Enzyme activity detection method of aminopeptidase
By drawing standard curves and controlling reaction conditions, an aminopeptidase enzyme activity detection method is provided, which solves the problem of lack of detection methods in the existing technology, and realizes the accurate determination of aminopeptidase enzyme activity, meets market demand and improves The safety and precision of the detection are achieved.
Patent Information
- Application Number
- CN202411239272.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-05
- Publication Date
- 2024-12-17
AI Technical Summary
The lack of detection methods of aminopeptidase in the prior art has led to a lack of product standards and enterprise standard detection methods on the market, making it difficult to effectively detect the enzyme activity of aminopeptidase.
Provide an enzyme activity detection method of aminopeptidase, including drawing a standard curve of p-nitrophenol concentration, extracting the aminopeptidase to be tested and diluted, adding substrate L-leucine p-nitroaniline and sodium carbonate solution, and determining absorbance Values, calculate enzyme activity, control reaction conditions such as temperature and pH, and ensure the accuracy and stability of the detection.
It realizes the simplicity, speed, stability and accuracy of the enzyme activity detection of aminopeptidase, improves the safety and precision of the detection, provides reliable product standards, and meets market demand.
Smart Images
Figure CN119144694A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of enzyme preparation detection, and particularly relates to a method for detecting the enzyme activity of aminopeptidase. Background Art
[0002] Aminopeptidase is a type of exoproteinase that can selectively remove amino acid residues from the N-terminus of proteins and polypeptides to produce free amino acids. It is widely present in organisms of different species, including mammals, plants, microorganisms, etc. Due to the complex composition of animals and plants and the low content of aminopeptidase, the extraction cost is high. With the rapid development of the enzyme preparation industry, in order to meet market demand, the production of enzymes by microbial fermentation has become the mainstream trend and research hotspot today. Microorganisms produce higher enzymes, the extraction steps are simple, and the production cost is lower. Aminopeptidase products on the market are basically produced by microbial fermentation. In view of the characteristics of the action of aminopeptidase, its scope of use is extremely wide. However, there is a gap in the detection method of aminopeptidase at present. There is still a lack of product standard machine detection method standards for aminopeptidase, and there is also a lack of relevant detection methods in enterprise standards. Summary of the invention
[0003] The purpose of the present invention is to overcome the problem of lack of aminopeptidase detection method in the prior art.
[0004] To this end, the present invention provides a method for detecting the enzyme activity of aminopeptidase, comprising the following steps:
[0005] (1) Draw a standard curve of p-nitrophenol concentration;
[0006] (2) weighing a mass m of the aminopeptidase to be tested, adding a buffer solution for extraction, and diluting the extract by n times to obtain a solution of the aminopeptidase to be tested;
[0007] (3) Preheating the aminopeptidase solution to be tested, adding the substrate L-leucine p-nitroaniline thereto, adding sodium carbonate solution after t minutes of reaction, and measuring the absorbance value A1 at 405 nm; Preheating the aminopeptidase solution to be tested, adding sodium carbonate solution after t minutes, and then adding the substrate L-leucine p-nitroaniline, and measuring the absorbance value A0 at 405 nm;
[0008] (4) Calculate the enzyme activity X of the aminopeptidase to be tested,
[0009]
[0010] Where a is the slope of the standard curve and k is the intercept of the standard curve.
[0011] Specifically, in the above step (3), A1-A0 is controlled to be 0.2-0.45.
[0012] Specifically, in the above step (3), a sodium carbonate solution with a concentration of 2-10% is added after t minutes.
[0013] Specifically, the reaction for t minutes in the above step (3) is specifically carried out by keeping the temperature at 40° C.-80° C. for t minutes.
[0014] Specifically, the preheating condition in the above step (3) is to keep warm in a 50° C. water bath for 2-5 minutes.
[0015] Specifically, in the above step (3), the substrate is 2.5 mmoL / L L-leucine p-nitroaniline.
[0016] Specifically, in the above step (3), the volume ratio of the aminopeptidase solution to be tested, L-leucine p-nitroaniline, and the sodium carbonate solution is 9:1:20.
