Coated immobilized nitrile hydratase, preparation method and application thereof, and preparation method of methacrylamide

Abstract

The invention relates to the field of immobilized enzymes, and discloses coated immobilized nitrile hydratase as well as a preparation method and application thereof and a preparation method of methacrylamide. The coated type immobilized nitrile hydratase comprises immobilized nitrile hydratase and polyethyleneimine coated on the surface of the immobilized nitrile hydratase. The coated immobilized nitrile hydratase provided by the invention has high recovery rate, can catalyze substrate hydration in a high-concentration ethylenically unsaturated nitrile substrate (such as methacrylonitrile), and can still maintain high relative residual enzyme activity after being repeatedly used for many times.

Description

Technical Field

[0001] The invention relates to the technical field of immobilized enzymes, and in particular to a coated immobilized nitrile hydratase, a preparation method and application thereof, and a preparation method of methacrylamide. Background Art

[0002] Amide compounds are widely used in industrial applications. For example, acrylamide and nicotinamide are widely used in daily chemical industry and petrochemical industry. Nitrile hydratase, as a green biocatalyst, can efficiently and environmentally friendly hydrate organic nitrile substrates to generate corresponding amide compounds. At present, various new processes for synthesizing high value-added amide compounds using nitrile hydratase have been continuously developed. However, the preparation of amide compounds using organic nitrile as substrate is an exothermic process, and most existing nitrile hydratases have the disadvantages of poor thermal stability, low catalytic activity, narrow substrate range or poor catalytic selectivity, which limits the application of nitrile hydratase in the synthesis of amide compounds. In addition, high concentration of organic nitrile solution will have toxic effects on cell catalysts and enzyme preparations, and seriously inhibit the catalytic activity of nitrile hydratase.

[0003] Therefore, developing enzyme immobilization technology, improving the stability and substrate tolerance of nitrile hydratase preparations, and increasing the number of times the enzyme preparations can be recycled are of great significance and application prospects in the field of green chemical manufacturing of amide compounds. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems of poor stability, low catalytic activity, narrow substrate range and poor catalytic selectivity of nitrile hydratase used in catalytic synthesis (for example, used in the manufacture of amide compounds) in the prior art, and to provide a coated immobilized nitrile hydratase and a preparation method and application thereof, as well as a preparation method of methacrylamide. The coated immobilized nitrile hydratase has high stability, substrate tolerance and number of recycling uses, and is of great significance and application prospect in the field of green chemical manufacturing of amide compounds.

[0005] A first aspect of the present invention provides a coated immobilized nitrile hydratase, which comprises immobilized nitrile hydratase and polyethyleneimine coated on the surface of the immobilized nitrile hydratase.

[0006] The second aspect of the present invention provides a method for preparing the above-mentioned coated immobilized nitrile hydratase, wherein the preparation method comprises: contacting a boric acid-borax buffer containing immobilized nitrile hydratase with a polyethyleneimine solution, and then performing post-treatment to obtain the coated immobilized nitrile hydratase.

[0007] The third aspect of the present invention provides the use of the above-mentioned coated immobilized nitrile hydratase in a hydration reaction.

[0008] A fourth aspect of the present invention provides a method for preparing methacrylamide, which comprises: subjecting methacrylonitrile to a hydration reaction in the presence of an aqueous medium and the above-mentioned coated immobilized nitrile hydratase of the present invention.

[0009] Compared with the prior art, the present invention has at least the following beneficial effects:

[0010] The coated immobilized nitrile hydratase of the present invention has a high recovery rate and catalytic stability. When it is used in a high-concentration ethylenically unsaturated nitrile substrate (such as methacrylonitrile) to catalyze substrate hydration, it can be reused, and the coated immobilized nitrile hydratase can still maintain a high relative residual enzyme activity after multiple uses. The coated immobilized nitrile hydratase of the present invention has application potential in industrial production fields such as establishing a clean and safe production process and constructing a green synthesis system for ethylenically unsaturated amides (such as methacrylamide). BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a schematic diagram of the structure of the coated immobilized nitrile hydratase in Example 1;

[0012] Figure 2 is a scanning electron micrograph of the coated immobilized nitrile hydratase in Example 1;

[0013] Figure 3 is the immobilized enzyme fluorescence image of the coated immobilized nitrile hydratase in Example 1;

[0014] Figure 4 is the reaction batch determination test result of the coated immobilized nitrile hydratase in Example 1;

[0015] Figure 5 This is a reaction process diagram for preparing methacrylamide using the coated immobilized nitrile hydratase in Example 1. DETAILED DESCRIPTION

[0016] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0017] As a biocatalyst, nitrile hydratase can catalyze the hydration of ethylenically unsaturated nitriles into ethylenically unsaturated amides. However, when used alone, nitrile hydratase has disadvantages such as poor thermal stability, low catalytic activity, narrow substrate range or poor catalytic selectivity, which limits the application of nitrile hydratase in the synthesis of organic matter, especially amide compounds.

[0018] A first aspect of the present invention provides a coated immobilized nitrile hydratase, comprising immobilized nitrile hydratase and polyethyleneimine coated on the surface of the immobilized nitrile hydratase.

