Immobilized enzymes and their use in continuous production

Abstract

The present invention provides an immobilized enzyme and its use in continuous production. The immobilized enzyme is a polyethyleneimine-modified immobilized enzyme, which includes an enzyme including amine dehydrogenase and / or formate dehydrogenase, and a support which is an amino-based support activated with cyanuric chloride. The immobilized enzyme solves the problem of the poor performance of the immobilized enzyme in the prior art, improves the catalytic activity and reusability of the immobilized enzyme, and is suitable for the field of enzyme immobilization.

Description

[Technical field]

[0001] The present invention relates to the field of enzyme immobilization, and in particular to immobilized enzymes and their use in continuous production. [Background technology]

[0002] Chiral amines are important intermediates for the synthesis of chiral drugs, agrochemicals, and fine chemicals. In recent years, amine dehydrogenases (AmDHs) have been increasingly used to avoid many chemical synthesis processes for producing key compounds and meet the requirements of green chemistry. Amine dehydrogenase refers to enzymes that can catalyze the reduction of ketone-based compounds to chiral amines. Unlike transaminases, amine dehydrogenases use inexpensive ammonium salts as ammonia sources and do not rely on expensive ammonia donors such as isopropylamine or alanine. Since reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotinamide adenine dinucleotide (NADH) is usually required for the reduction process to transfer hydrogen, amine dehydrogenases are often combined with formate dehydrogenase (FDH) to reduce production costs. Compared with chemical catalytic synthesis, amine dehydrogenases not only have milder reaction conditions, but also have higher catalytic activity, selectivity, and substrate specificity. However, water-soluble free enzymes face many problems in their production and application, such as poor stability, lack of reusability, and problems in product separation and purification.

[0003] The main task of enzyme immobilization is to select an appropriate immobilization method to design a biocatalyst that meets both the catalytic (selectivity, stability) and non-catalytic (cost, separation process) needs of an application. Currently, covalent immobilization of enzymes is widely used in production applications, and the commercially available supports used include amino-based and epoxy-based supports. Taking amino-based resins as an example, they usually need to be activated by a bifunctional crosslinker and then linked to the enzyme through the terminal functional group of the crosslinker. The most commonly used activation method is to activate the amino with glutaraldehyde, and then use the active aldehyde group to link the enzyme molecule to achieve immobilization. There have also been reports that after immobilizing the enzyme with glutaraldehyde, the stability of the immobilized enzyme can be improved by post-modification with amino acids or small molecule compounds such as NaBH4.

[0004] The present inventors have studied the immobilization of amine dehydrogenase and found that the immobilization of amine dehydrogenase on the conventional glutaraldehyde-activated amino- or epoxy-based supports is not very successful. The immobilized enzyme obtained has the disadvantages of low catalytic activity and poor reusability. Adding a protective agent such as mannitol during the immobilization process or post-modifying the immobilized enzyme obtained with small molecules cannot overcome the above-mentioned disadvantages. Summary of the Invention [Problem to be solved by the invention]

[0005] The main object of the present invention is to provide an immobilized enzyme and its use in continuous production to improve the catalytic activity and reusability of the immobilized enzyme. [Means for solving the problem]

[0006] In order to achieve the above object, according to a first aspect of the present invention, there is provided an immobilized enzyme, comprising: a polyethyleneimine-modified immobilized enzyme, the enzyme comprising amine dehydrogenase and / or formate dehydrogenase; and a support for immobilizing the enzyme, the support being an amino-based support activated with cyanuric chloride.

[0007] Furthermore, the amine dehydrogenase includes an amine dehydrogenase as shown in the sequence of SEQ ID NO: 1, preferably the formate dehydrogenase includes a formate dehydrogenase as shown in the sequence of SEQ ID NO: 2, preferably the amino carrier includes ECR8409, LX-1000HA or LX-1000EPHA, and the polyethyleneimine preferably has a molecular weight of 1.8 to 10 KDa.

[0008] In order to achieve the above object, according to a second aspect of the present invention, there is provided a method for producing an immobilized enzyme, comprising the steps of activating an amino-based support with cyanuric chloride to obtain an activated support, mixing an enzyme with the activated support, incubating and immobilizing the enzyme, and then modifying the immobilized product with polyethyleneimine to obtain an immobilized enzyme, wherein the enzyme includes amine dehydrogenase and / or formate dehydrogenase.

[0009] Further, the step of activating the amino support with cyanuric chloride to obtain an activated support includes a step of dispersing the amino support in an organic solvent to obtain a support suspension, and a step of activating the amino support by reacting the support suspension with cyanuric chloride to obtain an activated support, preferably the organic solvent is a polar organic solvent, preferably the polar organic solvent comprises any one or more of tetrahydrofuran, acetone, dimethylformamide or dimethylsulfoxide, preferably the activation temperature is 0 to 30°C, more preferably 0 to 5°C, preferably the activation time is 2 to 5h, preferably after activation, the activated support is washed with an organic solvent to remove unreacted cyanuric chloride, preferably after washing, the organic solvent on the activated support is blown dry with nitrogen gas, preferably the amino support is a dry support, preferably the amino support comprises ECR8409, LX-1000HA or LX-1000EPHA.

