobilized millimeter ball catalyst prepared by the method, the chemical bonding between the amine and the cofactor, between the amine and the solid nanoparticles, and between the enzyme and the solid nanoparticles encapsulated inside the millimeter ball, can not only effectively fix the enzyme and the cofactor, but also enhance the mechanical strength of the millimeter ball. At the same time, the amine can form a network structure between the solid nanoparticles, which can ensure the effective contact between the cofactor and the enzyme to improve the catalytic activity of the enzyme. The entire preparation process is mild, and the enzyme and cofactor can be immobilized in situ. While retaining the initial activity of the enzyme, there will be no obvious loss of the cofactor in the immobilization process. The hydrophobic silicon oxide shell on the surface of the millimeter ball can not only effectively reduce the contact between the hydrophilic substance and the cofactor, effectively reduce the loss of the cofactor, but also help to increase the contact between the organic reaction substrate and the enzyme and the cofactor, thereby increasing the reaction rate.

[0011] Furthermore, in step 1, the dosage ratio of the solid nanoparticles, the biological enzyme, the cofactor, the amine and the deionized water is 0.1-0.4 g: 5-40 mg: 2-15 mg: 5-100 mg: 1-3 mL.

[0012] Furthermore, in step 1, the solid nanoparticles are any one of mesoporous alumina, mesoporous silica, solid spherical alumina, solid spherical silica or hollow silica, or a mixture of two of them in any ratio; the biological enzyme is one or more of transaminase ATA-101 to ATA-165 and their mutants; the cofactor is pyridoxal phosphate (PLP), nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADP + ) any one of; the amine is any one of polyethyleneimine, polypropyleneamine, pentaethylenehexamine or polyethylene glycol diamine.

[0013] Furthermore, in step 2, the flat surface is any one of a glass surface, a ceramic surface, a cardboard surface, a plastic surface, and a metal surface.

[0014] Furthermore, in step 2, the inner diameter of the needle of the syringe is 0.1 to 3.0 mm, and the flow rate is 0.05 to 1.0 mL / min.

[0015] Furthermore, the hydrophobic silica powder in step 2 includes any one of Wacker H18 hydrophobic fumed silica, Evonik Degussa R974 hydrophobic fumed silica or homemade hydrophobic mesoporous silica; the thickness of the hydrophobic silica powder is 1.0 to 3.0 mm; the homemade hydrophobic mesoporous silica is obtained by hydrophilic mesoporous silica being grafted and modified with hydrophobic silane.

[0016] Furthermore, the temperature of the slow drying in step 3 is 20 to 40° C. and the time is 4 to 12 hours.

[0017] On the other hand, the present invention provides the application of the above-mentioned millimeter-sphere catalyst co-immobilized with the enzyme and cofactor, wherein the millimeter-sphere catalyst is directly loaded into a fixed bed reactor, the reactant solution is transported from the upper end of the fixed bed by a constant flow pump, and the coenzyme regeneration product is added to the reactant solution, and the product is collected from the lower end of the fixed bed.

[0018] Further, the coenzyme regenerator is any one of isopropylamine, α-phenylethylamine or 2-octylamine

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The enzyme and cofactor co-immobilized millimeter ball catalyst prepared by the present invention realizes the in-situ immobilization of biological enzymes and cofactors. The enzymes and cofactors are distributed in the network structure formed by the internal nanoparticles and amines. The strong chemical bonding between the amines and the cofactors can not only adsorb the cofactors, but also ensure that the cofactors have sufficient degrees of freedom to participate in the enzyme catalysis process; the solid nanoparticles are tightly combined with the amines and enzymes through non-covalent interactions, which enhances the stability of the millimeter ball skeleton structure, and obtains a millimeter ball catalyst with a mechanical strength of up to 3 to 12N;

[0021] 2. The enzyme and cofactor co-immobilized catalyst prepared by the present invention is a microsphere with a particle size of millimeters, which can be directly filled into a fixed bed reactor for continuous flow enzyme catalysis without post-treatment;

[0022] 3. The enzyme and cofactor co-immobilized millimeter ball catalyst prepared by the present invention has a simple preparation method and mild conditions, does not affect the initial activity of the enzyme during the preparation process, and does not significantly lose the cofactor during the immobilization process;

[0023] 4. The enzyme and cofactor co-immobilized millimeter ball catalyst prepared by the present invention has a hydrophobic silicon oxide shell layer on the surface that can effectively reduce the loss of cofactors, effectively ensure the distribution of reaction substrates and the diffusion of products, and achieve efficient mass transfer;