[0017] Specifically, the buffer in the above step (2) is 0.05-0.1 mol / L Tris-HCl buffer.
[0018] Specifically, in the above step (2), after the aminopeptidase to be tested is added into the buffer solution, the pH value is 5.0-11.0.
[0019] Specifically, in the above step (2), the aminopeptidase to be tested is added with a buffer solution and then placed in a water bath shaker for extraction for 20-30 minutes. After standing for 10-20 minutes, the supernatant is diluted n times to obtain the aminopeptidase solution to be tested.
[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0021] The aminopeptidase enzyme activity detection method provided by the present invention optimizes the aminopeptidase detection steps, utilizes the reaction between the enzyme and the substrate, can simply, quickly, stably and accurately determine the enzyme activity of the aminopeptidase, and improves the safety of the aminopeptidase activity detection.
[0022] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 It is a standard curve diagram of the relationship between the p-nitrophenol content and the absorbance value in Example 1 of the present invention.
[0024] Figure 2 It is a curve diagram showing the change of aminopeptidase activity with pH value in Example 1 of the present invention.
[0025] Figure 3 It is a curve diagram showing the change of aminopeptidase activity with temperature in Example 1 of the present invention. DETAILED DESCRIPTION
[0026] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.
[0027] The present invention provides a method for detecting the enzyme activity of aminopeptidase, comprising the following steps:
[0028] (1) Draw a standard curve of p-nitrophenol concentration.
[0029] Take p-nitrophenol solutions of different concentrations, add the buffer solution, substrate and sodium carbonate solution respectively, mix thoroughly and measure the absorbance at 405 nm, and draw a standard curve with the absorbance as the abscissa and the p-nitrophenol content as the ordinate.
[0030] (2) Weigh the mass m of the aminopeptidase to be tested, add a buffer for extraction, preferably a Tris-HCl buffer, and after adding the buffer, the pH is 5.0-11.0, preferably pH 5.0-9.0, more preferably pH 6.0-8.0, more preferably pH 7-8, and most preferably pH 7.5. Then place in a water bath shaker for extraction for 20-30 minutes, let stand for 10-20 minutes, and take the supernatant and dilute it n times to obtain the aminopeptidase solution to be tested.
[0031] (3) Preheat the aminopeptidase solution to be tested, preferably keep it in a water bath at 50°C for 5 minutes, add the substrate L-leucine p-nitroaniline, preferably the substrate concentration is 2.5mmoL / L, keep it in a temperature range of 40°C-80°C, preferably 45°C-65°C for t minutes, then add a sodium carbonate solution, preferably a sodium carbonate solution with a concentration of 2-10%, and measure the absorbance value A1 at 405nm; Preheat the aminopeptidase solution to be tested, keep it in a temperature range of t minutes under the same conditions, then add a sodium carbonate solution of the same concentration, then add L-leucine p-nitroaniline, and measure the absorbance value A0 at 405nm. t minute is preferably 5 minutes.
[0032] (4) Calculate the enzyme activity X of the aminopeptidase to be tested,
[0033]
[0034] Where a is the slope of the standard curve and k is the intercept of the standard curve.
[0035] The effect of the method for detecting the activity of aminopeptidase of the present invention is studied by specific examples. The enzyme activity of aminopeptidase in the following examples is defined as: 1g solid enzyme or 1mL liquid enzyme, at 50°C, pH 7.5, hydrolyzes 2.5mmol / L L-leucine p-nitroaniline (L-Leu-pNA) per minute to produce 1μmol p-nitrophenol, which is 1 enzyme activity unit, expressed as U / g or U / mL.
[0036] Embodiment 1:
[0037] This embodiment provides a method for detecting the enzyme activity of aminopeptidase, which specifically includes the following steps.
[0038] 1. Prepare reagents
[0039] 0.1mol / LTris-HCl buffer (pH 7.5): Accurately weigh 12.11g Tris, dissolve in 900mL water, add dilute hydrochloric acid solution to adjust the pH to 7.5, and add water to make up to 1L.
