Method for detecting enzyme activity of papain in hypersensitive protein compound enzyme

Abstract

The invention provides a method for detecting enzyme activity of papain in a hypersensitive protein complex enzyme, which comprises the following steps: mixing the hypersensitive protein complex enzyme with an L-cysteine hydrochloride buffer solution to obtain a sample solution to be detected; adding the to-be-tested sample solution into a substrate solution containing casein, and testing a change curve of a voltage value of the to-be-tested sample solution along with time under the condition that the casein can be decomposed by papain; and determining the enzyme activity of the papain in the hypersensitive protein compound enzyme according to the change curve of the voltage value of the to-be-detected sample solution along with time. The method can solve the problem that the enzyme activity of papain in the hypersensitive protein complex enzyme cannot be accurately detected in the prior art.

Description

Technical Field

[0001] This invention relates to a method for detecting papain enzyme activity in hypersensitive protein complex enzymes, belonging to the field of detecting papain enzyme activity in hypersensitive protein complex enzymes. Background Technology

[0002] Hypersensitive protein complex enzymes are biological agents containing specific bioactive components used in agriculture. They are produced using plant monomeric enzymes or plant extracts as the main raw materials, with or without excipients, through advanced low-temperature fermentation and chelation processes. The plant monomeric enzymes include three or more plant monomeric enzymes, such as bromelain, papain, and phytase. Currently, hypersensitive protein complex enzymes are widely used in agriculture, animal husbandry, environmental protection, and daily chemicals, exhibiting significant characteristics such as high stability, high activity, rapid reaction speed, and wide applicability.

[0003] Papain is a protease that can decompose proteins in acidic, neutral, and alkaline environments. Among the techniques related to the detection of papain activity, T / CCCMHPIE 1.18-2016 provides a spectrophotometric method for detecting papain activity, while GB / T 23527.1-2023 provides the Folin method, ultraviolet spectrophotometer method, fully automated biochemical analysis method, and micro-colorimetric method for detecting papain activity. However, existing detection methods cannot accurately detect the papain activity in hypersensitive protein complex enzymes. Summary of the Invention

[0004] This invention provides a method for detecting papain activity in hypersensitive protein complex enzymes, thereby at least solving the problem of the inability of existing technologies to accurately detect papain activity in hypersensitive protein complex enzymes.

[0005] This invention provides a method for detecting papain enzyme activity in a hypersensitive protein complex enzyme, comprising the following steps: mixing a hypersensitive protein complex enzyme and L-cysteine ​​hydrochloride buffer to obtain a test sample solution; adding the test sample solution to a substrate solution containing casein, and testing the voltage change curve of the test sample solution over time under the condition that casein can be decomposed by papain; and determining the papain enzyme activity in the hypersensitive protein complex enzyme based on the voltage change curve of the test sample solution over time.

[0006] According to one embodiment of the present invention, the volume ratio of the hypersensitive protein complex enzyme to the L-cysteine ​​hydrochloride buffer is 100 μl:(800-1000) μL; and / or, the preparation process of the L-cysteine ​​hydrochloride buffer includes: mixing L-cysteine ​​hydrochloride, sodium chloride, disodium ethylenediaminetetraacetate and water to obtain a third solution; wherein the mass ratio of L-cysteine ​​hydrochloride to sodium chloride is 1:(4-5); the mass ratio of L-cysteine ​​hydrochloride to disodium ethylenediaminetetraacetate is 1:(0.35-0.5); adjusting the pH of the third solution to 7-7.5 using sodium hydroxide solution, and then adding water to make up the volume to obtain the L-cysteine ​​hydrochloride buffer; wherein the volume ratio of the third solution to the L-cysteine ​​hydrochloride buffer is 60%-80%.

[0007] According to one embodiment of the present invention, the volume ratio of the sample solution to the casein-containing substrate solution is 200 μL: (1.5–2.5) mL; and / or, the preparation process of the casein-containing substrate solution includes: wetting the casein with sodium hydroxide solution and mixing it with disodium hydrogen phosphate solution to obtain a fourth solution; adjusting the pH of the fourth solution to 7–7.5 with hydrochloric acid solution, and then adding water to make up the volume to obtain the casein-containing substrate solution; wherein, the volume ratio of the fourth solution to the casein-containing substrate solution is 70%–90%.