[0024] 5. The enzyme and cofactor co-immobilized millimeter ball catalyst prepared by the present invention can be directly loaded into a fixed bed reactor for continuous synthesis of chiral amine products. The continuous reaction can last for more than 1500 hours, and the reaction conversion rate remains basically unchanged, the transaminase activity remains basically unchanged, and there is basically no loss of cofactors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the structure of the millimeter ball in which the enzyme and the cofactor are co-immobilized in Example 1 of the present invention;

[0026] Figure 2 The scanning electron microscope image of the cross section and overall morphology of the millimeter ball on which the enzyme and the cofactor are co-immobilized in Example 1 of the present invention;

[0027] Figure 3 This is a particle size diagram of the millimeter spheres on which the enzyme and cofactor are co-immobilized in Example 1 of the present invention;

[0028] Figure 4 This is a particle size diagram of the millimeter spheres on which the enzyme and cofactor are co-immobilized in Example 2 of the present invention;

[0029] Figure 5 This is a cross-sectional view of the millimeter ball in which the enzyme and cofactor are co-immobilized in Example 2 of the present invention.

[0030] Figure 6 This is a particle size diagram of the millimeter spheres on which the enzyme and cofactor are co-immobilized in Example 3 of the present invention;

[0031] Figure 7 This is a particle size diagram of the millimeter spheres on which the enzyme and cofactor are co-immobilized in Example 4 of the present invention;

[0032] Figure 8 This is a diagram showing the particle size of the millimeter-sized spheres in which the enzyme and cofactor are co-immobilized in Example 5 of the present invention. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0034] Example 1

[0035] like Figure 1 As shown, a method for preparing a millimeter ball catalyst co-immobilized with an enzyme and a cofactor comprises the following steps:

[0036] (1) Weigh 0.4 g of mesoporous silica nanoparticles, 0.1 g of polyethyleneimine, 600 uL of transaminase ATA-117 solution (40 mg of enzyme), and 10 mg of PLP into 1400 uL of deionized water, and stir to obtain a mixed solution.

[0037] (2) The mixed solution was added to a syringe with a needle inner diameter of 1.0 mm. The mixed solution was continuously dropped onto R974 hydrophobic silica powder with a thickness of 3.0 mm spread on the surface of a glass culture dish at a flow rate of 0.5 mL / min through a syringe pump. The droplets rolled slowly under the action of a shaker to form droplet millimeter balls with a silica hydrophobic shell.

[0038] (3) Collect the droplet millimeter balls, and then put them into a constant temperature shaker to slowly dry them during the rolling process. The drying temperature is controlled at 20°C. During the drying process, the droplets gradually shrink and the mechanical strength gradually increases through the interaction between the amine and the silanol group on the surface of the silicon oxide, and the interaction between the enzyme and the silicon oxide particles. After 12 hours, the millimeter balls with a mechanical strength of 6N are obtained. Figure 2 The cross-section and overall morphology of the prepared millimeter ball are shown in the scanning electron microscope image. It can be seen from the image that the prepared millimeter ball is a spherical particle with a smooth surface and a mesoporous-macroporous structure formed by the accumulation of silicon oxide nanoparticles of about 80nm in the interior. Figure 3 This is the particle size diagram of the prepared millimeter spheres. From the figure, it can be seen that millimeter spheres with a size of about 2 mm were prepared.

[0039] Example 2

[0040] A method for preparing a millimeter ball catalyst co-immobilized with an enzyme and a cofactor comprises the following steps:

[0041] (1) Weigh 0.4 g of solid spherical alumina nanoparticles, 0.1 g of polyethyleneimine, 600 uL of transaminase ATA-119 solution (20 mg of enzyme), and 15 mg of PLP into 2400 uL of deionized water, and stir to obtain a mixed solution.

[0042] (2) The mixed solution was added to a syringe with a needle inner diameter of 2 mm. The mixed solution was continuously dropped onto H18 hydrophobic silica powder with a thickness of 3.0 mm spread on the surface of a flat-bottomed ceramic evaporating dish at a flow rate of 0.8 mL / min through a syringe pump. The droplets rolled slowly under the action of a shaker to form droplet millimeter balls with a silica hydrophobic shell.

[0043] (3) Collect the droplet millimeter balls, and then put them into a constant temperature shaker to slowly dry them during the rolling process. The drying temperature is controlled at 25°C. During the drying process, the droplets gradually shrink and the mechanical strength gradually increases through the interaction between the amine and the hydroxyl groups on the surface of the alumina, and the interaction between the enzyme and the alumina particles. After 12 hours, the millimeter balls with a mechanical strength of 12N are obtained. Figure 4 is the particle size diagram of the prepared millimeter spheres, Figure 5 This is a cross-sectional view of the prepared millimeter ball. It can be seen from the figure that the millimeter ball is a spherical particle with a size of 3.5 mm, and its interior is a mesoporous-macroporous structure formed by the accumulation of nano-alumina particles of about 20 nm.