[0040] 2.5mmoL / L aminopeptidase substrate solution: L-leucine p-nitroaniline was purchased from alfa aesar A11994. Accurately weigh 0.0314g of L-leucine p-nitroaniline and heat it in buffer for 10 minutes to dissolve it. After cooling, dilute to 50mL with 0.1mol / L Tris-HCl buffer. It can be stored at 4℃ for 5 days and at -18℃ for 20 days. Thaw naturally at room temperature.
[0041] 25mmol / L p-nitrophenol standard solution: Accurately weigh 1.7390g p-nitrophenol and dissolve it in 400mL buffer, then add 0.1mol / LTris-HCl buffer to make up to 500mL.
[0042] 10% sodium carbonate solution: Accurately weigh 10 g of anhydrous Na2CO3, add 80 mL of water to dissolve it, and finally make up to 100 mL with water.
[0043] 2. Draw a standard curve
[0044] Prepare a p-nitrophenol standard solution with a concentration of 200-1400 μmol / L according to Table 1, add equal amounts of Tris-HCl buffer, L-leucine p-nitroaniline solution and sodium carbonate solution, mix thoroughly and measure the absorbance at 405 nm, and draw a standard curve with the absorbance as the horizontal axis and the p-nitrophenol content as the vertical axis, as shown in Figure 1. Figure 1 As shown. Figure 1 It can be seen that when the concentration of p-nitrophenol in the solution is in the range of 0-1400 μmol / L (corresponding to an absorbance value of 0.1-0.95), there is an excellent linear relationship between the absorbance value of the solution and the p-nitrophenol concentration.
[0045] Table 1 Proportions of p-nitrophenol standard solution
[0046]
[0047] 3. Prepare the aminopeptidase solution to be tested
[0048] Weigh multiple portions of aminopeptidase to be tested with a mass of m grams, accurate to 0.0001g. Place each portion of aminopeptidase in a conical flask, add 100mL of 0.1mol / L pH=7.5 Tris-HCl buffer, extract for 30min in a waterbath shaker, let stand for 10min, take the supernatant and dilute it n times with 0.1mol / L pH=7.5 Tris-HCl buffer as the aminopeptidase solution to be tested. The m and n of each group are shown in Table 2.
[0049] 4. Absorbance determination
[0050] Experimental group: Take 0.2mL of the aminopeptidase solution to be tested and preheat it in a 50℃ water bath for 5min, add 1.8mL of aminopeptidase substrate, react at 50℃ for 10min, add 4mL of 10% sodium carbonate to terminate the reaction, and obtain the first mixed solution. Cool it to room temperature with tap water, and measure the absorbance value A1 of the first mixed solution at 405nm.
[0051] Control group: Take 0.2mL of the aminopeptidase solution to be tested and preheat it in a 50℃ water bath for 5min, react accurately at 50℃ for 10min, first add 4mL of 10% sodium carbonate, then add 1.8mL of aminopeptidase substrate to obtain a second mixed solution, cool it to room temperature with tap water, and measure the absorbance value A0 of the second mixed solution at 405nm.
[0052] In order to make the test result more accurate, in this embodiment, the concentration of p-nitrophenol in the first mixed solution is controlled within the concentration range corresponding to the absorbance value of 0.2 to 0.45, that is, the enzyme activity of the aminopeptidase in the aminopeptidase solution to be tested is controlled within the range of A1-A0=0.2 to 0.45.
[0053] The enzyme activity X of the aminopeptidase to be tested was calculated according to the following formula.
[0054]
[0055] The slope a=1495.3, k=7.58, t=10min, and the values of m and n are shown in Table 2.
[0056] Table 2 Aminopeptidase activity test results
[0057]
[0058] It can be seen from the test results in Table 2 that when the enzyme activity range of the aminopeptidase to be tested is A1-A0=0.2-0.45, the results of the enzyme activity of the aminopeptidase tested by the detection method of the present invention have good repeatability and high precision.
[0059] Embodiment 2:
[0060] This example adopts a method for detecting the activity of aminopeptidase similar to that of Example 1, except that the dilution factor n of the aminopeptidase solution to be tested in step 3 and the reaction temperature in step 4 are different. The specific dilution factor and reaction temperature in this example are shown in Table 3.