[0010] Furthermore, before mixing the enzyme with the activating carrier and incubating, the production method further includes a step of replacing the organic solvent by washing the activating carrier with a buffer solution, preferably the enzyme is prepared using a buffer solution, preferably the buffer solution includes a phosphate buffer solution, a glycine buffer solution or a borax buffer solution, preferably the concentration of the phosphate buffer solution is 20-50 mM, preferably the pH of the phosphate buffer solution is 6.5-9.0, preferably the immobilization temperature is 0-30°C, more preferably 4-30°C, and preferably the immobilization time is 2-5 hours.

[0011] Further, the step of mixing the enzyme with an activating carrier, incubating and immobilizing, and then modifying the immobilized product with polyethyleneimine to obtain an immobilized enzyme includes the steps of mixing the enzyme with an activating carrier, incubating and immobilizing, to obtain a pre-immobilized enzyme, and post-modifying the pre-immobilized enzyme with polyethyleneimine to obtain an immobilized enzyme, and preferably the enzyme includes amine dehydrogenase and / or formate dehydrogenase, preferably the amine dehydrogenase includes an amine dehydrogenase as shown in the sequence of SEQ ID NO: 1, and preferably the formate dehydrogenase includes an amine dehydrogenase as shown in the sequence of SEQ ID NO: The enzyme comprises formate dehydrogenase as shown in sequence No. 2, and preferably the post-modification is carried out by adding a solution of polyethyleneimine to the pre-immobilized enzyme, preferably the polyethyleneimine comprises polyethyleneimine, preferably the molecular weight of the polyethyleneimine is 1.8 to 10 KDa, preferably the pH of the polyethyleneimine solution is 5 to 8, preferably the final concentration of the polyethyleneimine added is 0.5 to 3% W / V, preferably the temperature of the post-modification is 0 to 30°C, more preferably 10 to 30°C, and preferably the time of post-modification is 8 to 24 hours.

[0012] In order to achieve the above object, according to a third aspect of the present invention, there is provided use of the above immobilized enzyme, or the immobilized enzyme produced by the above production method, in a continuous reaction.

[0013] Further, the continuous reaction includes carrying out the continuous reaction in a packed bed reactor, preferably the packed bed reactor is manufactured by packing the immobilized enzyme into an empty chromatography column or a stainless steel chromatography column, preferably the volume of the packed bed reactor is 5-50 mL, and preferably the packing method of the packed bed reactor is wet packing.

[0014] Furthermore, the continuous reaction includes pumping the reaction system containing the substrate from bottom to top into a packed bed reactor and remixing the reaction product into the reaction system, the pump is a constant flow pump, preferably the pressure of the constant flow pump is ≦20 MPa, and preferably the flow rate of the continuous reaction is 0.01-10 mL / min.

[0015] Further, the enzyme is a main enzyme or a mixture of a main enzyme and a coenzyme, preferably the main enzyme is selected from amine dehydrogenase and correspondingly the coenzyme is selected from formate dehydrogenase, preferably the immobilized enzyme comprises an immobilized amine dehydrogenase or a co-immobilized system of amine dehydrogenase and formate dehydrogenase, preferably the amine dehydrogenase comprises an amine dehydrogenase as shown in the sequence of SEQ ID NO:1, preferably the formate dehydrogenase comprises a formate dehydrogenase as shown in the sequence of SEQ ID NO:2, preferably the reaction system comprises a substrate or a mixture of a substrate and a coenzyme, preferably the immobilized enzyme is an immobilized amine dehydrogenase and the reaction system comprises a mixture of a substrate and formate dehydrogenase. Effect of the Invention

[0016] According to the technical solution of the present invention, the immobilized amine dehydrogenase and formate dehydrogenase having high catalytic activity and good operational stability are prepared by using an amino-based carrier activated with cyanuric chloride, which has higher catalytic activity and reusability than the immobilized enzymes prepared by the conventional immobilization method. [Brief description of the drawings]

[0017] The accompanying drawings of the specification forming a part of this application are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and the description thereof are used to interpret the present invention and are not to be construed as unduly limiting the present invention. The drawings are as follows: [Figure 1] FIG. 5 is a schematic diagram of a continuous reaction apparatus according to Example 5 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In addition, the embodiments and features of the embodiments of the present application may be combined with each other without causing any contradiction.

[0019] As mentioned in the background art, the immobilization of amine dehydrogenase cannot be completed well using conventional glutaraldehyde-activated amino- or epoxy-based supports. The immobilized enzyme obtained has the disadvantages of low catalytic activity and poor reusability. Similarly, adding a protective agent such as mannitol during the immobilization process or post-modifying the immobilized enzyme obtained with small molecules cannot overcome the above-mentioned disadvantages.

[0020] Therefore, in this application, the inventors have studied immobilized enzymes in depth and tried many alternatives to glutaraldehyde-activated amino carriers, and found that only the alternative activated with cyanuric chloride has excellent effects. That is, they found an amino carrier activated with a cyanuric chloride carrier activator, and used it to prepare an immobilized amine dehydrogenase with high catalytic activity and good operational stability. This immobilized amine dehydrogenase has higher catalytic activity and reusability than immobilized amine dehydrogenase prepared by conventional immobilization methods. Although the use of cyanuric chloride-activated resins has been reported so far, in the field of enzyme immobilization, only in the 1970s, a few documents reported that enzyme immobilization was performed by activating a carrier having hydroxyl groups, such as a polysaccharide carrier, with cyanuric chloride, but there has been no report on enzyme immobilization by activating an amino resin with cyanuric chloride. Therefore, the series of protection methods of this application have been proposed.

