An enzyme protective agent and its application

Abstract

The present application discloses an enzyme protectant and its application. The protectant uses pH 6.5-7.5 buffer as solvent and includes the following components: glycerol, 25%-35%; serine, 25-45mmol / L; CaCl2, 5-15mmol / L. The present application adopts a relatively simple protectant to significantly improve the stability of the enzyme at room temperature and under heat stress, which not only prolongs the service life of the enzyme, but also does not contain any heavy metal salts and reducing agents, effectively avoiding the interference of the protectant with the detection reaction.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an enzyme protecting agent and application thereof. Background Art

[0002] Horseradish peroxidase (HRP, EC 1.11.1.7) is a type III peroxidase with heme as the redox center, with a molecular weight of about 44kD. It is a classic peroxidase secreted by plants. Horseradish peroxidase catalyzes the redox reaction of peroxide and hydrogen donor (DH2). Due to its high specific activity, stability, small molecular weight, and easy preparation of pure enzyme, horseradish peroxidase has a wide range of applications. It is not only used in multiple biochemical detection projects such as blood sugar and urine, but also used in enzyme-linked immunosorbent assay (ELISA) kits, with rapid reaction and high sensitivity.

[0003] Since protein enzymes are usually less stable and easily inactivated at room temperature, most diagnostic enzymes including HRP and corresponding diagnostic reagents need to be stored and transported at low temperature (4°C) to ensure that the test kit has a sufficiently long shelf life and has normal detection performance within the validity period. This is not only inconvenient for the transportation, sales, storage and use of diagnostic enzymes and corresponding diagnostic reagents, but also significantly increases the use cost of the detection products and increases the economic burden on patients. If a protective agent that enhances the thermal stability of the diagnostic enzyme can be developed so that the diagnostic enzyme can remain stable for a long time at room temperature, it will not only free the use of the diagnostic enzyme from the constraints of the cold chain, facilitate its storage, transportation and use, but also reduce the cost of use and reduce the testing costs of patients.

[0004] Currently, many scientific research teams have studied thermal protectants for diagnostic enzymes and invented reagents that help enhance the stability of HRP. For example, the Chinese invention patent application with publication number CN 103642781A discloses a horseradish peroxidase protective agent that can significantly improve the stability of the HRP conjugate after coupling with HRP and antibodies. However, the protective agent disclosed in this application has a relatively complex composition, containing 10 substances. Not only is the composition relatively complex, but the ZnSO4 contained therein is a heavy metal salt that is likely to cause denaturation and inactivation of the enzyme or antibody. Therefore, the protective agent may interfere with the binding of the antibody to the antigen or the chemical reaction of the enzyme to the detection substrate, thereby causing inaccurate test results. The Chinese invention patent application with publication number CN103031291A discloses a room temperature storage buffer for proteinase K, the main components of which are water, Tris, and Ca. 2+ and glycerol, and the optimal concentration of glycerol is 50%. Although such a high concentration of glycerol can play a good enzyme protection effect, it will also cause the viscosity of the enzyme solution to increase significantly, which is not conducive to the packaging, removal and use of the enzyme solution. Summary of the invention

[0005] The present application provides an enzyme protectant, which has simple components and low glycerol content, and the presence of serine can significantly improve the stability of the enzyme.

[0006] An enzyme protectant, using pH 6.5-7.5 buffer as solvent, comprises the following components:

[0007] Glycerol volume percentage 25% to 35%;

[0008] Serine 25-45 mmol / L;

[0009] Ca 2+ 5~15mmol / L.

[0010] Optionally, the buffer is sodium phosphate buffer, pH 6.5-7.5, 0.05-0.25 mol / L.

[0011] Furthermore, the buffer is sodium phosphate buffer, pH 6.5, 0.2 mol / L.

[0012] Optionally, the content of serine is 38-42 mmol / L. Further, the content of serine is 40 mmol / L;

[0013] Optionally, the volume percentage of glycerol in the enzyme protecting agent is 30% to 35%, and further 32.5%.

[0014] Optionally, the Ca 2+ The content of Ca is 8-10 mmol / L; 2+ It is CaCl2.

[0015] Most preferably, the content of glycerol is 32.5%; the content of serine is 40 mmol / L; and the content of CaCl2 is 9 mmol / L.

[0016] Glycerol is a common enzyme protectant that can prevent protein unfolding through spatial repulsion with polypeptide chains. However, due to the limitation of its mechanism of action, glycerol is often used at high concentrations when used as a room temperature protectant for enzymes, generally at a concentration of about 40% to 60%, and the optimal concentration is even as high as 50%. This will make the enzyme solution with the added protectant extremely viscous, making it extremely inconvenient to dispense and pipette. 2+ It is a known HRP stabilizer. HRP and Ca 2+ After binding, the strength of the three-dimensional structure of HRP can be improved. Serine has not been used in enzyme protectants.

