A method for improving the stability of horseradish peroxidase

Abstract

The present invention provides a method for improving the stability of horseradish peroxidase, belonging to the field of biological detection technology. 7-chloroindole is used to prepare a reagent containing horseradish peroxidase, and lysine on the horseradish peroxidase is modified by 7-chloroindole to improve the stability of the horseradish peroxidase. This method greatly solves the problem of HRP being unstable in a heated environment. Moreover, through the action of a protective liquid, its thermal stability at 37°C for 4 days is increased from the initial 40% to more than 90%. The method provided by the present invention greatly improves the stability of horseradish peroxidase in use, storage and transportation, and relates to the use of HRP in various fields such as biochemistry, immunochemistry, medical diagnosis, and food safety.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for improving the stability of horseradish peroxidase. Background Art

[0002] Horseradish peroxidase (HRP) is the most commonly used enzyme due to its high specific activity, stability, small molecular weight, high specificity, and easy preparation of pure enzyme. HRP is widely distributed in the plant kingdom and is found in high concentrations in horseradish. It is a glycoprotein composed of a colorless enzyme protein bound to a brown iron porphyrin, with a sugar content of 18%. HRP is a typical peroxidase, and its structure and mechanism are well-studied. HRP has a wide range of applications in biochemistry, immunochemistry, medical diagnostics, food safety, and other fields.

[0003] HRP has the advantage of being easy to purify and modify. Modern biotechnology allows for the convenient acquisition of high-purity HRP and its chemical modification to enhance its catalytic activity, change substrate properties, and improve its stability in solution and at high temperatures.

[0004] In extreme environments, such as high temperatures, strong acids, and strong bases, HRP's activity may be affected, thereby affecting its catalytic effectiveness. Improving the stability of HRP so that it maintains high catalytic activity under a wider range of environmental conditions is a pressing issue for researchers. Summary of the Invention

[0005] The object of the present invention is to address the above-mentioned deficiencies in the prior art and provide a method for improving the stability of horseradish peroxidase, wherein 7-chloroindole is used to prepare a reagent containing horseradish peroxidase, and lysine on the horseradish peroxidase is modified by 7-chloroindole to improve the stability of the horseradish peroxidase.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The invention provides a method for improving the stability of horseradish peroxidase. 7-chloroindole is used to prepare a reagent containing horseradish peroxidase. The reagent containing horseradish peroxidase comprises 7-chloroindole and horseradish peroxidase.

[0008] Furthermore, in the horseradish peroxidase-containing reagent, the mass percentage of 7-chloroindole is 0.09% to 0.47%.

[0009] Furthermore, the horseradish peroxidase-containing reagent further comprises a protective solution, which is prepared from a buffer solution, bovine serum albumin, trehalose, polyethylene glycol, glycerol, MgSO4, casein, galactosidol dihydrate, cynomolgus monkey serum and Proclin 300.

[0010] Furthermore, the buffer comprises one or more of phosphate buffer, MES buffer and acetate buffer.

[0011] Furthermore, the buffer solution is a phosphate buffer solution, and the concentration of the buffer solution is 0.1 mol / L.

[0012] Furthermore, the protective solution comprises, by weight, PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 0.5-2 g / L; trehalose 0.5-2 g / L; polyethylene glycol 1-5%; glycerol 0.5-2 g / L; MgSO4 8-10 mM; casein 0.5-2 g / L; inositol galactosidase dihydrate 0.5-2 g / L; cynomolgus monkey serum 5-20%; and Proclin 300 0.5-1 g / L.

[0013] Furthermore, the protective solution comprises, by weight, PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 1 g / L; trehalose 1 g / L; polyethylene glycol 2%; glycerol 1 g / L; MgSO4 8 mM; casein 0.5 g / L; inositol galactosidase dihydrate 1 g / L; cynomolgus monkey serum 5%; and Proclin 300 1 g / L.

