Immobilized enzyme as well as preparation and application thereof

Abstract

The invention discloses an immobilized enzyme and a preparation method and application thereof.The immobilized enzyme comprises a carrier, protease and an immobilization layer, the immobilization layer comprises amino molecules and aldehyde molecules, the amino molecules comprise dendritic low polyetherimide, the amino molecules are formed through secondary hydroformylation of the aldehyde molecules, and the immobilized enzyme is rich in protease binding sites, high in enzyme activity and high in enzyme digestion efficiency; the method is suitable for a sample pretreatment process of chromatographic detection.

Description

Technical Field

[0001] This invention relates to the field of immobilized enzymes, and more specifically, to an immobilized enzyme, its preparation method, and its applications. Background Technology

[0002] Proteases effectively break down proteins, breaking down peptide bonds that link amino acids into polypeptide chains, and are widely found in various organisms. With the development of modern science and technology, proteases have become industrial enzyme preparations used in biotechnology, analytical testing, and other fields. Due to their high enzymatic digestion efficiency and specificity, proteases are widely used in the production of polypeptide / protein drugs.

[0003] Currently, proteases are often used by directly adding them to the protein drug precursor solution. Once added, these exogenous enzymes are difficult to separate and reuse. Enzyme preparations are expensive, and each use increases the cost burden. This operation has significant follow-up problems, as it easily leads to the self-degradation of proteases, resulting in low overall enzyme activity and low utilization. More seriously, the products of protease self-degradation, such as peptide chains or amino acids, enter downstream products, making the separation process more difficult and even affecting product quality.

[0004] Immobilizing enzymes is one of the effective means to improve the above-mentioned problems of enzyme preparations, increase enzyme utilization efficiency and reduce costs. While maintaining its high efficiency, specificity and mild enzyme catalytic reaction characteristics, immobilized enzymes overcome the above-mentioned shortcomings of free enzymes. Moreover, immobilized proteases have high storage stability and are easy to separate and recover for multiple uses.

[0005] For example, existing Chinese patent document CN111220680 discloses a protein adsorbent material and its application. Using graphene oxide as a carrier, it modifies the surface with polyethyleneimine through hydrogen bonding and electrostatic interactions. Polyethyleneimine acts as a reducing agent and stabilizer. Through a redox reaction with chloroauric acid at high temperature, nano-gold particles are immobilized on the graphene oxide surface. Finally, polymer microspheres are added, and by stirring at room temperature, the graphene oxide nano-gold composite material is modified on the polymer microsphere surface using a non-covalent bonding method, ultimately achieving the preparation of the protein adsorbent material. Its adsorption is mainly physical adsorption, not covalent adsorption. If used as a carrier for immobilizing enzymes, this non-specific adsorption can lead to the adsorption of drug proteins, and the adsorption density of the enzyme protein is low, resulting in low enzymatic hydrolysis efficiency.

[0006] Another Chinese patent document, CN1975429B, discloses a type of magnetic nanosphere with a protease immobilized on its surface. The magnetic nanosphere is prepared by a one-step hydrothermal synthesis method, using glutaraldehyde as a crosslinking agent. The surface has amino groups, which can reduce non-specific protein adsorption. However, it still has technical problems such as low enzyme-protein density, unstable binding, and low enzymatic hydrolysis efficiency. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the low protein density, unstable binding, and low enzymatic hydrolysis efficiency of immobilized enzymes in the prior art, thereby providing an immobilized enzyme and its preparation and application.

[0008] Therefore, the present invention discloses an immobilized enzyme, comprising a carrier, a protease, and an immobilization layer, wherein the immobilization layer connects the carrier and the protease, the immobilization layer comprises amino molecules and aldehyde molecules, the amino molecules comprising dendritic oligoether imide with a molecular weight of 400-800, and the immobilized enzyme having a particle size of 50nm-30μm.

[0009] The amino molecule also includes PEI, the molecular weight of which is 400-1000, and the aldehyde molecule is glutaraldehyde.