[0021] In a first embodiment of the present invention, there is provided an immobilized enzyme, comprising: a polyethyleneimine-modified immobilized enzyme, the enzyme comprising amine dehydrogenase and / or formate dehydrogenase; and a support for immobilizing the enzyme, the support being an amino-based support activated with cyanuric chloride.

[0022] The amino of the amino support can be activated with cyanuric chloride, providing a resin structure different from that of conventional glutaraldehyde-activated amino resins, providing a new support for enzymes that cannot be effectively immobilized on glutaraldehyde-activated amino resins due to structure or other reasons. After the enzyme is bound to the activated support to form the immobilized enzyme, in order to further improve the operational stability of the immobilized enzyme, the immobilized enzyme is modified with polyethyleneimine, thereby utilizing the polymer to "tie" or protect the enzyme and achieve a more stable immobilization. This results in an immobilized enzyme with improved catalytic activity and reusability.

[0023] The enzymes in the above immobilized enzymes are mainly targeted at enzymes that are difficult to immobilize using glutaraldehyde-activated amino carriers. Specifically, they include amine dehydrogenase, formate dehydrogenase, or other enzymes, and combinations of these enzymes. The activation principle of the above cyanuric chloride-activated amino carrier is as follows. Taking advantage of the fact that cyanuric chloride has three halogen active sites, one of the active sites is first used to bind with amino during activation, and then the remaining active sites can be reacted with functional groups such as amino and hydroxy on the enzyme molecule to complete immobilization. Preferably, the amine dehydrogenase includes, but is not limited to, amine dehydrogenase AmDH, and AmDH in this application specifically refers to amine dehydrogenase whose amino acid sequence is SEQ ID NO: 1, and does not refer to various types of amine dehydrogenase. Preferably, the formate dehydrogenase includes, but is not limited to, formate dehydrogenase AIY34662.1. Preferably, the specifically activated amino-based carrier includes, but is not limited to, LX-1000EPHA, ECR8409 or LX-1000HA. Preferably, the molecular weight of the polyethyleneimine is 1.8-10 KDa.

[0024] The amino acid sequence of the above amine dehydrogenase AmDH, SEQ ID NO:1, is as follows: TIFF2025500687000002.tif35164 The formate dehydrogenase described above is AIY34662.1 wild type. The amino acid sequence of SEQ ID NO:2 is as follows: TIFF2025500687000003.tif34164 Amine dehydrogenase refers to an enzyme that can catalyze the reduction of ketone-based compounds to chiral amines, using inexpensive ammonium salts as the ammonia source and not relying on expensive ammonia donors. Usually, the reduction process requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotinamide adenine dinucleotide (NADH) to transfer hydrogen, so amine dehydrogenase and formate dehydrogenase are often combined to reduce production costs. Compared with chemical catalytic synthesis, amine dehydrogenase not only has milder reaction conditions, but also has higher catalytic activity, selectivity, and substrate specificity. By using the above-mentioned activating carrier, free enzymes, especially water-soluble free enzymes, can be immobilized to form immobilized enzymes, thereby solving problems such as poor stability of free enzymes, inability to be reused, and difficulty in separating and purifying products. The enzyme to be immobilized may be one type, or multiple enzymes may be immobilized simultaneously to achieve the purpose of combined use.

[0025] In the conventional manufacturing method of immobilized enzyme carriers, taking amino resins as an example, they usually need to be activated by a bifunctional crosslinker and then bonded with enzymes through the terminal functional groups of the crosslinker. The most commonly used activation method is to activate the amino with glutaraldehyde, and then use the active aldehyde group to link to the enzyme molecule to achieve immobilization. There are also reports that the stability of the immobilized enzyme can be increased by post-modification with amino acids or small molecule compounds such as NaBH4 after immobilizing the enzyme using glutaraldehyde. Cyanuric chloride has an activating effect on amino resins, and is suitable for use in amino resin carriers, providing a resin structure different from the conventional glutaraldehyde activation. When amino resins are activated with cyanuric chloride, the active halogen (chlorine) sites remaining on the cyanuric chloride groups can react with enzyme molecules, and the binding ability of different resin structures to different enzymes is greatly different. The use of a carrier activator assists in the immobilization of enzymes that were difficult to bind to conventional immobilized enzyme carriers. The amino support may include many commercially available amino resins, such as ECR8409 resin from Purolite, LX-1000HA resin from Xi'an Lanxiao Technology Co., Ltd.

[0026] Polyethyleneimine (PEI) can be used by preparing it as an aqueous solution of an appropriate concentration, and the final concentration during use can be adjusted depending on factors such as the properties and content of the immobilized enzyme, and may be 0.5 to 3% W / V. Polyethyleneimine of different molecular weights can also be selected and used to modify and protect different immobilized enzymes, and the molecular weight of PEI may be 1.8 to 10 KDa.

[0027] In a second embodiment of the present invention, there is provided a method for producing an immobilized enzyme, the method including the steps of activating an amino-based support with cyanuric chloride to obtain an activated support, mixing an enzyme with the activated support, incubating and immobilizing the enzyme, and then modifying the immobilized product with polyethyleneimine to obtain an immobilized enzyme, the enzyme including amine dehydrogenase and / or formate dehydrogenase.

[0028] The activated amino-based carrier produced using cyanuric chloride has a structure and binding site different from conventional activated carriers, providing a new selectable carrier for the production of immobilized enzymes and supporting the immobilization of enzymes that were difficult to bind to conventional immobilized enzyme carriers. Immobilized enzymes can be obtained by immobilizing the enzyme with an activated carrier at appropriate temperature, rotation speed, etc.