[0014] Furthermore, the protective solution comprises PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 0.5 g / L; trehalose 2 g / L; polyethylene glycol 1%; glycerol 2 g / L; MgSO4 10 mM; casein 2 g / L; inositol galactosidase dihydrate 2 g / L; cynomolgus monkey serum 5%; and Proclin 300 0.5 g / L.

[0015] Furthermore, the protective solution comprises PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 2 g / L; trehalose 0.5 g / L; polyethylene glycol 5%; glycerol 0.5 g / L; MgSO4 8 mM; casein 1 g / L; inositol galactosidase dihydrate 0.5 g / L; cynomolgus monkey serum 20%; and Proclin 300 1 g / L.

[0016] Furthermore, the protective solution comprises, by weight, PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 1 g / L; trehalose 1 g / L; polyethylene glycol 2%; glycerol 1 g / L; MgSO4 8 mM; casein 1 g / L; inositol galactosidase dihydrate 1 g / L; cynomolgus monkey serum 10%; and Proclin 300 1 g / L.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention provides a method for improving the stability of horseradish peroxidase. 7-chloroindole is used to prepare a reagent containing horseradish peroxidase. 7-chloroindole modifies the lysine residue on the horseradish peroxidase to improve the stability of the horseradish peroxidase. This method significantly solves the problem of HRP being unstable under heating.

[0019] (2) The method for improving the stability of horseradish peroxidase provided by the present invention increases its thermal stability at 37°C for 4 days from the initial 40% to more than 90% through the action of a protective solution.

[0020] (3) The present invention greatly improves the stability of horseradish peroxidase in use, storage and transportation, and involves the use of HRP in various fields such as biochemistry, immunochemistry, medical diagnosis, and food safety. DETAILED DESCRIPTION

[0021] To make the objectives, technical solutions and advantages of the present invention more clear, embodiments of the present invention are described in detail below.

[0022] 7-Chloroindole (Chinese National Pharmaceutical Group, Product No.: XW015392405303); Citraconic anhydride (Chinese National Pharmaceutical Group, Product No.: C154790010); Horseradish peroxidase (Thermo, Product No.: 31490); Casein (Chinese National Pharmaceutical Group, Product No.: 69006227); Inositol galactoside dihydrate (Chinese National Pharmaceutical Group, Product No.: TG0298100MG); Cynomolgus monkey serum (Nanjing Senbega Biotechnology Co., Ltd., product number: P406395-100ml); BSA (Sinopharm, product number: C134730100); Trehalose (Sinopharm, product number: LA1943401); Polyethylene glycol 4000 (Sinopharm, product number: 30151627); Glycerol (Sinopharm, product number: C158922500); MgSO4 (Sinopharm, product number: 20025117); Proclin 300 (Aladdin, product number: P406395-100ml).

[0023] Example 1

[0024] This example provides a process for modifying horseradish peroxidase with 7-chloroindole as follows:

[0025] Dissolve 7-chloroindole (148 mmol) in methanol (60 mL) and prepare 20 mL of 1 mg / mL horseradish peroxidase in phosphate buffer.

[0026] Add 2 ml of horseradish peroxidase to each of 5 small beakers, and then add (5 μl, 10 μl, 15 μl, 20 μl, 25 μl) of the prepared 7-chloroindole solution in sequence. Adjust the pH to 7.4 with sodium hydroxide and react in an ice bath for one hour. Stir continuously with a magnetic stirrer during the reaction.

[0027] After the reaction, the reaction solutions were placed in dialysis bags and dialyzed at 2-8°C for two days using phosphate buffer, with the buffer changed three times a day.

[0028] After dialysis, the solution in the dialysis bag was carefully poured into a 10 ml volumetric flask and fixed to volume with phosphate buffer to a concentration of 0.2 mg / ml.