[0019] In the present invention, an immobilized nitrile hydratase is coated with a polyethyleneimine layer to form a coated immobilized nitrile hydratase. The coated immobilized nitrile hydratase has a high recovery rate after catalyzing ethylenically unsaturated nitrile, and can catalyze substrate hydration in a high concentration of ethylenically unsaturated nitrile substrate (such as methacrylonitrile), has high catalytic activity, and also has excellent reusability.

[0020] According to the present invention, as long as the purpose of the present invention can be achieved, the coating amount of the polyethyleneimine coated on the surface of the immobilized nitrile hydratase is not particularly limited. In some embodiments, the coating amount of the polyethyleneimine is 2-10wt% of the mass of the coated immobilized nitrile hydratase, such as 2wt%, 3wt%, 3.7wt%, 4.5wt%, 6.7wt%, 7wt%, 8wt%, 9wt% or 10wt%, preferably 3-8wt%. The above embodiment can further increase the stability, substrate tolerance and number of cycles of the coated immobilized nitrile hydratase, and at the same time better ensure the relative enzyme activity of the coated immobilized nitrile hydratase.

[0021] According to the present invention, as long as the purpose of the present invention can be achieved, the structural form of the polyethyleneimine is not particularly limited. In some embodiments, the polyethyleneimine includes branched polyethyleneimine and / or linear polyethyleneimine. By adopting the above-mentioned embodiment, the stability, substrate tolerance, number of cycles and relative enzyme activity of the coated immobilized nitrile hydratase are enhanced.

[0022] According to the present invention, based on the consideration of raw material cost, in some preferred embodiments, the polyethyleneimine is a linear polyethyleneimine. By adopting the above embodiment, the coated immobilized nitrile hydratase can more stably catalyze the hydration of ethylenically unsaturated nitriles to synthesize ethylenically unsaturated amides.

[0023] According to the present invention, as long as the purpose of the present invention can be achieved, there is no particular limitation on the molecular size of polyethyleneimine. In some embodiments, the molecular weight of the polyethyleneimine is greater than 1000 g / mol, preferably 1200-2000 g / mol, such as 1200 g / mol, 1400 g / mol, 1600 g / mol, 1800 g / mol, 1900 g / mol or 2000 g / mol. The above embodiment can better increase the stability and catalytic activity of the coated immobilized nitrile hydratase.

[0024] According to the present invention, immobilized nitrile hydratase refers to using nitrile hydratase to block solid materials or confine them in a certain area. In some embodiments, the immobilized nitrile hydratase includes an immobilized enzyme carrier and a nitrile hydratase loaded on the immobilized enzyme carrier. The above-mentioned embodiment can better increase the stability and catalytic activity of the coated immobilized nitrile hydratase.

[0025] According to the present invention, in some preferred embodiments, the immobilized enzyme carrier and the nitrile hydratase are connected by a covalent bond. The above embodiment is adopted to make the coated immobilized nitrile hydratase have better stability.

[0026] According to the present invention, based on the enzymatic activity of the coated immobilized nitrile hydratase, in some preferred embodiments, the loading amount of nitrile hydratase per gram of immobilized enzyme carrier is not less than 40 mg. The above embodiment can better increase the catalytic activity of the coated immobilized nitrile hydratase.

[0027] According to the present invention, based on cost and enzyme activity considerations, in some more preferred embodiments, the loading amount of nitrile hydratase per gram of immobilized enzyme carrier is 40-50 mg, such as 40 mg, 41 mg, 44 mg, 45 mg, 46 mg, 48 mg or 50 mg. The above embodiment can better increase the catalytic activity of the coated immobilized nitrile hydratase.

[0028] According to the present invention, as long as the purpose of the present invention can be achieved, the size of the immobilized enzyme carrier in the immobilized nitrile hydratase is not particularly limited. In some embodiments, the particle size of the immobilized enzyme carrier is 10-1000 μm, such as 10 μm, 50 μm, 100 μm, 110 μm, 125 μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm or 1000 μm, preferably 100-500 μm. The above embodiment can make the coated immobilized nitrile hydratase have better enzyme activity and stability.

[0029] According to the present invention, as long as the purpose of the present invention can be achieved, the specific type of the immobilized enzyme carrier is not particularly limited. In some embodiments, the immobilized enzyme carrier includes epoxy resin. Using the above embodiment, the epoxy group in the epoxy resin can form a covalent bond with the amino group in the nitrile hydratase, further ensuring the stability of the coated immobilized nitrile hydratase.

[0030] According to the present invention, as long as the purpose of the present invention can be achieved, the source of the epoxy resin is not particularly limited. In some preferred embodiments, the epoxy resin includes at least one of LX-1000HA, LX-103B, LX-1000EP F, LX-1000EP, LX-1000HFA, ES-103B(a), ES-103B(b), ES-1, ECR8214 and ECR4204. The epoxy resins used in the present invention can be obtained from the market, wherein the particle size range of LX-1000HA is 300-500 μm, the particle size range of LX-103B is 100-300 μm, and the particle size range of LX-1000EP is The particle size range of F is 125-300μm, the particle size range of LX-1000EP is 150-300μm, the particle size range of LX-1000HFA is 100-200μm, the particle size range of ES-103B(a) is 110-250μm, the particle size range of ES-103B(b) is 100-300μm; the particle size range of ES-1 is 150-300μm; the particle size range of ECR8214 is 150-300μm; the particle size range of ECR4204 is 150-300μm.

