Method for accurately measuring mass concentration of protease in enzymatic reaction
By preparing protease solutions of different mass concentrations and reacting with casein solution, and combining absorbance measurement and standard curves, the mass concentration of protease is accurately measured, which solves the problem of error in the determination of protease mass concentration in the enzymatic reaction and improves the accuracy of the determination.
Patent Information
- Application Number
- CN202510324306.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-19
- Publication Date
- 2025-05-30
AI Technical Summary
During the enzymatic reaction, it is difficult for the prior art to accurately determine the mass concentration of proteases, and it is easily disturbed by factors such as temperature, pH and salt concentration, resulting in experimental errors.
By preparing protease solutions of different mass concentrations and reacting with casein solution, the absorbance of the reaction solution is measured, and the relationship curve between the L-tyrosine standard curve and the relationship curve between the protease mass concentration and L-tyrosine yield is accurately measured.
This method can accurately determine the mass concentration of protease, reduce experimental errors, and improve the accuracy of enzymatic reaction kinetics research.
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Figure CN120064179A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of enzymatic reactions, and specifically to a method for accurately measuring the mass concentration of protease in an enzymatic reaction. Background Art
[0002] Enzymatic reaction, also known as enzyme catalysis or zymogenic catalysis, refers to a chemical reaction catalyzed by an enzyme as a catalyst. Enzymatic reaction kinetics mainly studies the reaction rate catalyzed by an enzyme and various factors affecting the reaction rate. Protease is an enzyme that can act in a neutral or weak acid or weak alkaline environment and can be used for the hydrolysis treatment of various proteins, and is widely used in the fields of food industry, leather industry, pharmaceutical industry, etc.
[0003] In the process of studying the adsorption reaction kinetics of an enzyme, it is of great significance to measure the mass concentration of protease in real time and then determine the consumption of protease. At present, basically, the method of measuring protease activity is used to reflect the consumption of protease in the enzymatic reaction process. However, protease has poor stability and is easily interfered by physical and chemical factors such as temperature, pH, and salt concentration. Therefore, using the activity of protease to determine the content of protease in a solution will inevitably cause experimental errors and affect the study of the adsorption reaction kinetics of the enzyme. Summary of the Invention
[0004] Based on the above technical problems, the present invention proposes a method for accurately measuring the mass concentration of protease in an enzymatic reaction.
[0005] The technical solution adopted by the present invention is as follows:
[0006] A method for accurately measuring the mass concentration of protease in an enzymatic reaction, comprising the following steps:
[0007] (1) Prepare L-tyrosine standard solutions with different concentrations, and measure the absorbance respectively. Taking the absorbance value as the ordinate and the L-tyrosine concentration as the abscissa, draw an L-tyrosine standard curve;
[0008] (2) Prepare a casein solution and divide it into multiple groups as substrate solutions;
[0009] (3) Prepare protease solutions with different mass concentrations;
[0010] (4) Add the protease solutions with different mass concentrations prepared in step (3) to the respective groups of casein solutions prepared in step (2) for reaction to obtain reaction solutions; then measure the absorbance of the reaction solutions;
[0011] (5) Based on the absorbance measured in step (4) and combined with the L-tyrosine standard curve plotted in step (1), determine the concentration of L-tyrosine in the reaction solution; and then plot the relationship curve between the protease mass concentration and the L-tyrosine yield.
[0012] (6) During the enzymatic reaction or adsorption reaction process, by measuring the absorbance and combining it with the L-tyrosine standard curve and the relationship curve between the protease mass concentration and the L-tyrosine yield, obtain the protease mass concentration at different time points.