[0029] Similarly, 2 ml of original enzyme solution (1 mg / ml HRP) was diluted to 10 ml with phosphate buffer, with a concentration of 0.2 mg / ml.

[0030] Comparative Example 1

[0031] This comparative example provides a process for modifying horseradish peroxidase with citraconic anhydride as follows:

[0032] Dilute citraconic anhydride by half with phosphate buffer (0.1 mol / L, pH=7.4), and prepare 20 ml of 1 mg / ml horseradish peroxidase with phosphate buffer.

[0033] Add 2 ml of horseradish peroxidase to each of 5 small beakers, and then add (5 μl, 10 μl, 15 μl, 20 μl, 25 μl) of the prepared citraconic anhydride solution in sequence. Adjust the pH to 7.4 with sodium hydroxide and react in an ice bath for one hour. Stir continuously with a magnetic stirrer during the reaction.

[0034] After the reaction, the reaction solutions were placed in dialysis bags and dialyzed at 2-8°C for two days using phosphate buffer, with the buffer changed three times a day.

[0035] After dialysis, the solution in the dialysis bag was carefully poured into a 10 ml volumetric flask and fixed to volume with phosphate buffer to a concentration of 0.2 mg / ml.

[0036] Similarly, 2 ml of original enzyme solution (1 mg / ml HRP) was diluted to 10 ml with phosphate buffer, with a concentration of 0.2 mg / ml.

[0037] The heat stability test of the horseradish peroxidase-containing reagents prepared in Example 1 and Comparative Example 1 was performed, and the specific process is as follows:

[0038] Five different concentrations of 7-chloroindole-modified 7-Chloroindole-HRP and HRP (1 mg / ml) were each aliquoted into six vials (100 μl / vial) and stored at 4°C and 37°C for 12, 24, 72, and 96 hours for stability testing. There were six different HRP types, each with five storage conditions, for a total of 30 conditions.

[0039] Five different concentrations of citraconic acid-modified CA-HRP (1 mg / ml) were each aliquoted into six vials (100 μl / vial) and stored at 4°C and 37°C for 12, 24, 72, and 96 hours for stability testing. There were five storage conditions for each of the six HRP types, for a total of 25 conditions.

[0040] 7-Chloroindole-HRP, CA-HRP, and HRP under 55 conditions were diluted 100,000 times with PBS buffer, and 10 μl was added to a blank ELISA plate. 100 μl of TMB substrate was added and color was developed at 37°C for 5 min. The OD value was measured with an ELISA reader at a wavelength of 450 nm.

[0041] The OD values ​​measured at 37°C for 12 h, 24 h, 72 h, and 96 h were compared with the OD value at 4°C as the activity retention value of each HRP.

[0042] The results are shown in Table 1. HRP stability was best when 10 μl of 7-Chloroindole solution was added. After 96 h at 37°C, 7-Chloroindole-HRP activity retained 72%, CA-HRP activity retained 58%, and HRP activity retained 39%.

[0043] Table 1.

[0044]

[0045] Example 2

[0046] In this embodiment, the proportions of the protective solution 1 are as follows: PBS buffer, pH = 7.4, 0.1 mol / L; BSA 1 g / L; trehalose 1 g / L; polyethylene glycol 2%; glycerol 1 g / L; MgSO4 8 mM; casein 0.5 g / L; inositol galactosidase dihydrate 1 g / L; cynomolgus monkey serum 5%; and Proclin 300 1 g / L.

[0047] The protective solution described in this example was prepared as follows: 0.1 mol / L pH 7.4 phosphate buffer was prepared, and the corresponding components were weighed or measured into the buffer and dissolved one by one by stirring. The solution was then filtered through a 0.8 μm filter membrane, bottled, and stored at 2-8°C.