[0031] The second aspect of the present invention provides a method for preparing the above-mentioned coated immobilized nitrile hydratase, wherein the preparation method comprises: contacting a weak alkaline buffer solution containing immobilized nitrile hydratase with a polyethyleneimine solution, and then performing post-treatment to obtain the coated immobilized nitrile hydratase.

[0032] In the present invention, by including a layer of polyethyleneimine on the surface of the immobilized nitrile hydratase, the enzyme activity of the reaction substrate can be further increased, and the stability and the number of recycling times of the coated immobilized nitrile hydratase can be better maintained. In addition, the method of the present invention can also prepare the coated immobilized nitrile hydratase of the present invention without using glutaraldehyde.

[0033] According to the present invention, when preparing the coated immobilized nitrile hydratase, the polyethyleneimine solution can be added dropwise into the weak alkaline buffer solution containing the immobilized nitrile hydratase.

[0034] According to the present invention, as long as the purpose of the present invention can be achieved, the amount of immobilized nitrile hydratase and polyethyleneimine is not particularly limited. In some embodiments, the mass ratio of the immobilized nitrile hydratase to polyethyleneimine is 100:(2-10), for example, 100:2, 100:3, 100:4, 100:, 100:7.5, 100:8 or 100:10, preferably 100:(3-8). The above-mentioned embodiment can further increase the stability, substrate tolerance and number of cycles of the coated immobilized nitrile hydratase, and at the same time, better ensure the relative enzyme activity of the coated immobilized nitrile hydratase.

[0035] According to the present invention, as long as the purpose of the present invention can be achieved, the concentration of immobilized nitrile hydratase in the weak alkaline buffer solution containing immobilized nitrile hydratase is not particularly limited. In some preferred embodiments, the concentration of immobilized nitrile hydratase in the weak alkaline buffer solution containing immobilized nitrile hydratase is not less than 100 mg / mL, such as 100 mg / mL, 116 mg / mL, 120 mg / mL, 150 mg / mL or 200 mg / mL, preferably 100-120 mg / mL. The above embodiment can better ensure the formation of coated immobilized nitrile hydratase.

[0036] According to the present invention, polyethyleneimine is a water-soluble polymer. Polyethyleneimine sold on the market generally exists in the form of an aqueous solution. The polyethyleneimine solution in the present invention refers to polyethyleneimine dissolved in water. In some embodiments, the concentration of polyethyleneimine in the polyethyleneimine solution is 30-60g / L.

[0037] According to the present invention, in some embodiments, the weak alkaline buffer solution containing immobilized nitrile hydratase is obtained by incubating a nitrile hydratase raw material with an immobilized enzyme carrier in a weak alkaline buffer to obtain a weak alkaline buffer solution containing immobilized nitrile hydratase. Using the above embodiment, the nitrile hydratase can be connected to the immobilized enzyme carrier in the form of a covalent bond, so that the coated immobilized nitrile hydratase has better stability.

[0038] According to the present invention, in some preferred embodiments, the pH value of the weak alkaline buffer solution is 7.5-8.5. The above embodiment is more conducive to the solidification between the nitrile hydratase raw material and the immobilized enzyme carrier.

[0039] According to the present invention, as long as the purpose of the present invention can be achieved, the specific type of the weak alkaline buffer solution is not particularly limited. In the present invention, a boric acid-borax buffer solution with a pH value of 8 is used as a weak alkaline buffer solution to illustrate the advantages of the present invention, but the present invention is not limited to this.

[0040] According to the present invention, in some preferred embodiments, the incubation condition includes: room temperature. The temperature condition of the aforementioned embodiment can effectively avoid the inactivation of the nitrile hydratase raw material and can better improve the stability of the immobilized nitrile hydratase and the coated immobilized nitrile hydratase.

[0041] In the present invention, room temperature refers to "10-25°C".

[0042] According to the present invention, in some preferred embodiments, the incubation conditions include: a rotation speed of 50-200 rpm. The above embodiment can better improve the efficiency of nitrile hydratase immobilization.

[0043] According to the present invention, in some preferred embodiments, the incubation conditions include: the incubation time is 1-3 hours. The above embodiment can better achieve the immobilization of nitrile hydratase.

[0044] According to the present invention, the nitrile hydratase raw material is a raw material for providing nitrile hydratase, and the loading amount of nitrile hydratase per gram of immobilized enzyme carrier mentioned in the first aspect of the present invention depends to a certain extent on the amount of pure nitrile hydratase provided in the nitrile hydratase raw material and the immobilized enzyme carrier. In some embodiments, the mass ratio of the nitrile hydratase raw material to the immobilized enzyme carrier is not less than 40 mg / g, preferably 50-80 mg / g, for example 50 mg / g, 60 mg / g, 70 mg / g or 80 mg / g, based on the pure nitrile hydratase. The above-mentioned embodiment can better increase the catalytic activity of the final product.