[0013] The beneficial technical effects of the present invention are as follows:
[0014] The present invention provides a method for accurately measuring the protease mass concentration in an enzymatic reaction. This method prepares protease solutions with different mass concentrations by directly weighing different masses of protease, and obtains the corresponding L-tyrosine yields through experiments to obtain the relationship curve between the two; in practical applications, by measuring the L-tyrosine yield and combining with the relationship curve, the mass concentration of the enzyme at different time points can be obtained, and thus the accurate consumption of protease during the enzymatic reaction or adsorption reaction process can be determined. Compared with measuring enzyme activity, the experimental error is greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The present invention will be further described below in conjunction with the drawings and specific embodiments:
[0016] Figure 1 It is a schematic flow chart of the method for accurately measuring the protease mass concentration in the enzymatic reaction of the present invention;
[0017] Figure 2 It is a specific flow chart of the present invention for reacting the protease solution with the substrate solution and measuring the absorbance;
[0018] Figure 3 It is the relationship curve between the protease mass concentration and the L-tyrosine yield established in Example 1 of the present invention;
[0019] Figure 4 It is the relationship curve between the protease mass concentration and the L-tyrosine yield in Comparative Example 1 of the present invention;
[0020] Figure 5 It is the relationship curve between the protease mass concentration and the L-tyrosine yield in Comparative Example 2 of the present invention;
[0021] Figure 6 It is the relationship curve between the protease mass concentration and the L-tyrosine yield in Comparative Example 3 of the present invention;
[0022] Figure 7 It is the relationship curve between the protease mass concentration and the L-tyrosine yield in Comparative Example 4 of the present invention. Detailed implementation mode
[0023] Example 1
[0024] As Figure 1 shown, this example provides a method for accurately measuring the mass concentration of protease in an enzymatic reaction, including the following steps:
[0025] (1) Prepare a series of standard working solutions of L-tyrosine according to Table 1. Take 1.00 mL of the standard working solutions in Table 1 into glass test tubes respectively, add 5.00 mL of sodium carbonate solution and 1.00 mL of Folin working solution (the volume ratio of Folin reagent to water is 1:2), shake well, place in a water bath at 40°C ± 0.2°C for 20 min, take out and cool to room temperature. Using the 0 tube as the blank, measure the absorbance values of the series of standard working solutions at 680 nm using a 10 mm cuvette respectively. Plot a standard curve with the absorbance value as the ordinate and the L-tyrosine concentration as the abscissa.
[0026] (2) Weigh 1.00 g of the casein substrate for protease preparation (accurate to 0.001 g) into a beaker, add about 80 mL of the corresponding buffer solution, place in a boiling water bath and keep for 30 min, continuously stir during this period until the casein is completely dissolved. After cooling to room temperature, transfer it to a 100 mL volumetric flask, adjust the pH to 7.5 with hydrochloric acid solution or sodium hydroxide solution, and then make up the volume with the buffer solution. This casein solution is stored at 4°C and the validity period is 3 days. Reconfirm and adjust the pH to 7.5 before use.
[0027] (3) Prepare protease solutions with different mass concentrations. Weigh 0 mg, 5 mg, 10 mg, 20 mg and 40 mg of protease preparation samples respectively, add 800 mL of the corresponding buffer solution, stir and dissolve for 30 min, then transfer to a 1000 mL volumetric flask, make up the volume with the corresponding buffer solution and mix well.
[0028] (4) Place the casein solution in a constant temperature water bath at 40°C ± 0.2°C for preheating, and measure the absorbance at 680 nm according to the operation process as Figure 2 shown.
[0029] The specific steps are as follows: Take six empty test tubes. One test tube is used as a blank sample, and the other five are parallel samples of the protease sample. Add 1.00 mL of protease solution to each. The blank sample is added with 1.00 mL of the corresponding buffer solution. At the same time, water bath at 40 °C for 2 min. Add 2.00 mL of trichloroacetic acid to the blank sample and shake well. Add 1.00 mL of casein solution to the parallel samples and shake well. At the same time, water bath at 40 °C for 10 min. Add 1.00 mL of casein solution to the blank sample and shake well. Add 2.00 mL of trichloroacetic acid to the parallel samples and shake well. Then take them out and let them stand for 10 min and filter with slow qualitative filter paper. Take 1.00 mL of the filtrate, add 5.00 mL of sodium carbonate solution and 1.00 mL of Folin reagent solution, and water bath at 40 °C for color development for 20 min; using the blank sample as the blank, measure the absorbance of the parallel samples at a wavelength of 680 nm with a 10 nm cuvette.
[0030] (5) Taking the protease mass concentration as the ordinate and the L-tyrosine concentration as the abscissa, plot the relationship curve between the protease mass concentration and the L-tyrosine yield.
[0031] By substituting the measured OD 680 value into the L-tyrosine standard curve to obtain the concentration of L-tyrosine. Taking the protease mass concentration as the ordinate and the L-tyrosine yield as the abscissa, plot the curve to obtain the relationship between the protease mass concentration and the L-tyrosine yield. Substituting the L-tyrosine yield into the known curve can calculate the protease mass concentration.
[0032] (6) During the enzymatic reaction or adsorption reaction process, by measuring the absorbance and combining with the L-tyrosine standard curve and the relationship curve between the protease mass concentration and the L-tyrosine yield, obtain the protease mass concentration at different time points.