[0010] The protease includes one or more of trypsin, pepsin, carboxypeptidase B, chymotrypsin, gamma-glutamyl protease, lysine protease, proteinase K, and TEV protease.

[0011] The carrier is a silicon sphere or a magnetic nanosphere, and the surface of the carrier contains amino groups, which are attached to the molecule before synthesis and have amino groups on the surface after synthesis. The particle size is 45nm-30μm.

[0012] The present invention also discloses a method for preparing the immobilized enzyme, comprising the following steps:

[0013] S1. After washing the carrier with buffer solution, it undergoes a first reaction with aldehyde solution, resulting in aldehyde groups on the carrier surface;

[0014] S2. The aldehyde-modified support further reacts with amino molecules, resulting in amino modification on the support surface;

[0015] S3. The support obtained in S2 is reacted with the aldehyde solution for a second reaction;

[0016] S4. React the carrier obtained in S3 with the protease solution to achieve protease immobilization.

[0017] The aldehyde solution contains 5%-20% glutaraldehyde by mass and has a pH of 6.8-8.0. The reaction time between the carrier and the aldehyde solution is 30-60 min, and the reaction temperature is 20-25℃.

[0018] The concentration of the amino solution is 50-200 mg / mL, the reaction temperature of the carrier and the amino molecule is 20-25℃, and the reaction time is 12-18 h.

[0019] In step S4, an enzyme solution with a concentration of 20-25 mg / mL is added to the modified vector and incubated at 20-25°C for 16-24 h.

[0020] The mass ratio of the amino molecule to the aldehyde molecule is 1:(1-2).

[0021] The present invention also discloses the application of an immobilized enzyme prepared by the method described above in the field of processing proteins in cells, tissues, plasma, and urine and performing mass spectrometry analysis.

[0022] The technical solution of this invention has the following advantages:

[0023] 1. The immobilized enzyme of this invention uses dendritic polyether amide with a molecular weight of 400-800 as a linker. Through a secondary aldehyde-relation reaction with glutaraldehyde, the surface of the immobilized enzyme has abundant enzyme binding sites. The trypsin binding amount on a 5μm ordinary silicon sphere is 0.5 mg / 0.1g; after PEI is immobilized on the silicon sphere, the trypsin binding amount can reach 10 mg / 0.1g, resulting in a high protease concentration per unit volume. Furthermore, the high concentration of protease on the surface of the solid enzyme also helps to improve production efficiency. It can hydrolyze the target protein as much as possible, improving the enzymatic digestion efficiency.

[0024] 2. The immobilized enzyme described in this invention enables rapid recovery and reuse of proteases. Even after multiple reuses, it retains relatively high enzyme activity, thereby reducing production costs, minimizing the self-degradation of proteases, improving product quality, and preventing the introduction of degradation products that could affect subsequent analysis.

[0025] 3. The immobilized enzyme described in this invention uses the aldehyde group of glutaraldehyde to covalently bind with the amino group of dendritic oligoether imide. Through layer-by-layer modification, a network structure with abundant aldehyde, amino, and hydroxyl groups is formed. It can bind to the protease through various mechanisms such as covalent interaction, hydrogen bonding, ionic interaction, and hydrophobic interaction, resulting in higher protease activity and stronger enzymatic digestion efficiency.

[0026] 4. Compared to traditional liquid enzymes, the solid enzyme provided by this invention can be recovered by continuously adding new samples that need to be enzymatically hydrolyzed; in addition to the analytical field, it can also be used in production, which can eliminate impurities generated by enzyme self-degradation, improve product quality, and reduce the cost of enzyme use. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1To evaluate the distribution of missing cut rates in Example 2 using the solution method and the solid enzyme method: the solution method was used for 30 min of enzymatic digestion; the solid enzyme method was used for 1 min of enzymatic digestion.