[0029] In the above-mentioned manufacturing method, the step of activating the amino-based support with cyanuric chloride to obtain an activated support includes the steps of dissolving the amino-based support in an organic solvent to obtain a support suspension, and activating the amino-based support by mixing cyanuric chloride with the support suspension to obtain an activated support, preferably, the organic solvent may be a polar organic solvent, preferably, the polar organic solvent includes any one or more of tetrahydrofuran, acetone, dimethylformamide or dimethylsulfoxide, preferably, the activation temperature is 0 to 30°C, more preferably 0 to 5°C, preferably, the activation time is 2 to 5h, preferably, after activation, the activated support is washed with an organic solvent to remove unreacted cyanuric chloride, preferably, after washing, the activated support is blown dry with nitrogen gas, i.e., the organic solvent on the support is blown dry, preferably, the amino-based support is a dry support, preferably, the amino-based support includes, but is not limited to, ECR8409, LX-1000HA or LX-1000EPHA.

[0030] A polar organic solvent such as tetrahydrofuran, acetone, dimethylformamide, or dimethyl sulfoxide is used to disperse the amino-based carrier, and cyanuric chloride is used to activate the amino-based carrier. Depending on factors such as different enzymes and carriers, the activation temperature may be 0 to 30°C, more preferably 0 to 5°C, and the activation time may be 2 to 5h. Low-temperature activation can protect the activity of the carrier and slow down the activation rate, thereby preventing the activation rate from being too fast and generating a large amount of heat, which affects the activation effect of the carrier. The activation time can be appropriately adjusted according to the conditions such as the enzyme, carrier, or activation temperature to obtain a better activation effect. By making the amino-based carrier before activation a dry carrier, the influence of liquid such as residual moisture in the carrier on the composition of the reaction organic system can be reduced, and the contact of the residual liquid with the amino group on the carrier or the encapsulation of the amino group on the carrier also affects the activation efficiency. After activation, the activated support may be washed with the same organic solvent to remove unreacted cyanuric chloride, and the organic solvent on the activated support may be blown dry with nitrogen gas to avoid residual cyanuric chloride and organic solvent from affecting the enzyme activity in the subsequent immobilization step and adversely affecting the preparation yield and performance of the immobilized enzyme.

[0031] The above-mentioned production method further includes a step of replacing the organic solvent by washing the activating carrier with a buffer solution before mixing and incubating the enzyme with the activating carrier, i.e., before immobilization. Preferably, the enzyme is prepared using a buffer solution. Preferably, the buffer solution includes, but is not limited to, a phosphate buffer solution, a glycine buffer solution, or a borax buffer solution. Preferably, the concentration of the phosphate buffer solution is 20 to 50 mM, and preferably, the pH of the phosphate buffer solution is 6.5 to 9.0. Preferably, the immobilization temperature is 4 to 30° C., and preferably, the immobilization time is 2 to 5 hours.

[0032] The activated carrier is washed with a buffer solution to replace the organic solvent remaining in the activated carrier and prevent the remaining organic solvent from affecting the enzyme activity. The enzyme is prepared using a buffer solution, which may be a commonly used phosphate buffer solution, glycine buffer solution, or other commonly used buffer solution, and can keep the pH of the solution relatively stable when a small amount of acid or alkali is added, and can prevent products such as carbon dioxide generated by the reaction from affecting the pH of the solution, thereby maintaining the enzyme activity. The immobilization time can be appropriately adjusted according to the conditions of the enzyme, carrier, immobilization temperature, etc., to obtain a better immobilization effect.

[0033] In the above-mentioned production method, the step of mixing the enzyme with an activating carrier, incubating and immobilizing, and then modifying the immobilized product with polyethyleneimine to obtain an immobilized enzyme includes the steps of mixing the enzyme with an activating carrier, incubating and immobilizing to obtain a pre-immobilized enzyme, and post-modifying the pre-immobilized enzyme with polyethyleneimine to obtain an immobilized enzyme, and preferably the enzyme includes one or more of amine dehydrogenase, formate dehydrogenase or other enzymes, and preferably the amine dehydrogenase is an amine dehydrogenase as shown in the sequence of SEQ ID NO:1. Preferably, the formate dehydrogenase comprises the formate dehydrogenase AIY34662.1 as shown in the sequence of SEQ ID NO: 2; preferably, the post-modification is carried out by adding a solution of polyethyleneimine to the pre-immobilized enzyme; preferably, the molecular weight of the polyethyleneimine is 1.8 to 10 KDa; preferably, the pH of the polyethyleneimine is 5 to 8; preferably, the final concentration of the polyethyleneimine is 0.5 to 3% W / V; preferably, the temperature of the post-modification may be 10 to 30° C.; and preferably, the time of the post-modification may be 8 to 24 hours.