[0033] Table 1
[0034] Tube number Concentration of L-tyrosine standard solution (mg / mL) Volume of L-tyrosine standard stock solution (mL) Volume of added water (mL) 0 0 0 10 1 10 1 9 2 20 2 8 3 30 3 7 4 40 4 6 5 50 5 5
[0035] Example 2
[0036] Based on the relationship between the protease mass concentration and the L-tyrosine yield established in Example 1, this example further obtains the change of the adsorption amount of protease on quartz sand with time, including the following steps:
[0037] (1) Prepare a series of standard working solutions of L-tyrosine according to Table 1. Take 1.00 mL of the standard solutions in Table 1 into glass test tubes respectively, add 5.00 mL of sodium carbonate solution and 1.00 mL of Folin reagent solution, shake well, place in a water bath at 40 °C ± 0.2 °C for reaction for 20 min, and take out and cool to room temperature. Using the 0 tube as the blank, measure the absorbance values of the series of standard working solutions at 680 nm respectively with a 10 mm colorimetric cuvette. Taking the absorbance value as the ordinate and the L-tyrosine concentration as the abscissa, draw a standard curve.
[0038] (2) Weigh 1.00 g of the special casein substrate for protease preparation (accurate to 0.001 g) into a beaker, add about 80 mL of the corresponding buffer solution, place in a boiling water bath and keep for 30 min, and continuously stir during this period until all the casein is dissolved. After cooling to room temperature, transfer it to a 100 mL volumetric flask, adjust the pH to 7.5 with hydrochloric acid solution or sodium hydroxide solution, and then make up the volume with the corresponding pH buffer solution. This solution is stored at 4 °C and the validity period is 3 d. Reconfirm and adjust the pH to 7.5 before use.
[0039] (3) Weigh an appropriate amount of protease preparation sample, add 800 mL of the corresponding buffer solution, stir and dissolve for 30 min, and then transfer it to a 1000 mL volumetric flask, make up the volume with the corresponding buffer solution and mix well.
[0040] (4) To determine the change in the adsorption amount of protease on quartz sand over time, add 20 mL of the prepared enzyme solution to a centrifuge tube containing 5 g of quartz sand. Set 6 time points, namely 0 min, 30 min, 60 min, 80 min, 90 min, and 100 min. Set one blank sample and five parallel samples for each time point. Place the samples in a shaker, and the conditions of the shaker are 25 °C and 180 rpm. According to the time sequence, take out the samples in turn, let them stand for 15 min and then take the supernatant, and repeat the operation in step (4) of Example 1.
[0041] That is, take six empty test tubes for each time point, one test tube is the blank sample, and the other five are parallel samples. Add 1.00 mL of the supernatant to each respectively. The blank sample adds 1.00 mL of the corresponding buffer solution, and at the same time, water bath at 40 °C for 2 min. The blank sample adds 2.00 mL of trichloroacetic acid and shakes well. The parallel samples add 1.00 mL of casein solution and shake well. At the same time, water bath at 40 °C for 10 min. The blank sample adds 1.00 mL of casein solution and shakes well. The parallel samples add 2.00 mL of trichloroacetic acid and shake well. Then take out and let stand for 10 min and filter with slow qualitative filter paper. Take 1.00 mL of the filtrate, add 5.00 mL of sodium carbonate solution and 1.00 mL of Folin reagent solution, and water bath for color development at 40 °C for 20 min; using the blank sample as the blank, measure the absorbance of the parallel samples at a wavelength of 680 nm with a 10 nm colorimetric cuvette.
[0042] (5) Determine the L-tyrosine concentration according to the L-tyrosine standard curve, and determine the protease mass concentration through the relationship curve between the protease mass concentration and the L-tyrosine yield. The results are shown in Table 2, and the change in the adsorption amount of protease on quartz sand is obtained.
[0043] Table 2
[0044]
[0045] To further illustrate this method, the following comparative experiments were also carried out:
[0046] Comparative Example 1
[0047] The preparation method is the same as that of Example 1, except that in step (4), the casein solution is placed in a constant temperature water bath at 30°C ± 0.2°C and preheated for 5 minutes, and the temperature in the operation procedure is also 30°C ± 0.2°C.
[0048] Comparative Example 2
[0049] The preparation method is the same as that of Example 1, except that in step (4), the casein solution is placed in a constant temperature water bath at 50°C ± 0.2°C and preheated for 5 minutes, and the temperature in the operation procedure is also 50°C ± 0.2°C.