[0048] Example 3

[0049] In this embodiment, the proportions of the protective solution 2 are as follows: PBS buffer, pH = 7.4, 0.1 mol / L; BSA 0.5 g / L; trehalose 2 g / L; polyethylene glycol 1%; glycerol 2 g / L; MgSO4 10 mM; casein 2 g / L; inositol galactosidase dihydrate 2 g / L; cynomolgus monkey serum 5%; Proclin 300 0.5 g / L.

[0050] The protective solution described in this example was prepared as follows: 0.1 mol / L pH 7.4 phosphate buffer was prepared, and the corresponding components were weighed or measured into the buffer and dissolved one by one by stirring. The solution was then filtered through a 0.8 μm filter membrane, bottled, and stored at 2-8°C.

[0051] Example 4

[0052] In this embodiment, the proportions of the protective solution 3 are as follows: PBS buffer, pH = 7.4, 0.1 mol / L; BSA 2 g / L; trehalose 0.5 g / L; polyethylene glycol 5%; glycerol 0.5 g / L; MgSO4 8 mM; casein 1 g / L; inositol galactosidase dihydrate 0.5 g / L; cynomolgus monkey serum 20%; Proclin 300 1 g / L.

[0053] The protective solution described in this example was prepared as follows: 0.1 mol / L pH 7.4 phosphate buffer was prepared, and the corresponding components were weighed or measured into the buffer and dissolved one by one by stirring. The solution was then filtered through a 0.8 μm filter membrane, bottled, and stored at 2-8°C.

[0054] Example 5

[0055] In this embodiment, the proportions of the protective solution 4 components are as follows: PBS buffer, pH = 7.4, 0.1 mol / L; BSA 1 g / L; trehalose 1 g / L; polyethylene glycol 2%; glycerol 1 g / L; MgSO4 8 mM; casein 1 g / L; inositol galactosidase dihydrate 1 g / L; cynomolgus monkey serum 10%; Proclin 300 1 g / L.

[0056] The protective solution described in this example was prepared as follows: 0.1 mol / L pH 7.4 phosphate buffer was prepared, and the corresponding components were weighed or measured into the buffer and dissolved one by one by stirring. The solution was then filtered through a 0.8 μm filter membrane, bottled, and stored at 2-8°C.

[0057] To investigate the stability of the horseradish peroxidase-containing reagent prepared in Example 1, the protective solutions prepared in Examples 2-5 were used to dilute HRP to 0.01 mg / ml, and then diluted to 0.01 mg / ml with PBS. Five vials of each of the five HRP types were stored at 4°C and 37°C for 12, 24, 72, and 96 hours, respectively, for stability testing. Five storage conditions were used for each of the five HRP types, for a total of 25 conditions.

[0058] HRP under 25 conditions was diluted 5000 times with PBS buffer, 10 μl was added to a blank ELISA plate, and 100 μl TMB substrate was added. The color was developed at 37°C for 5 min, and the OD value was measured with an ELISA reader at a wavelength of 450 nm.

[0059] The OD values ​​measured at 37°C for 12 h, 24 h, 72 h, and 96 h were compared with the OD value at 4°C as the activity retention value of each HRP.

[0060] The results are shown in Table 2. HRP diluted with Protective Solution 4 had the best stability. After 72 hours at 37°C, the activity of HRP after protection was 74%; the activity of HRP diluted with PBS was 41%.

[0061] Table 2.

[0062]

[0063] Example 6

[0064] The thermal stability of 7-Chloroindole-HRP protected by protective solution was investigated.

[0065] 7-Chloroindole-HRP was diluted to 0.01 mg / ml using the four protective solutions prepared in Examples 3, 4, 5, and 6, respectively. Then, the solution was diluted to 0.01 mg / ml using PBS. Five vials of each of the five 7-Chloroindole-HRP types were stored at 4°C and 37°C for 12, 24, 72, and 96 hours, respectively, for stability testing. This total of five 7-Chloroindole-HRP types was tested under five different storage conditions, for a total of 25 conditions.