[0045] According to the present invention, nitrile hydratase is a biocatalyst. The source of the nitrile hydratase raw material used as the raw material for providing nitrile hydratase is not particularly limited. In order to ensure that the catalytic activity is fully exerted, in some preferred embodiments, the nitrile hydratase raw material is selected from at least one of cells with nitrile hydratase activity, cell disrupted liquid with nitrile hydratase activity and nitrile hydratase purified preparation.

[0046] According to the present invention, based on the difficulty of the preparation method, in some more preferred embodiments, the nitrile hydratase raw material is a purified nitrile hydratase preparation (referred to as nitrile hydratase, i.e., the commercial nitrile hydratase protein used in the examples).

[0047] According to the present invention, in the present invention, the immobilized nitrile hydratase can be prepared first, that is, the nitrile hydratase raw material and the immobilized enzyme carrier are first incubated in a weak alkaline buffer to obtain a weak alkaline buffer solution containing the immobilized nitrile hydratase, and then the immobilized nitrile hydratase is separated, and then the weak alkaline buffer solution containing the immobilized nitrile hydratase is prepared, and the weak alkaline buffer solution containing the immobilized nitrile hydratase is contacted with a polyethyleneimine solution, and then post-processed to obtain the coated immobilized nitrile hydratase; the weak alkaline buffer solution containing the immobilized nitrile hydratase obtained by incubation can also be directly used to contact the polyethyleneimine solution. In the present invention, based on the convenience of operation, the weak alkaline buffer solution containing the immobilized nitrile hydratase obtained by incubation is directly used to contact the polyethyleneimine solution.

[0048] According to the present invention, when preparing the coated immobilized nitrile hydratase, there will be some residues of raw materials such as nitrile hydratase protein, and the residual protein can be removed by post-treatment. The post-treatment method is not limited, for example, centrifugation at 3000-5000 rpm and 2-7° C. for 5-15 min, collecting the precipitate and rinsing with a weak alkaline buffer solution until the supernatant has no protein residue, thereby obtaining the coated immobilized nitrile hydratase.

[0049] The third aspect of the present invention provides the use of the above-mentioned coated immobilized nitrile hydratase in a hydration reaction.

[0050] According to the present invention, in some embodiments, the coated immobilized nitrile is used in the preparation of ethylenically unsaturated amides by continuous hydration reaction of ethylenically unsaturated nitrile.

[0051] In the present invention, since the coated immobilized nitrile hydratase has good stability and enzyme catalytic activity, the high catalytic activity in the continuous preparation of ethylenically unsaturated amides from ethylenically unsaturated nitriles can be well maintained, so that the life of the coated immobilized nitrile hydratase can be extended, thereby ensuring the reaction efficiency when continuously preparing ethylenically unsaturated amides.

[0052] Methacrylonitrile is a kind of ethylenically unsaturated nitrile. The fourth aspect of the present invention provides a method for preparing methacrylamide, which comprises: in the presence of an aqueous medium and the above-mentioned coated immobilized nitrile hydratase of the present invention, hydrating methacrylonitrile.

[0053] In the present invention, methacrylonitrile is subjected to hydration reaction using the coated immobilized nitrile hydratase of the present invention, so that methacrylamide can be prepared stably and efficiently.

[0054] According to the present invention, in some embodiments, the pH value of the aqueous medium is 7.5-8.5.

[0055] According to the present invention, the aqueous medium may refer to water alone, or may refer to a buffer solution formed by dissolving some auxiliary agents in water. In some preferred embodiments, the aqueous medium includes a boric acid-borax buffer solution.

[0056] According to the present invention, in some embodiments, the coated immobilized nitrile hydratase is measured by mass and the methacrylonitrile is measured by mole, and the ratio of methacrylonitrile to the coated immobilized nitrile hydratase is not greater than 2 mol / g, preferably 0.1-1.4 mol / g, such as 0.1 mol / g, 0.5 mol / g, 1 mol / g, 1.2 mol / g or 1.4 mol / g. The coated immobilized nitrile hydratase of the present invention can perform continuous hydration reaction in a high concentration methacrylonitrile solution and can better exert its catalytic activity.

[0057] According to the present invention, as long as the purpose of the present invention can be achieved, the conditions of the hydration reaction are not particularly limited. In some embodiments, the conditions of the hydration reaction include: room temperature.

[0058] According to the present invention, in some embodiments, the conditions of the hydration reaction include: a rotation speed of 100-300 rpm.

[0059] According to the present invention, the time of the hydration reaction is not particularly limited, and the reaction endpoint is determined after sampling and testing.