[0017] The present application found that adding serine in the presence of glycerol and CaCl2 can significantly improve the stability of the enzyme. The mechanism is not yet fully understood, but it may be related to its ability to work together with glycerol and CaCl2 to further stabilize the three-dimensional structure of the protein. Because when glycerol is added alone, the glycerol concentration must be higher than 40% to have a protective effect on HRP. When serine and CaCl2 are present, a protective effect can be produced at a glycerol concentration of 30%.

[0018] The present application also provides a use of the enzyme protectant in the storage or transportation of enzymes at room temperature.

[0019] Specifically, the present application also provides a method for storing an enzyme at room temperature, wherein the enzyme protectant is mixed with the enzyme and the mixture is placed at room temperature.

[0020] Optionally, after the enzyme protectant is mixed with the enzyme, the final concentration of the enzyme is 2 to 50 mg / L.

[0021] Optionally, the enzyme is horseradish peroxidase or glutamate decarboxylase.

[0022] The present application also provides a use of serine in preparing a protective agent for horseradish peroxidase or glutamate decarboxylase.

[0023] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0024] (1) The use of a relatively simple protective agent can significantly improve the stability of horseradish peroxidase or glutamate decarboxylase at room temperature and under heat stress. This protective agent not only prolongs the service life of the enzyme, but also does not contain any heavy metal salts and reducing agents, effectively avoiding the interference of the protective agent with the detection reaction.

[0025] (2) The presence of serine can reduce the concentration of glycerol in the protective agent (the optimal concentration is less than 35%), so that the enzyme solution after adding the protective agent has normal viscosity and fluidity, does not affect the transfer of the enzyme solution, and is conducive to the packaging and use of the enzyme solution.

[0026] (3) The protection of the enzymatic activity of free enzymes during storage and transportation is a technical difficulty. The enzyme protectant of the present application can significantly improve the activity of free enzymes.

[0027] (4) After the enzyme protectant of the present application was mixed with horseradish peroxidase and incubated at 47° C. for 24 hours, the residual enzyme activity of HRP was still maintained at 85.12%.

[0028] (5) When the enzyme protectant of the present application was mixed with horseradish peroxidase and incubated at room temperature for 28 days, the residual enzyme activity of HRP was still maintained at 90.08%.

[0029] (6) The enzyme protectant of the present application also has a clear protective effect on amino acid decarboxylase, which is highly sensitive to heat. After the GAD enzyme solution containing the protectant of the present application was stored at 50° C. for 2 h, the residual activity of GAD was still maintained at 80.03%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a comparison chart of the residual enzyme activity of the protective agent of the present application and that of the enzyme without the protective agent when placed at 47°C for up to 24 hours.

[0031] Figure 2 This is a comparison chart of the residual enzyme activity of the protective agent of the present application and that of the enzyme without the protective agent when placed at room temperature for up to 28 days.

[0032] Figure 3 This is a comparison chart of the protective effect of the protective agent of the present application on HRP at room temperature when it contains serine and when it does not contain serine.

[0033] Figure 4 This is a graph showing the protective agent of the present application for glutamate decarboxylase at room temperature. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0036] The present application will be further described below in conjunction with specific embodiments.

[0037] Embodiment 1:

[0038] A HRP solution without a protective agent was prepared and placed in a 47° C. metal bath. Samples were taken at 0, 3, 6, 9, 12 and 24 hours of incubation to determine the residual enzyme activity of HRP in the sample.

[0039] Preparation method: Prepare a 0.2 M sodium phosphate buffer (pH = 6.5), dilute 50 mg / L HRP solution with the sodium phosphate buffer to prepare a final concentration of 2 mg / L HRP solution.

[0040] The residual enzyme activity of HRP can reflect the protective effect of HRP (horseradish peroxidase) protective agent. The reason for preparing 2mg / L HRP solution is that HRP at this concentration is more sensitive to inactivation: as long as HRP is inactivated to a certain extent, the amount of enzyme-catalyzed reaction products generated can be reduced and can be detected by spectrophotometer.

[0041] Embodiment 2:

[0042] The enzyme protective agent was prepared, wherein the main solvent was a 0.2 mol / L sodium phosphate buffer solution with a pH of 6.5, which contained: 32.5% (volume ratio) of glycerol, 40 mmol / L of serine, and 9 mmol / L of CaCl2.

[0043] 1920 μL of protective agent was mixed with 80 μL of HRP enzyme solution to prepare an HRP solution with a final concentration of 2 mg / L, and then placed in a 47°C metal bath. Samples were taken at 3, 6, 9, 12 and 24 hours of incubation to determine the residual enzyme activity.