[0034] When the enzyme is mixed with the activated carrier and incubated for immobilization, a pre-immobilized enzyme can be obtained. The enzyme used may be one or more selected, and different types of enzymes may be mixed in any ratio to prepare a pre-immobilized system of multiple enzymes. In order to improve the stability of the immobilized enzyme, the pre-immobilized enzyme can be further post-modified with polyethyleneimine, and the operation stability of the immobilized enzyme can be further improved by "binding" or protecting the enzyme with the polymer. By adding a certain volume of a polyethyleneimine solution dissolved and prepared in advance to the pre-immobilized enzyme, the final concentration of polyethyleneimine can be effectively controlled by efficient mixing, which is also favorable for uniform contact between the pre-immobilized enzyme and polyethyleneimine, and the performance of the obtained immobilized enzyme can be improved. The molecular weight of polyethyleneimine can be adjusted according to different carriers and enzymes to obtain better protection performance. Polyethyleneimine is made into a 10% W / V aqueous solution, and may also be adjusted with a 1M hydrochloric acid solution. An appropriate amount of polyethyleneimine aqueous solution is added to the pre-immobilized enzyme so that the final concentration is 0.5 to 3% W / V.

[0035] In a third embodiment of the present invention, there is provided a use of the above immobilized enzyme or the immobilized enzyme produced by the above production method in a continuous reaction. The above immobilized enzyme has a remarkably improved catalytic activity and reusability compared to free enzymes and immobilized enzymes produced by conventional processes, and can be used in a continuous reaction in a continuous apparatus, which is easier to operate and has higher production efficiency than a batch reaction.

[0036] In the use of the above immobilized enzyme in a continuous reaction, the continuous reaction includes carrying out the continuous reaction in a packed bed reactor, preferably the packed bed reactor is prepared by packing the immobilized enzyme into an empty chromatography column or a stainless steel chromatography column, preferably the volume of the packed bed reactor is 5-50 mL, and preferably the packing method of the packed bed reactor is wet packing.

[0037] The immobilized enzyme can be continuously reacted in a voltage-resistant and corrosion-resistant packed bed reactor such as an empty chromatography column or a stainless steel chromatography column. The volume of the packed bed reactor can be 5-50 mL to prevent the immobilized enzyme for catalyst from being wasted if the loading amount is too large, or the pressure on the immobilized enzyme at the bottom of the packed bed reactor is too high, which affects the mechanical structure and enzyme activity of the immobilized enzyme. In order to ensure the activity and loading uniformity of the immobilized enzyme, wet loading can be selected to load the immobilized enzyme into the packed bed reactor.

[0038] In the above use, the continuous reaction includes pumping the reaction system containing the substrate from bottom to top into the packed bed reactor and remixing the reaction product into the reaction system, the pump is a constant flow pump, preferably the pressure of the constant flow pump is ≦20 MPa, and preferably the flow rate of the continuous reaction is 0.01-10 mL / min.

[0039] The reaction system containing the substrate is pumped from the bottom to the top of the packed bed reactor, which prevents the flow rate of the reaction system in the reactor from becoming too fast due to gravity, resulting in a low conversion yield. Remixing the reaction product into the reaction system allows circulating catalysis for reactions with low primary catalysis yields, which maintains the homogeneity of the reaction system at all times and is favorable for the relative stabilization of the environment in which the immobilized enzyme is placed in the packed bed reactor. It is also favorable for the inspection and measurement of the reaction system, and is convenient for the replenishment of raw materials such as ammonium formate, and is convenient for the collection of the final product, and omits the process of separating the final product and the immobilized enzyme, which is required for batch reactions. The pump used in the continuous reaction can be a medium pressure constant flow pump or a low pressure constant flow pump, which can be reasonably selected according to the requirements of the packed bed reactor and the flow rate, and the resistance of the immobilized enzyme to pressure, and the pressure range provided can be 0-20 MPa.

[0040] In the above use, the enzyme may be a main enzyme or a mixture of a main enzyme and a coenzyme, preferably the main enzyme may be selected from amine dehydrogenases and correspondingly the coenzyme may be selected from formate dehydrogenases, the immobilized enzyme comprises immobilized amine dehydrogenase or a co-immobilized system of amine dehydrogenase and formate dehydrogenase, preferably the amine dehydrogenase comprises amine dehydrogenase AmDH as shown in sequence SEQ ID NO:1, preferably the formate dehydrogenase comprises formate dehydrogenase AIY34662.1 as shown in sequence SEQ ID NO:2, preferably the reaction system comprises a substrate or a mixture of substrate and coenzyme, preferably the immobilized enzyme is immobilized amine dehydrogenase and the reaction system comprises a mixture of substrate and formate dehydrogenase.

[0041] In the use of immobilized enzymes in continuous reactions, the enzymes may be the main enzyme alone or may be a mixture of the main enzyme and a coenzyme, so that the product of the coenzyme can be used to supply the catalytic reaction of the main enzyme. In the reduction process of amine dehydrogenase, NADPH or NADH is usually required to transfer hydrogen, so when amine dehydrogenase is used, it is often used in combination with formate dehydrogenase, which can produce NADH, thereby reducing production costs. The immobilized enzyme may be the immobilized amine dehydrogenase alone, in which case the reaction system needs to be supplemented with formate dehydrogenase, or the immobilized enzyme may be a co-immobilized system of amine dehydrogenase and formate dehydrogenase, in which case the reaction system may be a simple substrate solution.

[0042] The beneficial effects of the present invention will be described in more detail below with reference to specific examples.