[0050] Comparative Example 3
[0051] The preparation method is the same as that of Example 1, except that in step (4), the casein solution is placed in a constant temperature water bath at 60°C ± 0.2°C and preheated for 5 minutes, and the temperature in the operation procedure is also 60°C ± 0.2°C.
[0052] Comparative Example 4
[0053] The preparation method is the same as that of Example 1, except that alkaline protease is selected as the protease; in steps (2) and (3), the buffer solution is a borax-sodium hydroxide buffer solution with a pH of 11 suitable for alkaline protease (preparation method: Solution A: Weigh 9.54 g of borax and make up to 500 mL with distilled water; Solution B: Weigh 2.00 g of sodium hydroxide and make up to 500 mL with distilled water; Mix equal amounts of Solution A and Solution B).
[0054] From Figures 3 to 6 It can be seen that there is a linear relationship between the protease mass concentration and the L-tyrosine concentration at different reaction temperatures. As can be seen from the figure, when the reaction temperature is 40°C, the linear relationship is the strongest and the discrete points are few. Therefore, the optimal operating temperature range is about 40°C. Figure 3 Compared with Figure 7Different kinds of proteases were used respectively. It can be seen that at the same reaction temperature, there is a linear relationship between the mass concentration of different kinds of proteases and the concentration of L-tyrosine. Therefore, this method can also be applied to different kinds of proteases.
[0055] Those parts not described in the above manner can be achieved by adopting or referring to the existing technologies.
[0056] It should be noted that the above are only the preferred embodiments of the present invention and do not constitute any limitation to the present invention. Any simple modification, change or equivalent substitution made to the above embodiments according to the technical essence of the present invention shall be covered by the protection scope of the present invention.
Claims
1. A method for accurately determining the mass concentration of a protease in an enzymatic reaction, characterized in that The following steps are involved: (1) preparing L-tyrosine standard solutions of different concentrations, and measuring the absorbance of each solution, and drawing an L-tyrosine standard curve with the absorbance value as the ordinate and the L-tyrosine concentration as the abscissa; (2) preparing a casein solution and dividing it into multiple groups as a substrate solution; (3) preparing protease solutions with different mass concentrations; (4) adding the protease solutions of different mass concentrations prepared in step (3) to the casein solutions of each group prepared in step (2) to react and obtain a reaction solution; and then measuring the absorbance of the reaction solution; (5) determining the concentration of L-tyrosine in the reaction solution by using the absorbance measured in step (4) in combination with the L-tyrosine standard curve drawn in step (1); and then drawing a relationship curve between the mass concentration of the protease and the L-tyrosine yield; (6) During the enzymatic reaction or adsorption reaction, the mass concentration of the protease at different time points is obtained by measuring the absorbance and combining the L-tyrosine standard curve and the relationship curve between the mass concentration of the protease and the L-tyrosine yield.
2. The method for accurately determining the mass concentration of protease in an enzymatic reaction according to claim 1, characterized in that: In step (1), L-tyrosine standard solutions of different concentrations are respectively taken into glass test tubes, sodium carbonate solution and Folin working solution are added, the mixture is shaken and mixed, and the mixture is placed in a water bath for reaction. After the reaction is completed, the mixture is taken out and cooled to room temperature to obtain reaction solutions; and the absorbance of the reaction solutions is respectively measured.
3. The method for accurately determining the mass concentration of protease in an enzymatic reaction according to claim 1, characterized in that: In step (2): the concentration of the casein solution is 10.0 g / L.
4. The method for accurately determining the mass concentration of protease in an enzymatic reaction according to claim 1, characterized in that: In step (3): 0 mg, 5 mg, 10 mg, 20 mg and 40 mg of the protease preparation sample were weighed respectively, added to 800 mL of buffer solution, stirred to dissolve, then transferred to a 1000 mL volumetric flask, fixed to volume with buffer solution and mixed, to obtain protease solutions with mass concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 20 mg / L and 40 mg / L, respectively.
5. The method for accurately determining the mass concentration of protease in an enzymatic reaction according to claim 4, characterized in that: The buffer solution is a phosphate buffer solution with a pH of 7.
5.
6. The method for accurately determining the mass concentration of protease in an enzymatic reaction according to claim 1, characterized in that: The reaction temperature in step (4) is consistent with the temperature during the enzymatic reaction or adsorption reaction in step (6).
7. The method for accurately determining the mass concentration of protease in an enzymatic reaction according to claim 1, characterized in that: The wavelengths used to measure absorbance in steps (1), (4) and (6) are consistent.