[0044] Embodiment 3:

[0045] Prepare an HRP solution without a protective agent, filter it through a 0.22 mm sterile filter membrane and store it in a sterile centrifuge tube, then place it in a 25° C. incubator, and take samples on days 0, 7, 21, and 28 to determine the residual HRP enzyme activity.

[0046] Preparation method: Prepare a sodium phosphate buffer with a concentration of 0.2 mol / L and a pH of 6.5, and use the buffer to dilute a 50 mg / L HRP solution to prepare an HRP solution with a final concentration of 2 mg / L.

[0047] Embodiment 4:

[0048] The enzyme protective agent was prepared, wherein the main solvent was a 0.2 mol / L sodium phosphate buffer solution with a pH of 6.5, which contained: 32.5% (volume ratio) of glycerol, 40 mmol / L of serine, and 9 mmol / L of CaCl2.

[0049] 1920 μL of protective agent was mixed with 80 μL of HRP enzyme solution to prepare an HRP solution with a final concentration of 2 mg / L. The prepared HRP solution was filtered with a 0.22 mm sterile filter membrane and stored in a sterile centrifuge tube. Then, the solution was placed in a 25°C incubator and samples were taken on days 0, 7, 21 and 28 to measure the residual HRP enzyme activity.

[0050] Embodiment 5:

[0051] A protective agent without serine was prepared, wherein the main solvent was a 0.2 mol / L sodium phosphate buffer solution with a pH of 6.5, which contained: glycerol: 32.5% (volume ratio), CaCl2: 9 mmol / L.

[0052] 1920 μL of protective agent was mixed with 80 μL of HRP enzyme solution to prepare an HRP solution with a final concentration of 2 mg / L, and then placed in a 25°C incubator. Samples were taken on days 0, 7, 21, and 28 to measure the residual HRP enzyme activity.

[0053] The method for determining the residual HRP enzyme activity in the above embodiment is as follows:

[0054] Hydrogen peroxide (H2O2) was selected as the peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) was selected as the hydrogen donor. The reaction was carried out at room temperature (15-25°C) for 3 min. After the reaction was completed, 3 mol / L sulfuric acid (H2SO4) was added to terminate the reaction. The absorbance (OD) of the reaction system at 450 nm was measured by an enzyme marker. 450nm ) and calculate the residual enzyme activity of HRP.

[0055] Embodiment 6:

[0056] A glutamate decarboxylase (GAD) solution with electrophoretic purity and a concentration of 2 mg / L was diluted to 0.5 mg / L with a 0.2 mol / L, pH 6.5 sodium phosphate buffer without a protective agent and a 0.2 mol / L, pH 6.5 sodium phosphate buffer containing a protective agent (the protective agent formula is the same as in Example 2), and then 2 mL of the diluted enzyme solution was respectively taken and stored in a 50° C. metal bath, and samples were taken every 0.5 h to determine the residual enzyme activity of GAD.

[0057] The GAD enzyme activity detection method is as follows: take 10 μL of enzyme solution, add 400 μL of substrate solution (pH 4.4, 0.1 mol·L -1 Citric acid-sodium hydrogen phosphate buffer, containing 0.01mmol·L -1 PLP, 50mmol·L -1 Substrate L-MSG), quickly mix and react at 48℃ for 10min. After the reaction, quickly put it into boiling water for 10min to terminate the reaction, centrifuge and collect the supernatant. Then take 100μL of the reaction solution and add 100μL 0.5mol·L -1 Sodium bicarbonate solution, adjust pH>7.5, mix well and add 200 μL of dansyl chloride acetone solution (4g / L, about 15mmol·L -1 ), derivatized at 40°C in the dark for more than 1 hour. The derivatized sample was filtered through a 0.22 μm microporous membrane and the amount of GABA generated by the reaction was determined by high performance liquid chromatography, and the amount of GABA generated was used to characterize the catalytic activity of the enzyme.

[0058] The HPLC operating conditions were as follows: the chromatographic separation column was Hypersil ODS2 C18 (250 mm × 4.6 mm) (Thermo Company), the UV detection wavelength was 254 nm, the injection volume was 10 μL, the column temperature was controlled at 25°C, the mobile phase A was methanol, and the mobile phase B was tetrahydrofuran: methanol: sodium acetate (0.05 mol·L -1 , pH 6.2) (5:75:420, V / V / V). The gradient elution program is shown in Table 1.