[0043] Example 1: ECR8409 Immobilized Amine Dehydrogenase TIFF2025500687000004.tif52162Reaction scheme 1 Preparation of the substrate: 2-(1-adamantyl)-2-oxymethyl acetate shown in reaction formula 1 was used as the substrate. 10 g of the substrate was weighed into a 100 mL beaker, 50 mL of purified water and 1.6 g of K2HPO4·H2O were added, and the mixture was stirred uniformly. The pH was then adjusted to 8.0 with 10 M NaOH solution. 1 g of activated carbon was weighed and added to the prepared solution, and the mixture was stirred for 1 h to adsorb impurities and pigments. A filter paper was placed in a Buchner funnel, 2.5 g of diatomaceous earth was added, purified water was added, and the mixture was sucked until dry, obtaining a filter cake. The prepared substrate solution was filtered through the filter cake, and 20 mL of purified water was added to wash the filter cake, and finally, a clear filtrate was obtained. 5.6 g of ammonium formate and 15 mg of dithiothreitol (DTT) were weighed and added to the filtrate, and the pH was adjusted to 8.0 with 10 M NaOH solution. Purified water was added to the mixture up to 82 mL.

[0044] Activation of the support: 10 g of dried ECR8409 resin was placed in a four-neck flask, 250 mL of acetone was added under ice bath conditions, and the mixture was stirred for 0.5 h. Then, 5 g of cyanuric chloride was added, and the reaction was continued for 3 h under ice bath with stirring. After the reaction was completed, the modified support was filtered and washed with acetone three times, and was ready for use.

[0045] Preparation of enzyme solution: The frozen amine dehydrogenase AmDH enzyme solution was thawed at room temperature and then diluted to 8.5 mg / mL with 0.1 M pH 8.0 phosphate buffer for use.

[0046] Immobilization of enzyme: Immediately after washing 0.5 g of the activated carrier with 0.1 M pH 8.0 phosphate buffer three times, 4 mL of the prepared enzyme solution was added and immobilized in a shaker at 20°C and 100 rpm for 2 h. Then, 1 mL of the previously prepared 10% PEI (10 KDa) solution at pH 8.0 was added and the reaction was continued overnight. Finally, the immobilized enzyme was filtered and washed three times with buffer to obtain the immobilized amine dehydrogenase.

[0047] Reaction conditions: 0.15 g of immobilized amine dehydrogenase was weighed out, 0.82 mL of prepared substrate solution, 1 mg / mL NAD +25 μL of the solution and 50 μL of FDH were added. The reaction was allowed to proceed for 18 hours at 40° C. in a shaker at 100 rpm. The reaction conversion rate of the supernatant of the reaction system was measured by HPLC.

[0048] The results of detecting the activity of the immobilized amine dehydrogenase are shown in Table 1. For each reaction batch, the immobilized amine dehydrogenase was separated by filtration and washed three times with 0.1 M pH 8.0 phosphate buffer. [Table 1] Example 2: Co-immobilization of amine dehydrogenase and formate dehydrogenase on ECR8409 Except for adding 50 μL of formate dehydrogenase (FDH) AIY34662.1 crude enzyme solution to the enzyme immobilization process, the carrier activation step and the preparation of the enzyme solution were the same as in Example 1. Then, the immobilization was completed by the same immobilization method and post-modification process as in Example 1.

[0049] Amine dehydrogenase and FDH were co-immobilized on ECR8409, and the activity was detected. The results are shown in Table 2. The detailed operating conditions were the same as in Example 1, except that no FDH crude enzyme solution was added to the reaction system. [Table 2] Example 3: LX-1000HA immobilized amine dehydrogenase Support activation: 10 g of dried LX-1000HA resin was placed in a four-neck flask, 200 mL of tetrahydrofuran was added under ice bath conditions, and the mixture was stirred for 0.5 h. Then, 2.5 g of cyanuric chloride was added, and the reaction was continued for 3 h under ice bath with stirring. After the reaction was completed, the modified support was filtered and washed with tetrahydrofuran three times for preparation for use.

[0050] The preparation of the enzyme solution, the immobilization process, and the activity verification were the same as in Example 1.

[0051] The results of verifying the activity of the immobilized amine dehydrogenase are shown in Table 3. [Table 3] Example 4: LX-1000EPHA immobilized amine dehydrogenase Support activation: 10 g of dried LX-1000EPHA resin was placed in a four-neck flask, 200 mL of tetrahydrofuran was added under ice bath conditions, and the mixture was stirred for 0.5 h. Then, 2.5 g of cyanuric chloride was added, and the reaction was continued for 3 h under ice bath with stirring. After the reaction was completed, the modified support was filtered and washed with tetrahydrofuran three times for preparation for use.

[0052] The preparation of the enzyme solution, the immobilization process, and the activity verification were the same as in Example 1.

[0053] The results of verifying the activity of the immobilized amine dehydrogenase are shown in Table 4. [Table 4] Example 5: Use of immobilized amine dehydrogenase in continuous production Preparation of reaction system: Measure out 80 mL of substrate solution. For detailed steps of preparing the substrate solution, refer to Example 1. Then, add NAD + 2.5 mg of FDH and 5 mL of crude enzyme solution were added. The system was mixed uniformly with a magnetic stirrer and stirred slowly throughout the reaction process.

[0054] 3.5 g of the immobilized amine dehydrogenase prepared in Example 1 was weighed and packed into a 5 mL empty chromatography column according to the chromatography column packing workflow, and the chromatography column was placed in a 40° C. water bath.