[0066] 7-Chloroindole-HRP under 25 conditions was diluted 5000 times with PBS buffer, 10 μl was added to a blank ELISA plate, and 100 μl TMB substrate was added. The color was developed at 37°C for 5 min, and the OD value was measured with an ELISA reader at a wavelength of 450 nm.

[0067] The OD values ​​measured at 37°C for 12 h, 24 h, 72 h, and 96 h were compared with the OD value at 4°C as the activity retention value of each 7-Chloroindole-HRP.

[0068] The results are shown in Table 3. 7-Chloroindole-HRP diluted with Protective Solution 4 exhibited the best stability. After 72 hours at 37°C, the activity of the protected 7-Chloroindole-HRP remained at 94.34%, while that of the PBS-diluted 7-Chloroindole-HRP remained at 68.66%.

[0069] Table 3.

[0070]

[0071] Any matters not mentioned above shall be subject to the existing technology.

[0072] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art of the present invention may make various modifications or additions to the described specific embodiments or replace them in similar ways, but they will not deviate from the direction of the present invention or exceed the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent replacements, improvements, etc. made to the above embodiments based on the technical essence of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for improving the stability of horseradish peroxidase, characterized in that: 7-chloroindole is used to prepare a reagent containing horseradish peroxidase. The reagent containing horseradish peroxidase comprises 7-chloroindole and horseradish peroxidase, and the mass percentage of 7-chloroindole is 0.09%-0.47%.

2. The method according to claim 1, wherein The horseradish peroxidase-containing reagent further comprises a protective solution, which is prepared from a buffer solution, bovine serum albumin, trehalose, polyethylene glycol, glycerol, MgSO4, casein, galactosidol dihydrate, cynomolgus monkey serum and Proclin 300.

3. The method according to claim 2, wherein The buffer comprises one or more of phosphate buffer, MES buffer and acetate buffer.

4. The method according to claim 3, wherein The buffer solution is a phosphate buffer solution, and the concentration of the buffer solution is 0.1 mol / L.

5. The method according to claim 4, wherein By weight, the protective solution comprises PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 0.5-2 g / L; trehalose 0.5-2 g / L; polyethylene glycol 1-5%; glycerol 0.5-2 g / L; MgSO4 8-10 mM; casein 0.5-2 g / L; inositol galactoside dihydrate 0.5-2 g / L; cynomolgus monkey serum 5-20%; and Proclin 300 0.5-1 g / L.

6. The method according to claim 4, wherein By weight, the protective solution comprises PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 1 g / L; trehalose 1 g / L; polyethylene glycol 2%; glycerol 1 g / L; MgSO4 8 mM; casein 0.5 g / L; inositol galactoside dihydrate 1 g / L; cynomolgus monkey serum 5%; and Proclin 300 1 g / L.

7. The method according to claim 4, wherein By weight, the protective solution comprises PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 0.5 g / L; trehalose 2 g / L; polyethylene glycol 1%; glycerol 2 g / L; MgSO4 10 mM; casein 2 g / L; inositol galactosidase dihydrate 2 g / L; cynomolgus monkey serum 5%; and Proclin 300 0.5 g / L.

8. The method according to claim 4, wherein By weight, the protective solution comprises PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 2 g / L; trehalose 0.5 g / L; polyethylene glycol 5%; glycerol 0.5 g / L; MgSO4 8 mM; casein 1 g / L; inositol galactosidase dihydrate 0.5 g / L; cynomolgus monkey serum 20%; and Proclin 300 1 g / L.

9. The method according to claim 4, wherein By weight, the protective solution comprises PBS buffer, pH=7.4, 0.1 mol / L; bovine serum albumin 1 g / L; trehalose 1 g / L; polyethylene glycol 2%; glycerol 1 g / L; MgSO4 8 mM; casein 1 g / L; inositol galactosidase dihydrate 1 g / L; cynomolgus monkey serum 10%; and Proclin 300 1 g / L.

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