[0008] According to one embodiment of the present invention, the step of adding the test sample solution to a substrate solution containing casein and then testing the voltage value of the test sample solution over time under the condition that casein can be decomposed by papain specifically includes: adding the substrate solution containing casein to a first detection tube, placing the first detection tube in the detection channel of a biochemical process monitor, then adjusting the operating conditions of the biochemical process monitor to the condition that casein can be decomposed by papain, and then adding the test sample solution to the first detection tube and testing the voltage value of the test sample solution over time.

[0009] According to one embodiment of the present invention, after adjusting the operating conditions of the biochemical process monitor to a condition in which casein can be decomposed by papain, the condition is maintained for 50-70 seconds, and then the test sample solution is added to the first detection tube, and the voltage value of the test sample solution changes over time.

[0010] According to one embodiment of the present invention, the conditions under which the casein can be decomposed by papain include a temperature of 20–80°C.

[0011] According to one embodiment of the present invention, the process of testing the voltage value of the sample solution under the condition that casein can be decomposed by papain includes: collecting voltage values ​​at different times to obtain the voltage value change curve over time; wherein the voltage value collection period is 0.5 to 2 seconds, and the number of voltage value collections is 500 to 700 times.

[0012] According to one embodiment of the present invention, the method further includes testing the voltage change curve of the standard solution over time and / or the voltage change curve of the L-cysteine ​​hydrochloride buffer solution over time; wherein, the testing process of the voltage change curve of the standard solution over time includes: mixing papain and the L-cysteine ​​hydrochloride buffer solution to obtain a standard solution; adding the standard solution to the substrate solution containing casein, and testing the voltage change curve of the standard solution over time under the condition that the casein can be decomposed by papain; the testing process of the voltage change curve of the L-cysteine ​​hydrochloride buffer solution over time... The process includes: adding the L-cysteine ​​hydrochloride buffer to the substrate solution containing casein, and then testing the voltage change curve of the L-cysteine ​​hydrochloride buffer over time under the condition that the casein can be decomposed by papain; the process of determining the papain enzyme activity in the hypersensitive protein complex enzyme based on the voltage change curve of the test sample solution over time includes: determining the papain enzyme activity in the hypersensitive protein complex enzyme based on the voltage change curve of the test sample solution over time, and referring to the voltage change curve of the standard solution over time and / or the voltage change curve of the L-cysteine ​​hydrochloride buffer over time.

[0013] According to one embodiment of the present invention, in the standard solution, the mass-to-volume ratio of the papain and the L-cysteine ​​hydrochloride buffer is 0.05 g: (4-6) mL.

[0014] According to one embodiment of the present invention, the process of determining the papain enzyme activity in the hypersensitive protein complex enzyme based on the voltage value change curve of the test sample solution over time includes: based on the voltage value when the voltage value change curve over time reaches a stable state, using papain as a standard, determining the papain enzyme activity value in the hypersensitive protein complex enzyme by external standard method.

[0015] The implementation of this invention has at least the following beneficial effects:

[0016] By adding the sample solution to a substrate solution containing casein, and testing the voltage of the sample solution at different times under the condition that casein can be decomposed by papain, the voltage value of the sample solution as a function of time is obtained. Thus, the enzyme activity of papain in the hypersensitive protein complex enzyme can be determined based on the voltage value of the sample solution as a function of time. This allows for rapid and accurate measurement of the enzyme activity of papain in the hypersensitive protein complex enzyme. Detailed Implementation

[0017] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] This invention provides a method for detecting the papain activity in a hypersensitive protein complex enzyme, comprising the following steps: mixing the hypersensitive protein complex enzyme and L-cysteine ​​hydrochloride buffer to obtain a sample solution to be tested; adding the sample solution to be tested to a substrate solution containing casein, and testing the voltage change curve of the sample solution over time under the condition that casein can be decomposed by papain; determining the papain activity in the hypersensitive protein complex enzyme based on the voltage change curve of the sample solution over time.