[0055] A CP-M205 precision constant flow pump was connected to the reaction system, and the reaction system was pumped from bottom to top at a flow rate of 0.4 mL / min to a chromatography column filled with immobilized enzyme, and the effluent was returned to the reaction system to form a closed circuit, as shown in Figure 1. After 24 h of reaction, the supernatant of the reaction system was sampled and detected, and the substrate conversion rate reached 95%. Then, the reaction system was changed to a freshly prepared one and continuously added to the next cycle reaction. The measurement results showed that even after 10 cycles, the conversion rate of the reaction still reached 89%.

[0056] Use of the above described continuous system not only allows for easier production and operation and higher throughput, but also improves the operational stability of the immobilized amine dehydrogenase.

[0057] Example 6: Use of co-immobilized amine dehydrogenase and FDH in continuous production Preparation of reaction system: 80 mL of substrate solution was weighed out. The detailed steps of the substrate solution preparation were the same as in Example 1. Then, NAD + 2.5 mg was added. The system was mixed uniformly with a magnetic stirrer and stirred slowly throughout the reaction process.

[0058] 3.8 g of the immobilized amine dehydrogenase and FDH prepared in Example 2 were weighed and packed into a 5 mL empty chromatography column according to the chromatography column packing work procedure, and the chromatography column was placed in a 40° C. water bath.

[0059] The system was connected to a continuous apparatus as in Example 5. After 24 h reaction, the substrate conversion rate reached 94%, and even after 10 repeats, the conversion rate still reached 84%.

[0060] Use of the above continuous system not only allows for easier production and operation and higher throughput, but also improves the operational stability of the immobilized amine dehydrogenase and FDH.

[0061] Comparative Example 1: Glutaraldehyde-activated LX-1000HA immobilized amine dehydrogenase Glutaraldehyde activation of LX-1000HA: 1 g wet weight of LX-1000HA was added to 4 mL of 2% (w / v) glutaraldehyde solution, which was prepared in 20 mM pH 7.0 phosphate buffer. Activation was performed on a shaker at 30°C for 1 h.

[0062] The activated support was washed three times with 0.1 M pH 8.0 phosphate buffer, and then 4 mL of the prepared enzyme solution (prepared in the same manner as in Example 1) was added, followed by immobilization for 20 hours at 20°C and 100 rpm in a shaker. Finally, the immobilized enzyme was filtered and washed three times with the buffer to obtain immobilized amine dehydrogenase.

[0063] The results of verifying the activity of the immobilized amine dehydrogenase are shown in Table 5. [Table 5] Comparative Example 2: Hexamethylene diisocyanate-activated LX-1000HA immobilized amine dehydrogenase LX-1000HA resin hexamethylene diisocyanate activation: 10 g of dried LX-1000HA resin was placed in a four-neck flask, and 200 mL of dimethylformamide was added and stirred for 0.5 h under ice bath conditions. Then, 2.3 g of hexamethylene diisocyanate was added and the stirring reaction was continued for 3 h in an ice bath. After the reaction was completed, the modified support was filtered and washed with DMF three times for preparation for use.

[0064] The enzyme solution preparation, immobilization process, and activity verification were the same as in Example 1.

[0065] The results of verifying the activity of the immobilized amine dehydrogenase are shown in Table 6. [Table 6] Comparative Example 3: p-Benzoquinone-activated LX-1000HA immobilized amine dehydrogenase LX-1000HA resin activation with p-benzoquinone: 1 g of LX-1000HA (wet weight) was added to 4 mL of 0.02 g / mL p-benzoquinone ethanol solution, which is a 20% (V / V) ethanol solution. Activation was carried out for 1 h on a 30°C shaker. The activated support was then filtered and washed with a 20% (V / V) ethanol solution and 0.1 M pH 8.0 phosphate buffer solution in that order to obtain LX-1000HA activated with p-benzoquinone.

[0066] The activated support was added to 4 mL of the enzyme solution (prepared in the same manner as in Example 1) and immobilized in a shaker at 20°C and 100 rpm for 20 hours. Finally, the immobilized enzyme was filtered and washed three times with a buffer solution to obtain immobilized amine dehydrogenase.

[0067] The results of verifying the activity of the immobilized amine dehydrogenase are shown in Table 7. [Table 7] Comparative Example 4: LX-1000HA immobilized amine dehydrogenase (no post-modification) The carrier activation step and the preparation of the enzyme solution were the same as those in Example 3, but the enzyme immobilization process did not involve a post-modification step, and the enzyme was directly filtered after immobilization and washed three times with a buffer solution to obtain a pre-immobilized amine dehydrogenase, which was different from Example 3.

[0068] The results of verifying the activity of the immobilized amine dehydrogenase are shown in Table 8. [Table 8] Comparative Example 5: Co-immobilization of amine dehydrogenase and FDH on ECR8409 (without post-modification) The carrier activation step and the preparation of the enzyme solution were the same as in Example 2, but the enzyme immobilization process did not include a post-modification step, and instead was directly filtered after immobilization and washed three times with a buffer solution to obtain a preliminary co-immobilized system of amine dehydrogenase and FDH, which was different from Example 2.

[0069] The results of testing the activity of the immobilized amine dehydrogenase are shown in Table 9. [Table 9] From the above description, it can be seen that the above embodiment of the present invention has the following technical effects: an amino-based carrier is activated with cyanuric chloride, and the carrier is used to prepare immobilized amine dehydrogenase and immobilized formate dehydrogenase with high catalytic activity and good operational stability. The immobilized amine dehydrogenase and immobilized formate dehydrogenase have higher catalytic activity and reusability than immobilized enzymes prepared by conventional immobilization methods.