[0044] Example 3

[0045] A method for preparing a millimeter ball catalyst co-immobilized with an enzyme and a cofactor comprises the following steps:

[0046] (1) Weigh 0.2 g hollow silica nanoparticles, 0.1 g polyethyleneimine, 400 uL transaminase ATA-113 solution (20 mg enzyme), 10 mg NADP + Add into 600uL of deionized water and stir evenly to obtain a mixed solution.

[0047] (2) The mixed solution was added to a syringe with a needle inner diameter of 1.2 mm. The mixed solution was continuously dropped onto a 2.0 mm thick homemade hydrophobic mesoporous silica powder (made by hydrophilic mesoporous silica grafted with hydrophobic silane) spread on the surface of a polytetrafluoroethylene evaporating dish at a flow rate of 0.5 mL / min through a syringe pump. The droplets rolled slowly under the action of a shaker to form droplet millimeter balls with a hydrophobic silica shell.

[0048] (3) Collect the droplet millimeter balls, and then put them into a constant temperature shaker to slowly dry them during the rolling process. The drying temperature is controlled at 30°C. During the drying process, the droplets gradually shrink and the mechanical strength gradually increases through the interaction between the amine and the silanol group on the surface of the silicon oxide, and the interaction between the enzyme and the silicon oxide particles. After 8 hours, the millimeter balls with a mechanical strength of 7N are obtained. Figure 6 Figure 2 is the particle size diagram of the prepared millimeter spheres.

[0049] Example 4

[0050] A method for preparing a millimeter ball catalyst co-immobilized with an enzyme and a cofactor comprises the following steps:

[0051] (1) Weigh 0.2 g of solid spherical silica nanoparticles, 0.05 g of polyacrylamide, 300 uL of transaminase ATA-142 solution (10 mg of enzyme), and 10 mg of PLP into 1700 uL of deionized water, and stir to obtain a mixed solution.

[0052] (2) The mixed solution was added to a syringe with a needle inner diameter of 1.5 mm. The mixed solution was continuously dropped onto R974 hydrophobic silica powder with a thickness of 3.0 mm spread on a flat stainless steel disc at a flow rate of 0.6 mL / min through a syringe pump. The droplets rolled slowly under the action of a shaker to form droplet millimeter balls with a silica hydrophobic shell.

[0053] (3) Collect the droplet millimeter balls, and then put them into a constant temperature shaker to slowly dry them during the rolling process. The drying temperature is controlled at 40°C. During the drying process, the droplets gradually shrink and the mechanical strength gradually increases through the interaction between the amine and the silanol group on the surface of the silicon oxide, and the interaction between the enzyme and the silicon oxide particles. After 4 hours, the millimeter balls with a mechanical strength of 9N are obtained. Figure 7 Figure 2 is the particle size diagram of the prepared millimeter spheres.

[0054] Example 5

[0055] A method for preparing a millimeter ball catalyst co-immobilized with an enzyme and a cofactor comprises the following steps:

[0056] (1) Weigh 0.15 g of mesoporous silica nanoparticles, 0.15 g of solid spherical silica nanoparticles, 0.05 g of polyethylene glycol diamine, 200 uL of transaminase ATA-142 solution (15 mg of enzyme), and 15 mg of PLP, add them to 1300 uL of deionized water, and stir evenly to prepare a mixed solution.

[0057] (2) The mixed solution was added to a syringe with a needle inner diameter of 2.6 mm. The mixed solution was continuously dropped onto R974 hydrophobic silica powder with a thickness of 3.0 mm spread on the surface of coated paper at a flow rate of 1 mL / min through a syringe pump. The droplets rolled slowly under the action of a shaker to form droplet millimeter balls with a silica hydrophobic shell.

[0058] (3) Collect the droplet millimeter balls, and then put them into a constant temperature shaker to slowly dry them during the rolling process. The drying temperature is controlled at 25°C. During the drying process, the droplets gradually shrink and the mechanical strength gradually increases through the interaction between amine and silanol on the surface of silica, and the interaction between enzyme and silica particles. After 8 hours, the millimeter balls with a mechanical strength of 10N are obtained. Figure 8 Figure 2 is the particle size diagram of the prepared millimeter spheres.

[0059] Example 6

[0060] 800 mg of the millimeter ball catalyst prepared in Example 1 was directly loaded into a fixed bed reactor with an inner diameter of 1.34 cm. The reactor temperature was set to 45° C. A toluene solution with a concentration of 0.1 mol / L p-nitroacetophenone and 0.2 mol / L α-phenylethylamine was introduced into the reactor at a flow rate of 0.6 mL / h through a constant flow pump. The reaction was continued for 1500 h, the conversion rate was maintained above 90%, and the ee value of the generated chiral amine was greater than 99%.