[0029] Figure 2 To evaluate the complete sequence coverage of bovine serum albumin (BSA) in Example 2;

[0030] Figure 3 To evaluate the sequence coverage of bovine serum albumin obtained in repeated trials in Example 2. Detailed Implementation

[0031] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0032] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0033] The aminosilicone balls are UniSil amino chromatography packing materials (Suzhou Nanomicro), using dendritic low polyether imide with a molecular weight distribution of 400-800. The product numbers can be found as sigma 408719 and 408700.

[0034] Example 1

[0035] This embodiment provides a specific implementation method for immobilized enzymes, including the following steps:

[0036] 20 mg of aminosilicone spheres with an average particle size of 100 nm were washed with PB buffer containing 0.5 M sodium chloride at pH 8.0 and then dispersed in 1 mL of glutaraldehyde solution with a mass fraction of 20% and a pH of 7. The mixture was shaken at 25 °C for 60 min and then centrifuged at 8000 rpm for 10 min to remove the supernatant.

[0037] The obtained carrier was placed in 4 mL of dendritic polyether amide solution with a concentration of 100 mg / mL and a molecular weight of 400, and the reaction was carried out at 25 °C with shaking at 1400 rpm for 18 h. The supernatant was removed by centrifugation at 10000 rpm for 10 min.

[0038] The carrier modified with dendritic polyether amide solution was divided into two identical portions:

[0039] One portion of the solution was dispersed in 4 ml of trypsin solution with a concentration of 25 mg / mL, incubated at 25°C for 24 hours, centrifuged at 8000 rpm for 5 min to remove the supernatant, dispersed with PB buffer, and washed by centrifugation at 8000 rpm for 3 min to obtain the immobilized vector A1.

[0040] Another sample was reacted again with 1 mL of glutaraldehyde solution with a mass fraction of 20% and a pH of 7. The reaction was carried out with shaking at room temperature for 60 min. After the reaction, the sample was centrifuged at 8000 rpm for 15 min to remove the supernatant. Then, it was dispersed in 4 mL of trypsin solution with a concentration of 25 mg / mL and incubated at 20 °C for 24 h. After centrifugation at 8000 rpm for 5 min to remove the supernatant, the sample was dispersed with PB buffer and washed by centrifugation at 8000 rpm for 3 min to obtain the immobilized vector A2.

[0041] Example 2

[0042] This embodiment provides a specific implementation method for immobilized enzymes, including the following steps:

[0043] 20 mg of aminosilicone spheres with an average particle size of 45 nm were washed with PB buffer containing 0.4% sodium chloride and pH 6.8 and then dispersed in 1 mL of glutaraldehyde solution with a mass fraction of 10% and pH 7. The mixture was shaken at 25 °C for 30 min and then centrifuged at 8000 rpm for 10 min to remove the supernatant.

[0044] The obtained carrier was placed in 4 mL of dendritic polyether amide solution with a concentration of 100 mg / mL and a molecular weight of 800, and the reaction was carried out at 25 °C with shaking at 1400 rpm for 18 h. The supernatant was removed by centrifugation at 10000 rpm for 10 min.

[0045] The carrier modified with dendritic polyether amide solution was divided into two identical portions:

[0046] One sample was dispersed in 4 ml of lysinase solution with a concentration of 20 mg / mL, incubated at 25°C for 16 hours, centrifuged at 8000 rpm for 5 min to remove the supernatant, dispersed in PB buffer, and washed by centrifugation at 8000 rpm for 3 min to obtain immobilized vector B1.

[0047] Another sample was reacted again with 1 mL of glutaraldehyde solution with a mass fraction of 10% and 20% and a pH of 7. The reaction was carried out with shaking at room temperature for 60 min. After the reaction, the sample was centrifuged at 8000 rpm for 15 min to remove the supernatant. Then, it was dispersed in 4 mL of lysinase solution with a concentration of 25 mg / mL and incubated at 20 °C for 24 h. After centrifugation at 8000 rpm for 5 min to remove the supernatant, the sample was dispersed with PB buffer and washed by centrifugation at 8000 rpm for 3 min to obtain the immobilized carrier B2.