[0061] Table 3 Dilution multiple and reaction temperature of aminopeptidase solution in each experimental group
[0062] Serial number Reaction temperature (℃) Aminopeptidase dilution factor n 11 30 20000 12 40 38000 13 50 70000 14 60 130000 15 65 150000 16 70 170000 17 75 100000 18 80 65000
[0063] The curve is drawn with temperature as the horizontal axis and the calculated enzyme activity retention rate as the vertical axis, and the following is obtained: Figure 2 The curve of aminopeptidase activity changing with temperature is shown in the figure. Figure 2 It can be seen that the enzyme activity of aminopeptidase is strongest at 70℃; when the reaction temperature is lower than 60℃, the enzyme activity increases with the increase of temperature; when the reaction temperature is higher than 70℃, the enzyme activity gradually decreases with the increase of temperature. When the reaction temperature is in the range of 50℃~75℃, the enzyme activity retention rate of aminopeptidase is more than 50%; at 70℃, the enzyme activity retention rate of aminopeptidase reaches 100%. When aminopeptidase is used as an animal feed additive, in order to maintain the accuracy of the test results, 50℃ is selected as the temperature for defining enzyme activity.
[0064] Embodiment 3:
[0065] This example adopts an aminopeptidase activity detection method similar to that of Example 1, except that the pH of the 0.1 mol / L Tris-HCl buffer and the dilution factor n of the aminopeptidase solution to be tested in step 3 are different from those of Example 1. The dilution factor and pH value of this example are shown in Table 4.
[0066] Table 4 Dilution multiples of aminopeptidase solution and pH value of Tris-HCl buffer in each experimental group
[0067] Serial number Reaction pH Aminopeptidase dilution factor n 19 5.0 2500 20 6.0 5000 21 7.0 50000 22 7.5 70000 23 8.0 60000 24 9.0 50000 25 10.0 25000
[0068] The curve is drawn with the pH value of the reaction system as the horizontal axis and the calculated enzyme activity retention rate as the vertical axis. Figure 3 The curve of the pH value change of aminopeptidase activity shown in Figure 3It can be seen that the enzyme activity of aminopeptidase is strongest at pH = 7.5; when the pH value is lower than 7.5, the enzyme activity increases with the increase of pH value; when the pH value is higher than 7.5, the enzyme activity decreases with the increase of pH value. When the pH value is 7.0-9.0, the enzyme activity retention rate of aminopeptidase is more than 60%; when the pH value is 7.0-8.0, the enzyme activity retention rate of aminopeptidase is more than 65%. In the present invention, pH = 5.5 is selected as the optimal pH value for detecting the enzyme activity of aminopeptidase.
[0069] Embodiment 4:
[0070] In this example, the accuracy of the method for detecting the enzyme activity of aminopeptidase provided by the present invention is verified. The detection method is similar to that in Example 1, except that the dilution factor n of the aminopeptidase solution to be tested in step 3 is different from that in Example 1.
[0071] Commercially available aminopeptidase with a specific enzyme activity of 1000-5000 U / g was diluted with 0.1 mol / L Tris-HcL buffer to prepare standard aminopeptidase solutions with different enzyme activities. The enzyme activities were detected using the method in Example 1. The specific dilution multiples and test results are shown in Table 5.
[0072] Table 5 Results of commercial aminopeptidase activity test
[0073]
[0074]
[0075] As can be seen from Table 5, when the absorbance value of commercial aminopeptidase with a specific enzyme activity of 1000-5000U / g is controlled at 0.2-0.45 by the method of the present invention, the test result has good reproducibility and high precision. Compared with the theoretical specific enzyme activity data, the error is small (relative error <5%), indicating that the method for detecting the enzyme activity of aminopeptidase in the present invention is very accurate.
[0076] Comparative Example 1:
[0077] In this comparative example, the enzyme activity of aminopeptidase was detected by a method similar to that in Example 1, except that no sodium carbonate solution was added when drawing the standard curve in step 2 of this comparative example, and the absorbance value in step 4 was determined as:
[0078] Experimental group: Take 0.2 mL of the aminopeptidase solution to be tested and preheat it in a 50°C water bath for 5 minutes, add 1.8 mL of aminopeptidase substrate, react at 50°C for 10 minutes to obtain the first mixed solution. Cool it to room temperature with tap water, and measure the absorbance value A2 of the first mixed solution at 405 nm.