[0060] According to the present invention, when preparing methacrylamide, there is no particular restriction on the order of adding the aqueous medium, the coated immobilized nitrile hydratase and methacrylonitrile. For example, in some embodiments, methacrylonitrile is dissolved in the aqueous medium to form a methacrylonitrile suspension with a concentration of 0.5-1.5 M, and then the coated immobilized nitrile hydratase is added to the methacrylonitrile suspension for hydration reaction.

[0061] According to the present invention, in other embodiments, methacrylonitrile is dissolved in an aqueous medium to form a methacrylonitrile suspension with a concentration of 1-2 M, and then added to a reactor containing 10-30 g of coated immobilized nitrile hydratase at a certain flow rate (e.g., 0.2 mL / min) to carry out a hydration reaction.

[0062] The present invention will be described in detail below by way of examples. In the following examples and comparative examples:

[0063] All analytically pure chemical reagents used were purchased from Sinopharm Chemical Reagent Co., Ltd. The commercial nitrile hydratase used in the immobilized experiments was purchased from Beijing Biolab Technology Co., Ltd.

[0064] Molecular biology experimental methods not specifically described were performed in accordance with the specific methods listed in the book Molecular Cloning Experiment Guide (3rd edition) by J. Sambrook, or in accordance with the kits and product instructions.

[0065] Performance Testing

[0066] The test samples were the coated immobilized nitrile hydratase in the example and the immobilized nitrile hydratase in the comparative example.

[0067] 1. Enzyme activity determination:

[0068] The substrate methacrylonitrile is dissolved in boric acid-borax buffer (pH 8.0) to a final concentration of 10 mM; the reaction system contains 50 μL of a test sample suspension (obtained by mixing the test sample with a boric acid-borax buffer (pH 8.0)), and 450 μL of a substrate with a final concentration of 10 mM; the substrate is preheated at room temperature for 5 min at the reaction temperature, and then the test sample suspension is added and reacted for another 10 min, and then 20 μL of 5M concentrated HCl is added to terminate the reaction; after filtering with a 0.22 μm filter membrane to remove impurities, the amount of product methacrylamide generated is detected by HPLC. The HPLC detection conditions were as follows: an Agilent InfinityLab Poroshell 120EC-C18 column (4.6×150 mm, 4 μm) was used, the column temperature was 36°C, the mobile phase was 10% (v / v) acetonitrile, the flow rate was 0.5 mL / min, and the detection wavelength was 215 nm; 1 enzyme activity unit (U) was defined as the amount of enzyme required to hydrate the substrate to produce 1 μmol of product per minute under given conditions;

[0069]

[0070]

[0071] 2. Reaction batch determination of test samples:

[0072] The substrate methacrylonitrile was dissolved in a boric acid-borax buffer (pH 8.0) to a final concentration of 1 M, and the test sample was added to the substrate solution at an addition amount of 1 g / L, and the reaction was shaken at a low speed of 100 rpm at room temperature. Samples were taken every 30 minutes, and the substrate and product were detected by HPLC to monitor the progress of the enzyme catalytic reaction. After the substrate methacrylonitrile was completely hydrated, the test sample was collected by centrifugation and rinsed with a boric acid-borax buffer (pH 8.0) to remove the residual reactants, and the residual enzyme activity of the test sample was determined, and the reaction batch of the immobilized nitrile hydratase was repeatedly determined using the same method.

[0073] The calculation formula of the relative residual activity relative to the initial enzyme activity is:

[0074] 3. Preparation of methacrylamide using test samples:

[0075] Methacrylonitrile was selected as the substrate test sample to catalyze the synthesis of methacrylamide. A methacrylonitrile solution (supersaturated solution exceeding substrate solubility was prepared in the form of suspension) was prepared with boric acid-borax buffer (pH 8.0) at a concentration of 1.5 M. The substrate suspension was added at a flow rate of 0.2 mL / min to a reactor containing 20 g of immobilized nitrile hydratase, followed by a low-speed oscillation reaction at 100 rpm at room temperature, and sampling was performed at regular intervals. The product concentration was detected using HPLC to monitor the progress of the enzyme-catalyzed reaction and each group of reactions was repeated three times.

[0076] Example 1

[0077] Preparation of coated immobilized nitrile hydratase:

[0078] 1 g of epoxy resin ES-103B (a) was added to 9 mL of boric acid-borax buffer (pH 8.0) containing 50 mg of commercial nitrile hydratase protein, and the mixture was incubated at room temperature with low-speed shaking at 100 rpm for 2 h; then 1 ml of a 50 g / L aqueous solution of polyethyleneimine (straight-chain type, relative molecular weight of 1800 g / mol) was added dropwise, and the mixture was continued to be incubated at room temperature with low-speed shaking at 100 rpm for 1 h. Finally, the mixture was centrifuged at 5000 rpm and 4°C for 10 min, and the precipitate was collected and rinsed with boric acid-borax buffer (pH 8.0) until there was no protein residue in the supernatant to obtain coated immobilized nitrile hydratase.

[0079] No polyethyleneimine was present in the rinse solution obtained after rinsing, indicating that the polyethyleneimine was completely coated on the surface of the immobilized nitrile hydratase.

[0080] The coating amount of polyethyleneimine was 4.5% of the mass of the coated immobilized nitrile hydratase.