[0048] Example 3

[0049] This embodiment provides a specific implementation method for immobilized enzymes, including the following steps:

[0050] 20 mg of aminosilicone spheres with an average particle size of 500 nm were washed with PB buffer containing 0.4% sodium chloride and pH 6.8 and then dispersed in 1 mL of glutaraldehyde solution with a mass fraction of 5% and pH 7. The mixture was shaken at 25 °C for 30 min and then centrifuged at 8000 rpm for 10 min to remove the supernatant.

[0051] The obtained carrier was placed in 4 mL of dendritic polyether amide solution with a concentration of 50 mg / mL and a molecular weight of 800, and the reaction was carried out at 25 °C with shaking at 1400 rpm for 18 h. The supernatant was removed by centrifugation at 10000 rpm for 10 min.

[0052] The carrier modified with dendritic polyether amide solution was divided into two identical portions:

[0053] One sample was dispersed in 4 ml of chymotrypsin solution with a concentration of 20 mg / mL, incubated at 25°C for 16 hours, centrifuged at 8000 rpm for 5 min to remove the supernatant, dispersed with PB buffer, and washed by centrifugation at 8000 rpm for 3 min to obtain the immobilized vector C1.

[0054] Another sample was reacted again with 1 mL of glutaraldehyde solution with a mass fraction of 5% and a pH of 7. The reaction was carried out with shaking at room temperature for 60 min. After the reaction, the sample was centrifuged at 8000 rpm for 15 min to remove the supernatant. Then, it was dispersed in 4 mL of chymotrypsin solution with a concentration of 25 mg / mL and incubated at 20 °C for 24 h. After centrifugation at 8000 rpm for 5 min to remove the supernatant, the sample was dispersed with PB buffer and washed by centrifugation at 8000 rpm for 3 min to obtain the immobilized vector C2.

[0055] Example 4

[0056] This embodiment provides a specific implementation method for immobilized enzymes, including the following steps:

[0057] 20 mg of aminosilicone spheres with an average particle size of 50 μm were washed with PB buffer containing 0.5 M sodium chloride at pH 8.0 and then dispersed in 2 mL of glutaraldehyde solution with a mass fraction of 20% and a pH of 7. The mixture was shaken at 25 °C for 60 min and then centrifuged at 8000 rpm for 10 min to remove the supernatant.

[0058] The obtained carrier was placed in 4 mL of dendritic polyether amide solution with a concentration of 200 mg / mL and a molecular weight of 600, and the reaction was carried out at 25 °C with shaking at 1400 rpm for 18 h. The supernatant was removed by centrifugation at 10000 rpm for 10 min.

[0059] The carrier modified with dendritic polyether amide solution was divided into two identical portions:

[0060] One portion was dispersed in 4 ml of pepsin solution with a concentration of 25 mg / mL, incubated at 25°C for 24 hours, centrifuged at 8000 rpm for 5 min to remove the supernatant, dispersed with PB buffer, and washed by centrifugation at 8000 rpm for 3 min to obtain the immobilized vector D1.

[0061] Another sample was reacted again with 2 mL of 20% glutaraldehyde solution at pH 7, and the reaction was carried out with shaking at room temperature for 60 min. After the reaction, the sample was centrifuged at 8000 rpm for 15 min to remove the supernatant. Then it was dispersed in 4 mL of pepsin solution with a concentration of 25 mg / mL, incubated at 20 °C for 24 hours, centrifuged at 8000 rpm for 5 min to remove the supernatant, and then washed with PB buffer and centrifuged at 8000 rpm for 3 min to obtain the immobilized vector D2.

[0062] Example 5

[0063] This embodiment provides a specific implementation method for immobilized enzymes, including the following steps:

[0064] 20 mg of aminosilicone spheres with an average particle size of 30 μm were washed with PB buffer containing 0.5 M sodium chloride at pH 8.0 and then dispersed in 2 mL of glutaraldehyde solution with a mass fraction of 15% and a pH of 7. The mixture was shaken at 25 °C for 60 min and then centrifuged at 8000 rpm for 10 min to remove the supernatant.