[0079] Control group: Take 0.2mL of the aminopeptidase solution to be tested and preheat it in a 50℃ water bath for 5min, react accurately at 50℃ for 10min, add 1.8mL of aminopeptidase substrate to obtain a second mixed solution, cool it to room temperature with tap water, and measure the absorbance value A0 of the second mixed solution at 405nm.
[0080] The enzyme activity X of the aminopeptidase to be tested was calculated according to the following formula.
[0081]
[0082] The slope a=1188, k=-75.957, R2=0.9905, t=10 min, and the values of m and n are shown in Table 6. The test was repeated 5 times and the results are shown in Table 6.
[0083] Table 6 Comparative Example Aminopeptidase Activity Detection Results
[0084]
[0085]
[0086] From the test results in Table 6, it can be seen that the test results without adding sodium carbonate solution are higher than those with adding sodium carbonate solution, but the test data reproducibility is poor and the fluctuation is large.
[0087] The R2 of the standard curve is 0.9905, and the linear relationship of the standard curve is not very good, which means that adding sodium carbonate solution to the reaction system makes the test results more stable.
[0088] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.
Claims
1. A method for detecting the enzyme activity of aminopeptidase, characterized in that: The following steps are involved: (1) Draw a standard curve of p-nitrophenol concentration; (2) weighing a mass m of the aminopeptidase to be tested, adding a buffer solution for extraction, and diluting the extract by n times to obtain a solution of the aminopeptidase to be tested; (3) Preheating the aminopeptidase solution to be tested, adding the substrate L-leucine p-nitroaniline thereto, adding sodium carbonate solution after t minutes of reaction, and measuring the absorbance value A1 at 405 nm; Preheating the aminopeptidase solution to be tested, adding sodium carbonate solution after t minutes, and then adding the substrate L-leucine p-nitroaniline, and measuring the absorbance value A0 at 405 nm; (4) Calculate the enzyme activity X of the aminopeptidase to be tested, Where a is the slope of the standard curve and k is the intercept of the standard curve.
2. The method for detecting the activity of aminopeptidase according to claim 1, wherein: In the step (3), A1-A0 is controlled to be 0.2-0.
45.
3. The method for detecting the activity of aminopeptidase according to claim 1, wherein: In the step (3), a sodium carbonate solution with a concentration of 2-10% is added after t minutes.
4. The method for detecting the activity of aminopeptidase according to claim 1, wherein: The reaction for t minutes in step (3) specifically comprises keeping the temperature at 40° C.-80° C. for t minutes.
5. The method for detecting the activity of aminopeptidase according to claim 1, characterized in that: The preheating condition in step (3) is to keep warm in a 50° C. water bath for 2-5 minutes.
6. The method for detecting the activity of aminopeptidase according to claim 1, characterized in that: In the step (3), the substrate is 2.5 mmoL / L of L-leucine p-nitroaniline.
7. The method for detecting the activity of aminopeptidase according to claim 6, characterized in that: In the step (3), the volume ratio of the aminopeptidase solution to be tested, L-leucine p-nitroaniline, and the sodium carbonate solution is 9:1:
20.
8. The method for detecting the activity of aminopeptidase according to claim 1, characterized in that: The buffer in step (2) is 0.05-0.1 mol / L Tris-HCl buffer.
9. The method for detecting the activity of aminopeptidase according to claim 1, wherein: In the step (2), after the aminopeptidase to be tested is added into the buffer solution, the pH value is 5.0-11.
0.
10. The method for detecting the activity of aminopeptidase according to claim 1, characterized in that: In the step (2), the aminopeptidase to be tested is added with a buffer solution and then placed in a water bath shaker for extraction for 20-30 minutes. After standing for 10-20 minutes, the supernatant is taken and diluted n times to obtain the aminopeptidase solution to be tested.