[0081] The schematic diagram of the structure of the coated immobilized nitrile hydratase is shown in Figure 1 shown.

[0082] Comparison of scanning electron microscopy images of epoxy resin ES-103B (a) and coated immobilized nitrile hydratase Figure 2 As shown, Figure 2 In the figure, a, b, and c are observations of the morphology and microstructure of the unloaded resin (epoxy resin ES-103B (a)) by field emission electron scanning microscopy; d, e, and f are observations of the morphology and microstructure of the coated immobilized nitrile hydratase by field emission electron scanning microscopy. Figure 2 This indicates that there are obvious attachments on the surface of the coated immobilized nitrile hydratase.

[0083] Laser confocal microscopy characterization of immobilized nitrile hydratase Figure 3 As shown, the green fluorescence (circle) in the figure is nitrile hydratase labeled with FITC. Figure 3The results showed that the average particle size of the coated immobilized nitrile hydratase was 120 μm, and there was a layer of evenly distributed green fluorescence on the surface of the microspheres, indicating that the free nitrile hydratase was evenly fixed on the resin surface by covalent bonding and was evenly distributed, further confirming that the nitrile hydratase molecules were fixed on the surface of the epoxy resin ES-103B(a) carrier.

[0084] The results of the enzyme specific activity and enzyme loading rate of the coated immobilized nitrile hydratase are shown in Table 1.

[0085] The reaction batch test results of coated immobilized nitrile hydratase are as follows Figure 4 As shown, through Figure 4 It can be seen that the catalytic activity of epoxy resin immobilized nitrile hydratase did not decrease significantly after repeated use for 6 batches; thereafter, as the number of reaction batches increased, the catalytic activity of immobilized nitrile hydratase gradually decayed, and after repeated use for 10 batches, more than 68% of the relative catalytic activity remained relative to the initial enzyme activity. The results of the relative residual activity of the 6th and 10th batches of coated immobilized nitrile hydratase reaction batch determination test results relative to the initial enzyme activity are shown in Table 1.

[0086] The reaction process of preparing methacrylamide using coated immobilized nitrile hydratase is shown in the figure Figure 5 As shown, the results show that the maximum final concentration of the product methacrylamide reaches 121.02 g / L. At 200 min, the final concentration of the product in the preparation of methacrylamide using coated immobilized nitrile hydratase is shown in Table 1.

[0087] Example 2

[0088] Preparation of coated immobilized nitrile hydratase:

[0089] 1 g of epoxy resin LX-1000HA was added to 9 mL of boric acid-borax buffer (pH 8.0) containing 60 mg of commercial nitrile hydratase protein, and the mixture was incubated at room temperature with low-speed shaking at 150 rpm for 2.5 h; then 1.5 ml of a 50 g / L aqueous solution of polyethyleneimine (straight chain type, relative molecular weight of 1800 g / mol) was added dropwise, and the mixture was continued to be incubated at room temperature with low-speed shaking at 150 rpm for 1 h. Finally, the mixture was centrifuged at 5000 rpm and 4°C for 10 min, and the precipitate was collected and rinsed with boric acid-borax buffer (pH 8.0) until there was no protein residue in the supernatant to obtain the coated immobilized nitrile hydratase.

[0090] No polyethyleneimine was present in the rinse solution obtained after rinsing, indicating that the polyethyleneimine was completely coated on the surface of the immobilized nitrile hydratase.

[0091] The coating amount of polyethyleneimine was 6.7% of the mass of the coated immobilized nitrile hydratase.

[0092] The results of the enzyme specific activity and enzyme loading rate of the coated immobilized nitrile hydratase are shown in Table 1.

[0093] The results of the reaction batch determination of the coated immobilized nitrile hydratase and the relative residual activity of the 6th and 10th batches relative to the initial enzyme activity are shown in Table 1.

[0094] At 200 min, the final concentration of the product in methacrylamide prepared by using the coated immobilized nitrile hydratase is shown in Table 1.

[0095] Example 3

[0096] Preparation of coated immobilized nitrile hydratase:

[0097] 1 g of epoxy resin ECR8214 was added to 9 mL of boric acid-borax buffer (pH 8.0) containing 80 mg of commercial nitrile hydratase protein, and the mixture was incubated at room temperature with low-speed shaking at 200 rpm for 2.5 h; then 0.8 ml of a 50 g / L aqueous solution of polyethyleneimine (straight chain type, relative molecular weight of 1800 g / mol) was added dropwise, and the mixture was continued to be incubated at room temperature with low-speed shaking at 200 rpm for 1 h. Finally, the mixture was centrifuged at 5000 rpm and 4°C for 10 min, the precipitate was collected and rinsed with boric acid-borax buffer (pH 8.0) until there was no protein residue in the supernatant, thereby obtaining a coated immobilized nitrile hydratase.

[0098] No polyethyleneimine was present in the rinse solution obtained after rinsing, indicating that the polyethyleneimine was completely coated on the surface of the immobilized nitrile hydratase.

[0099] The coating amount of polyethyleneimine was 3.7% of the mass of the coated immobilized nitrile hydratase.