[0059] Table 1 HPLC gradient elution program

[0060] T / min 0 5 20 21 27 28 30 A％ 20 20 50 100 100 20 20 B％ 80 80 50 0 0 80 80

[0061] The comparison of the measurement results of the above-mentioned embodiment 1 and embodiment 2 is as follows Figure 1 As shown, the results of Example 3 and Implementation 4 are compared. Figure 2 As shown, the comparison of the measurement results of Example 4 and Example 5 is as follows Figure 3 As shown, the measurement results of Example 6 are as follows Figure 4 shown.

[0062] Depend on Figure 1 It can be seen that after the HRP enzyme solution obtained in Example 2 was stored at 47°C for 24 hours, the residual enzyme activity of HRP was still maintained at 85.12%. Figure 2 It can be seen that after the HRP enzyme solution obtained in Example 4 was stored at room temperature for 28 days, the residual HRP enzyme activity was still maintained at 90.08%; Figure 3 It can be seen that serine is a key component of the protective agent. This substance, together with glycerol and CaCl2, significantly improves the stability of HRP. Figure 4 It can be seen that the protective agent also has a clear protective effect on GAD, an amino acid decarboxylase that is extremely sensitive to heat. After the GAD enzyme solution containing the protective agent obtained in Example 6 was stored at 50° C. for 2 hours, the residual activity of GAD was still maintained at 80.03%.

[0063] In summary, (1) the HRP (horseradish peroxidase) protective agent involved in the present application can improve the stability of HRP, extend the shelf life of the kit, and improve its commercial competitiveness; (2) the protective agent also has a significant protective effect on amino acid decarboxylase, which is highly heat-sensitive. After the GAD enzyme solution containing the protective agent obtained in Example 6 was stored at 50°C for 2 hours, the residual activity of GAD was still maintained at 80.03%.

[0064] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. Application of a horseradish peroxidase or glutamate decarboxylase protective agent in the storage or transportation of horseradish peroxidase or glutamate decarboxylase at room temperature, characterized in that: The horseradish peroxidase or glutamate decarboxylase protective agent uses a pH 6.5-7.5 buffer as a solvent, and the components in the solvent are as follows: Glycerol volume percentage 25% to 35%; Serine 25-45 mmol / L; Ca 2+ 5~15mmol / L。 2. The use according to claim 1, characterized in that: The content of serine is 38-42 mmol / L.

3. The use according to claim 1, characterized in that: The volume percentage content of glycerol in the enzyme protecting agent is 30% to 35%.

4. The use according to claim 1, characterized in that: The Ca 2+ The content of Ca is 8-10 mmol / L; 2+ It is CaCl2.

5. The use according to claim 1, characterized in that: The content of glycerol is 32.5%; the content of serine is 40mmol / L; the Ca 2+ The content is 9mmol / L.

6. A method for storing horseradish peroxidase or glutamate decarboxylase at room temperature, characterized in that: The horseradish peroxidase or glutamate decarboxylase protective agent is mixed with the enzyme and placed at room temperature, wherein the horseradish peroxidase or glutamate decarboxylase protective agent uses a pH 6.5-7.5 buffer as a solvent, and the components in the solvent are as follows: Glycerol volume percentage 25% to 35%; Serine 25-45 mmol / L; Ca 2+ 5~15mmol / L。 7. The method for storing horseradish peroxidase or glutamate decarboxylase at room temperature according to claim 6, characterized in that: The content of serine is 38-42 mmol / L.

8. The method for storing horseradish peroxidase or glutamate decarboxylase at room temperature according to claim 6, wherein: The volume percentage content of glycerol in the enzyme protecting agent is 30% to 35%.

9. The method for storing horseradish peroxidase or glutamate decarboxylase at room temperature according to claim 6, characterized in that: The Ca 2+ The content of Ca is 8-10 mmol / L; 2+ It is CaCl2.

10. The method for storing horseradish peroxidase or glutamate decarboxylase at room temperature according to claim 6, characterized in that: The content of glycerol is 32.5%; the content of serine is 40mmol / L; the Ca 2+ The content is 9mmol / L.

11. Use of serine in the preparation of a protective agent for horseradish peroxidase or glutamate decarboxylase, characterized in that: The horseradish peroxidase or glutamate decarboxylase protective agent uses a pH 6.5-7.5 buffer as a solvent, and the components in the solvent are as follows: Glycerol volume percentage 25% to 35%; Serine 25-45 mmol / L; Ca 2+ 5~15mmol / L。 12. The use according to claim 11, characterized in that The content of serine is 38-42 mmol / L.

13. The use according to claim 11, characterized in that The volume percentage content of glycerol in the enzyme protecting agent is 30% to 35%.

14. The use according to claim 11, characterized in that The Ca 2+ The content of Ca is 8-10 mmol / L; 2+ It is CaCl2.

15. The use according to claim 11, characterized in that The content of glycerol is 32.5%; the content of serine is 40mmol / L; the Ca 2+ The content is 9mmol / L.

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