[0065] The obtained carrier was placed in 3 mL of dendritic polyether amide solution with a concentration of 200 mg / mL and a molecular weight of 800, and the reaction was carried out at 25 °C with shaking at 1400 rpm for 18 h. The supernatant was removed by centrifugation at 10000 rpm for 10 min.

[0066] The carrier modified with dendritic polyether amide solution was divided into two identical portions:

[0067] One portion was dispersed in 4 ml of trypsin solution with a concentration of 25 mg / mL, incubated at 25°C for 24 hours, centrifuged at 8000 rpm for 5 min to remove the supernatant, dispersed with PB buffer, and washed by centrifugation at 8000 rpm for 3 min to obtain the immobilized vector E1.

[0068] Another sample was reacted again with 2 mL of 15% glutaraldehyde solution at pH 7, and the reaction was carried out with shaking at room temperature for 60 min. After the reaction, the sample was centrifuged at 8000 rpm for 15 min to remove the supernatant. Then it was dispersed in 4 mL of trypsin solution with a concentration of 25 mg / mL, incubated at 20 °C for 24 hours, centrifuged at 8000 rpm for 5 min to remove the supernatant, and then washed with PB buffer and centrifuged at 8000 rpm for 3 min to obtain the immobilized vector E2.

[0069] Evaluation of enzyme activity in Example 1

[0070] The enzyme activity evaluation indicators in this example include two aspects: enzyme bond amount and enzyme digestion efficiency. These can be evaluated by the time required to complete the enzyme digestion or the percentage of uncut sites remaining after the same digestion time.

[0071] The assay method was as follows: using 10 mg / mL casein solution as the reaction substrate, 1 mL of the immobilized enzyme solution that had undergone secondary aldehyde alteration as described in the examples was added, and the mixture was incubated at 40 °C for 20 min. Then, 2 mL of 0.4 M trichloroacetic acid was added, and the mixture was incubated for 5 min. 1 mL of the supernatant was taken and 5 mL of 0.4 M sodium carbonate solution and 1 mL of Folin reagent were added. The mixture was incubated at 40 °C for 20 min, and the OD value at 680 nm was measured. With A2 enzyme activity as 100%, the following results were obtained, as shown in Table 1.

[0072] After the immobilized enzyme was isolated and reused, and after being reused 5 times, its enzyme activity was measured, and it could still maintain more than 50% of the initial activity.

[0073] The residual activity of the immobilized enzyme after storage at 4°C for 10, 60, and 120 days was used to assess the storage stability of the immobilized enzyme. For this stability experiment, the initial activity of the immobilized enzyme was assumed to be 100%, while other activities were relative to the initial activity. As shown in Table 1, the immobilized enzyme after two aldehyde treatments retained more than 50% of its initial activity after 120 days of storage, while the immobilized enzyme after one aldehyde treatment retained only about 20% of its initial activity after 120 days of storage.

[0074] Table 1 Enzyme activity detection results

[0075]

[0076] The enzyme concentration added to the reaction system was known. After the reaction, the enzyme concentration in the reaction solution was determined using the BCA method; the decrease in concentration was the amount of trypsin bonded. A 5μm ordinary silicon sphere showed a trypsin bond of 0.5 mg / 0.1 g. However, testing showed that after PEI was immobilized on silicon spheres in Example 1, the trypsin bond amount could reach 10 mg / 0.1 g.

[0077] Evaluation Example 2: Enzyme digestion efficiency

[0078] IP magnetic beads immobilized with the target protein were washed with 200 μL of PBS, repeated 5 times; 100 μL of pH 8 buffer solution and 10 μL of 100 mM dithiothreitol reducing solution were added, and the mixture was incubated at 56°C with shaking for 1 h; after cooling to room temperature, 10 μL of 200 mM iodoacetamide alkylation solution was added, and the mixture was incubated at room temperature with shaking in the dark for 30 min; 1 μL of the A2 immobilized enzyme dispersion described in Example 5 was added, and the mixture was incubated at 37°C with shaking for 12-18 h; 5 μL of enzyme digestion terminator was added to terminate the digestion. Because the high concentration of protease is bonded to the silica gel surface, protein digestion can be achieved rapidly. Figure 1 and Figure 2 As stated, complete coverage of the BSA sequence can be achieved in just 1 minute.