[0100] The results of the enzyme specific activity and enzyme loading rate of the coated immobilized nitrile hydratase are shown in Table 1.

[0101] The results of the reaction batch determination of the coated immobilized nitrile hydratase and the relative residual activity of the 6th and 10th batches relative to the initial enzyme activity are shown in Table 1.

[0102] At 200 min, the final concentration of the product in methacrylamide prepared by using the coated immobilized nitrile hydratase is shown in Table 1.

[0103] Example 4

[0104] The method according to Example 1 is different in that:

[0105] The amount of polyethyleneimine aqueous solution added was 0.4 mL.

[0106] No polyethyleneimine was present in the rinse solution obtained after rinsing, indicating that the polyethyleneimine was completely coated on the surface of the immobilized nitrile hydratase.

[0107] The coating amount of polyethyleneimine was 1.9% of the mass of the coated immobilized nitrile hydratase.

[0108] The results of the enzyme specific activity and enzyme loading rate of the coated immobilized nitrile hydratase are shown in Table 1.

[0109] The results of the reaction batch determination of the coated immobilized nitrile hydratase and the relative residual activity of the 6th and 10th batches relative to the initial enzyme activity are shown in Table 1.

[0110] At 200 min, the final concentration of the product in methacrylamide prepared by using the coated immobilized nitrile hydratase is shown in Table 1.

[0111] Example 5

[0112] The method according to Example 1 is different in that:

[0113] The amount of polyethyleneimine aqueous solution added was 4 mL.

[0114] About 50 mg of polyethyleneimine was present in the rinse solution obtained after rinsing, indicating that polyethyleneimine was partially coated on the surface of the immobilized nitrile hydratase, and the coating amount of polyethyleneimine was 12.5% ​​of the mass of the coated immobilized nitrile hydratase.

[0115] The results of the enzyme specific activity and enzyme loading rate of the coated immobilized nitrile hydratase are shown in Table 1.

[0116] The results of the reaction batch determination of the coated immobilized nitrile hydratase and the relative residual activity of the 6th and 10th batches relative to the initial enzyme activity are shown in Table 1.

[0117] At 200 min, the final concentration of the product in methacrylamide prepared by using the coated immobilized nitrile hydratase is shown in Table 1.

[0118] Example 6

[0119] The method according to Example 1 is different in that:

[0120] 1 ml of a 50 g / L aqueous solution of polyethyleneimine (straight chain type, relative molecular weight of 3000 g / mol) was added dropwise.

[0121] The coating amount of polyethyleneimine was 4.5% of the mass of the coated immobilized nitrile hydratase.

[0122] The results of the enzyme specific activity and enzyme loading rate of the coated immobilized nitrile hydratase are shown in Table 1.

[0123] The results of the reaction batch determination of the coated immobilized nitrile hydratase and the relative residual activity of the 6th and 10th batches relative to the initial enzyme activity are shown in Table 1.

[0124] At 200 min, the final concentration of the product in methacrylamide prepared by using the coated immobilized nitrile hydratase is shown in Table 1.

[0125] Comparative Example 1

[0126] Preparation of immobilized nitrile hydratase:

[0127] 1 g of epoxy resin ES-103B (a) was added to a boric acid-borax buffer (pH 8.0) containing 50 mg of commercial nitrile hydratase protein, and the mixture was incubated at room temperature with low-speed shaking at 100 rpm for 2 h. Finally, the mixture was centrifuged at 5000 rpm and 4°C for 10 min. The precipitate was collected and rinsed with a boric acid-borax buffer (pH 8.0) until there was no protein residue in the supernatant to obtain immobilized nitrile hydratase.

[0128] The results of the enzyme specific activity and enzyme loading rate of immobilized nitrile hydratase are shown in Table 1.

[0129] Reaction batch assay results of immobilized nitrile hydratase The results of the relative residual activity relative to the initial enzyme activity of the 6th and 10th batches are shown in Table 1.

[0130] The final concentration results of the products in the preparation of methacrylamide using immobilized nitrile hydratase at 200 min are shown in Table 1.

[0131] Comparative Example 2

[0132] Preparation of non-encapsulated immobilized nitrile hydratase:

[0133] 2 mL of boric acid-borax buffer (pH 8.0) containing 50 mg of commercial nitrile hydratase protein was mixed with 1 mL of 50 g / L polyethyleneimine (straight chain type, relative molecular weight of 1800 g / mol) to obtain a mixed solution, 7 mL of boric acid-borax buffer (pH 8.0) was mixed with 1 g of epoxy resin ES-103B (a) to obtain a suspension, and then the mixed solution was added dropwise to the suspension, incubated at room temperature with low speed shaking at 100 rpm for 1 h, and finally centrifuged at 5000 rpm and 4°C for 10 min, the precipitate was collected and rinsed with boric acid-borax buffer (pH 8.0) until there was no protein residue in the supernatant to obtain immobilized nitrile hydratase.

[0134] The results of the enzyme specific activity and enzyme loading rate of immobilized nitrile hydratase are shown in Table 1.