[0061] Example 7

[0062] 900 mg of the millimeter ball catalyst prepared in Example 2 was directly loaded into a fixed bed reactor with an inner diameter of 1.34 cm. The reactor temperature was set to 50° C. A toluene solution with a concentration of 0.1 mol / L p-methoxyacetophenone and 0.4 mol / L 2-octylamine was introduced into the reactor at a flow rate of 0.8 mL / h through a constant flow pump. The reaction was continued for 500 h, the conversion rate was maintained above 85%, and the ee value of the generated chiral amine was greater than 99%.

[0063] Example 8

[0064] 900 mg of the millimeter ball catalyst prepared in Example 4 was directly loaded into a fixed bed reactor with an inner diameter of 1.34 cm. The reactor temperature was set to 45°C, and a methyl tert-butyl ether solution with a concentration of 0.1 mol / L methyl benzoylformate and 0.2 mol / L isopropylamine was introduced at a flow rate of 0.5 mL / h through a constant flow pump to carry out the reaction. The reaction was continuously carried out for 500 h, the conversion rate was maintained above 90%, and the ee value of the generated chiral amine was greater than 99%.

[0065] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0066] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a millimeter-sphere catalyst in which an enzyme and a cofactor are co-immobilized, characterized in that: The following steps are involved: Step 1, adding solid nanoparticles, biological enzymes, cofactors and amines into deionized water, and magnetically stirring to obtain a mixed solution; Step 2, adding the above mixed solution into a syringe, and continuously dropping the mixed solution onto a layer of hydrophobic silicon oxide powder spread on a flat surface through a syringe pump, and the droplets roll under the action of a shaker to form droplet millimeter balls with a silicon oxide hydrophobic shell layer; Step 3, placing the above-mentioned droplet millimeter ball into a constant temperature shaker, and slowly drying it during rolling to obtain the millimeter ball catalyst with enzyme and cofactor co-immobilized; In the step 1, the dosage ratio of the solid nanoparticles, the biological enzyme, the cofactor, the amine and the deionized water is 0.1-0.4 g: 5-40 mg: 2-15 mg: 5-100 mg: 1-3 mL; In step 1, the solid nanoparticles are any one of mesoporous alumina, mesoporous silica, solid spherical alumina, solid spherical silica, and hollow silica, or a mixture of two of them in any ratio; the biological enzyme is one or more of transaminase ATA-101 to ATA-165 and their mutants; the cofactor is any one of pyridoxal phosphate or nicotinamide adenine dinucleotide phosphate; and the amine is any one of polyethyleneimine, polyacrylamide, and polyethylene glycol diamine.

2. The method for preparing a millimeter-sphere catalyst with enzyme and cofactor co-immobilized according to claim 1, characterized in that: In step 2, the flat surface is any one of a glass surface, a ceramic surface, a cardboard surface, a plastic surface, and a metal surface.

3. The method for preparing a millimeter-sphere catalyst with enzyme and cofactor co-immobilized according to claim 1, characterized in that: In step 2, the inner diameter of the needle of the syringe is 0.1-3.0 mm, and the flow rate is 0.05-1.0 mL / min.

4. The method for preparing a millimeter-sphere catalyst with enzyme and cofactor co-immobilized according to claim 1, characterized in that: The hydrophobic silicon oxide powder in step 2 includes any one of hydrophobic gas-phase silicon oxide or self-made hydrophobic mesoporous silicon oxide; the thickness of the hydrophobic silicon oxide powder is 1.0-3.0 mm; the self-made hydrophobic mesoporous silicon oxide is obtained by hydrophilic mesoporous silicon oxide being modified by hydrophobic silane grafting.

5. The method for preparing a millimeter-sphere catalyst with enzyme and cofactor co-immobilized according to claim 1, characterized in that: The temperature of the slow drying in step 3 is 20-40° C. and the time is 4-12 hours.

6. An application of the enzyme and cofactor co-immobilized millimeter ball catalyst prepared by the preparation method according to claim 1, characterized in that: The millimeter ball catalyst is directly loaded into a fixed bed reactor, a reactant solution is transported from the upper end of the fixed bed by a constant flow pump, a coenzyme regenerator is added to the reactant solution, and the product is collected from the lower end of the fixed bed.

7. Use of a millimeter-sphere catalyst with enzyme and cofactor co-immobilized obtained by the preparation method according to claim 6, characterized in that: The coenzyme regenerator is any one of isopropylamine, α-phenylethylamine or 2-octylamine.

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