[0079] Using a 0.1% (w / w) formic acid aqueous solution as phase A and an 80% (v / v) acetonitrile aqueous solution as phase B, the following operations were performed:

[0080] The desalting column was washed with 200 μL of phase B, and then equilibrated with 200 μL of phase A. The supernatant was loaded onto the desalting column, and the column was washed again with 200 μL of phase A. The peptide was then eluted with 200 μL of phase B. The eluent was collected, freeze-dried, and then dissolved in 20 μL of phase A before being loaded onto the instrument. The test results are as follows: Figure 2 and Figure 3 As stated above.

[0081] Enzymatic digestion efficiency calculation method: Trypsin only cleaves the C-terminus of K / R. After enzymatic digestion using standard protein and identification by mass spectrometry, the proportion of cleaved K / R to the total K / R of the protein is analyzed. The proportion of uncleaved K / R to the total K / R of the protein is called the missed cleavage rate.

[0082] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An immobilized enzyme, characterized in that, The invention includes a carrier, a protease, and an immobilization layer. The immobilization layer connects the carrier and the protease. The immobilization layer includes amino molecules and aldehyde molecules. The amino molecules include dendritic oligoether imide with a molecular weight of 400-800. The immobilized enzyme has a particle size of 50 nm-30 μm.

2. The immobilized enzyme according to claim 1, characterized in that, The amino molecule also includes PEI, the molecular weight of which is 400-1000, and the aldehyde molecule is glutaraldehyde.

3. The immobilized enzyme according to claim 1 or 2, characterized in that, The protease includes one or more of trypsin, pepsin, carboxypeptidase B, chymotrypsin, gamma-glutamyl protease, lysine protease, proteinase K, and TEV protease.

4. The immobilized enzyme according to any one of claims 1-3, characterized in that, The carrier is a silicon sphere or a magnetic nanosphere, and the surface of the carrier contains amino groups, which are attached to the molecule before synthesis and have amino groups on the surface after synthesis. The particle size is 45nm-30μm.

5. A method for preparing the immobilized enzyme according to any one of claims 1-4, characterized in that, Includes the following steps: S1. After washing the carrier with buffer solution, it undergoes a first reaction with aldehyde solution, resulting in aldehyde groups on the carrier surface; S2. The aldehyde-modified support further reacts with amino molecules, resulting in amino modification on the support surface; S3. The support obtained in S2 is reacted with the aldehyde solution for a second reaction; S4. React the carrier obtained in S3 with the protease solution to achieve protease immobilization.

6. The method for preparing immobilized enzymes according to claim 5 or 6, characterized in that, The aldehyde solution contains 5%-20% glutaraldehyde by mass and has a pH of 6.8-8.

0. The reaction time between the carrier and the aldehyde solution is 30-60 min, and the reaction temperature is 20-25℃.

7. The method for preparing immobilized enzymes according to claim 5 or 6, characterized in that, The concentration of the amino solution is 50-200 mg / mL, the reaction temperature of the carrier and the amino molecule is 20-25℃, and the reaction time is 12-18 h.

8. The method for preparing immobilized enzymes according to any one of claims 5-7, characterized in that, In step S4, an enzyme solution with a concentration of 20-25 mg / mL is added to the modified vector and incubated at 20-25°C for 16-24 h.

9. The method for preparing immobilized enzyme according to any one of claims 5-8, characterized in that, The mass ratio of the amino molecule to the aldehyde molecule is 1:(1-2).

10. The application of an immobilized enzyme according to any one of claims 1-4 or an immobilized enzyme prepared by the method of immobilized enzyme preparation according to any one of claims 5-9 in the field of processing proteins in cells, tissues, plasma, and urine and performing mass spectrometry analysis.

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