[0135] Reaction batch assay results of immobilized nitrile hydratase The results of the relative residual activity relative to the initial enzyme activity of the 6th and 10th batches are shown in Table 1.

[0136] The final concentration results of the products in the preparation of methacrylamide using immobilized nitrile hydratase at 200 min are shown in Table 1.

[0137] Table 1

[0138]

[0139]

[0140] It can be seen from the results in Table 1 that the coated immobilized nitrile hydratase of the present invention is used to catalyze the hydration of the substrate in an ethylenically unsaturated nitrile substrate (such as methacrylonitrile), which not only has a high initial catalytic activity, but also can maintain a high relative residual enzyme activity after repeatedly using the coated immobilized nitrile hydratase for multiple times.

[0141] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A coated immobilized nitrile hydratase, characterized in that: The invention comprises immobilized nitrile hydratase and polyethyleneimine coated on the surface of the immobilized nitrile hydratase.

2. The coated immobilized nitrile hydratase according to claim 1, wherein The coating amount of the polyethyleneimine is 2-10wt% of the mass of the coated immobilized nitrile hydratase, preferably 3-8wt%.

3. The coated immobilized nitrile hydratase according to claim 1 or 2, wherein The polyethyleneimine comprises branched polyethyleneimine and / or linear polyethyleneimine, preferably linear polyethyleneimine; and / or The molecular weight of the polyethyleneimine is greater than 1000 g / mol, preferably 1200-2000 g / mol.

4. The coated immobilized nitrile hydratase according to any one of claims 1 to 3, wherein The immobilized nitrile hydratase comprises an immobilized enzyme carrier and a nitrile hydratase loaded on the immobilized enzyme carrier, and preferably the immobilized enzyme carrier and the nitrile hydratase are connected by a covalent bond; Preferably, the loading amount of nitrile hydratase per gram of the immobilized enzyme carrier is not less than 40 mg, preferably 40-50 mg.

5. The coated immobilized nitrile hydratase according to claim 4, wherein The particle size of the immobilized enzyme carrier is 10-1000 μm, preferably 100-500 μm; and / or The immobilized enzyme carrier comprises epoxy resin; Preferably, the epoxy resin includes at least one of LX-1000HA, LX-103B, LX-1000EP F, LX-1000EP, LX-1000HFA, ES-103B(a), ES-103B(b), ES-1, ECR8214 and ECR4204.

6. A method for preparing the coated immobilized nitrile hydratase according to any one of claims 1 to 5, characterized in that: The preparation method comprises: The weak alkaline buffer solution containing the immobilized nitrile hydratase is contacted with the polyethyleneimine solution, and then post-treated to obtain the coated immobilized nitrile hydratase.

7. The preparation method according to claim 6, wherein: The mass ratio of the immobilized nitrile hydratase to polyethyleneimine is 100:(2-10), preferably 100:(3-8); Preferably, in the weakly alkaline buffer solution containing the immobilized nitrile hydratase, the concentration of the immobilized nitrile hydratase is not less than 100 mg / mL, preferably 100-120 mg / mL.

8. The preparation method according to claim 6 or 7, wherein: The weak alkaline buffer solution containing immobilized nitrile hydratase is obtained by incubating a nitrile hydratase raw material and an immobilized enzyme carrier in a weak alkaline buffer solution to obtain the weak alkaline buffer solution containing immobilized nitrile hydratase; Preferably, the pH value of the weakly alkaline buffer solution is 7.5-8.5; and / or The incubation conditions include: room temperature; and / or the rotation speed is 50-200 rpm; and / or the incubation time is 1-3 h.

9. The preparation method according to claim 8, wherein: The mass ratio of the nitrile hydratase raw material to the immobilized enzyme carrier is not less than 40 mg / g, preferably 50-80 mg / g, based on the pure nitrile hydratase agent; and / or The nitrile hydratase raw material is selected from at least one of cells having nitrile hydratase activity, cell disrupted liquid having nitrile hydratase activity and purified nitrile hydratase preparation, preferably purified nitrile hydratase preparation.

10. Use of the coated immobilized nitrile hydratase according to any one of claims 1 to 5 in a hydration reaction; Preferably, the coated immobilized nitrile is used in the preparation of ethylenically unsaturated amides by continuous hydration reaction of ethylenically unsaturated nitrile.

11. A method for preparing methacrylamide, characterized in that: The preparation method comprises: In the presence of an aqueous medium and the coated immobilized nitrile hydratase according to any one of claims 1 to 5, methacrylonitrile undergoes a hydration reaction.

12. The preparation method according to claim 11, wherein The pH value of the aqueous medium is 7.5-8.5, and preferably the aqueous medium comprises a boric acid-borax buffer; and / or The coated immobilized nitrile hydratase is calculated by mass and the methacrylonitrile is calculated by mole, and the ratio of methacrylonitrile to the coated immobilized nitrile hydratase is not greater than 2 mol / g, preferably 0.1-1.4 mol / g; and / or The conditions of the hydration reaction include: room temperature; and / or a rotation speed of 100-300 rpm.

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