[0070] The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Various modifications and variations of the present invention are possible for those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and principle of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Polyethylenimine modified immobilized enzyme, an enzyme comprising amine dehydrogenase and / or formate dehydrogenase; and a support which is an amino-based support activated with cyanuric chloride.

2. The amine dehydrogenase comprises an amine dehydrogenase as shown in the sequence of SEQ ID NO:1, Preferably, the formate dehydrogenase comprises a formate dehydrogenase as shown in the sequence of SEQ ID NO:2, Preferably, the amino carrier comprises ECR8409, LX-1000HA or LX-1000EPHA; The immobilized enzyme according to claim 1, characterized in that the molecular weight of the polyethyleneimine is preferably from 1.8 KDa to 10 KDa.

3. Activating the amino-based support with cyanuric chloride to obtain an activated support; mixing and incubating the enzyme with the activated carrier to immobilize it, and then modifying the immobilized product with polyethyleneimine to obtain the immobilized enzyme; 2. A method for producing an immobilized enzyme, wherein the enzyme comprises amine dehydrogenase and / or formate dehydrogenase.

4. The step of activating an amino-based support with cyanuric chloride to obtain an activated support includes: Dispersing the amino-based carrier in an organic solvent to obtain a carrier suspension; and activating the amino-based support by reacting the cyanuric chloride with the support suspension to obtain the activated support; Preferably, the organic solvent is a polar organic solvent, Preferably, the polar organic solvent comprises any one or more of tetrahydrofuran, acetone, dimethylformamide or dimethylsulfoxide; Preferably, the activation temperature is between 0° C. and 30° C., more preferably between 0° C. and 5° C. Preferably, the activation time is between 2 h and 5 h; Preferably, after said activation, said activated support is washed with said organic solvent to remove unreacted said cyanuric chloride; Preferably, the method further comprises blowing the activating support dry with nitrogen gas after washing; Preferably, the amino-based carrier is a dry carrier, The method according to claim 3, wherein the amino-based carrier preferably includes ECR8409, LX-1000HA or LX-1000EPHA.

5. The method further comprises the step of replacing the organic solvent by washing the activated carrier with a buffer solution prior to the immobilization; Preferably, the enzyme is formulated with the buffer; Preferably, the buffer comprises a phosphate buffer, a glycine buffer or a borax buffer; Preferably, the concentration of the phosphate buffer is between 20 mM and 50 mM; Preferably, the pH of the phosphate buffer is 6.5 to 9.0; Preferably, the immobilization temperature is between 0° C. and 30° C., more preferably between 4° C. and 30° C. The method according to claim 4, characterized in that the immobilization time is preferably 2 hours to 5 hours.

6. The steps of mixing the enzyme with the activated carrier, incubating and immobilizing the enzyme, and then modifying the immobilized product with polyethyleneimine to obtain the immobilized enzyme include: mixing said enzyme with said activating carrier and incubating and immobilizing it to obtain a pre-immobilized enzyme; post-modifying the pre-immobilized enzyme with the polyethyleneimine to obtain the immobilized enzyme; Preferably, the amine dehydrogenase comprises an amine dehydrogenase as shown in the sequence SEQ ID NO:1, Preferably, the formate dehydrogenase comprises a formate dehydrogenase as shown in the sequence of SEQ ID NO:2, Preferably, the post-modification is carried out by adding a solution of the polyethyleneimine to the pre-immobilized enzyme, Preferably, the molecular weight of the polyethyleneimine is between 1.8 KDa and 10 KDa; Preferably, the pH of the solution of polyethyleneimine is between 5 and 8; Preferably, the final concentration of the polyethyleneimine added is 0.5% W / V to 3% W / V; Preferably, the temperature of the post-modification is between 0° C. and 30° C., more preferably between 10° C. and 30° C.; The method according to any one of claims 3 to 5, wherein the post-modification time is preferably 8 hours to 24 hours.

7. Use of the immobilized enzyme according to claim 1 or 2, or the immobilized enzyme produced by the process according to any one of claims 3 to 6, in a continuous reaction.

8. The continuous reaction comprises conducting the continuous reaction in a packed bed reactor; Preferably, the packed bed reactor is prepared by packing the immobilized enzyme into an empty or stainless steel chromatography column, Preferably, the volume of the packed bed reactor is between 5 mL and 50 mL; 8. The use according to claim 7, characterized in that preferably the method of packing the packed bed reactor is wet packing.

9. The continuous reaction is pumping a reaction system containing a substrate from bottom to top into said packed bed reactor and remixing a reaction product into said reaction system; The pump is a constant flow pump, and preferably the pressure of the constant flow pump is ≦20 MPa; The use according to claim 8, characterized in that the flow rate of the continuous reaction is preferably between 0.01 mL / min and 10 mL / min.

10. the immobilized enzyme comprises an immobilized amine dehydrogenase or a co-immobilized system of an amine dehydrogenase and a formate dehydrogenase; Preferably, the amine dehydrogenase comprises an amine dehydrogenase as shown in the sequence SEQ ID NO:1, Preferably, the formate dehydrogenase comprises a formate dehydrogenase as shown in the sequence of SEQ ID NO:2, Preferably, the reaction system comprises a substrate or a mixture of a substrate and the formate dehydrogenase, 10. The use according to claim 9, characterized in that the immobilized enzyme is the immobilized amine dehydrogenase and the reaction system comprises a mixture of a substrate and the formate dehydrogenase.

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