[0019] The test sample solution contains papain. During the measurement of the voltage value of the test sample solution (i.e., the voltage value of the mixture of the test sample solution and the substrate solution containing casein) over time, under conditions where casein can be decomposed by papain, papain catalyzes the decomposition of casein, i.e., an enzymatic reaction occurs. As the enzymatic reaction proceeds (i.e., as the reaction time increases), casein is gradually decomposed, causing a change in the conductivity of the test sample solution, which in turn causes a change in the voltage value of the test sample solution. When the casein is basically completely decomposed... After the reaction, the voltage value of the sample solution reaches a plateau (i.e., the voltage value of the sample solution essentially stops changing with increasing reaction time). Therefore, the voltage value of the sample solution over time (enzyme-catalyzed reaction time) can be measured. This curve is a single parabola (i.e., in a coordinate system with time on the x-axis and voltage on the y-axis, the curve initially shows an upward trend until it reaches a plateau). In other words, the voltage value of the sample solution over time has a peak (i.e., the voltage value at which the curve reaches a plateau). Based on the voltage value of the sample solution over time, the enzyme activity of papain in the hypersensitive protein complex enzyme can be obtained.

[0020] In some embodiments, the volume ratio of the hypersensitive protein complex enzyme to the L-cysteine ​​hydrochloride buffer can be 100 μl: (800-1000) μL, for example, 100 μl: 800 μl, 100 μl: 850 μl, 100 μl: 900 μl, 100 μl: 950 μl, 100 μl: 1000 μl or any combination thereof, which can ensure that the hypersensitive protein complex enzyme is fully dissolved and diluted, thereby improving the accuracy of the papain enzyme activity assay results in the hypersensitive protein complex enzyme.

[0021] In addition, the preparation process of L-cysteine ​​hydrochloride buffer may include: mixing L-cysteine ​​hydrochloride, sodium chloride, disodium ethylenediaminetetraacetate and water to obtain a third solution.

[0022] The mass ratio of L-cysteine ​​hydrochloride to sodium chloride can be 1:(4-5), for example, 1:4, 1:4.2, 1:4.4, 1:4.6, 1:4.8, 1:5, or any combination thereof; the mass ratio of L-cysteine ​​hydrochloride to disodium ethylenediaminetetraacetate is 1:(0.35-0.5), for example, 1:0.35, 1:0.4, 1:0.45, 1:0.5, or any combination thereof; the pH of the third solution is adjusted to 7-7.5 using sodium hydroxide solution, for example, 7.1, 7.2, 7.3, 7.4, 7.5, or any combination thereof; then water is added to make up the volume to obtain the L-cysteine ​​hydrochloride buffer solution.

[0023] The volume ratio of the third solution to the L-cysteine ​​hydrochloride buffer is 60% to 80%, for example, a range of 60%, 65%, 70%, 75%, 80%, or any two of these.

[0024] Specifically, the concentration of the sodium hydroxide solution in the third solution can be adjusted to be 0.5–1.5 mol / L, for example, a range of 0.5 mol / L, 0.8 mol / L, 1 mol / L, 1.2 mol / L, 1.5 mol / L, or any combination thereof.

[0025] In specific implementation, L-cysteine ​​hydrochloride, sodium chloride, and water can be mixed to obtain a first solution. The volume of the first solution can be 400-600 mL, for example, 400 mL, 450 mL, 500 mL, 550 mL, 600 mL, or any combination thereof. Disodium ethylenediaminetetraacetate and water can be mixed to obtain a second solution. The volume of the second solution can be 150-250 mL, for example, 150 mL, 175 mL, 200 mL, 225 mL, 250 mL, or any combination thereof. The third solution can be diluted with water to a final volume of 1000 mL. The volume of water added during the dilution process can be 200 mL-400 mL, for example, 200 mL, 250 mL, 300 mL, 350 mL, 400 mL, or any combination thereof.

[0026] In some embodiments, the volume ratio of the sample solution to the substrate solution containing casein can be 200 μL: (1.5 to 2.5) mL, for example, 200 μL: 1.5 mL, 100 μL: 1.8 mL, 200 μL: 2 mL, 200 μL: 2.2 mL, 200 μL: 2.5 mL, or any combination thereof.

[0027] In addition, the preparation process of the substrate solution containing casein may include: wetting casein with sodium hydroxide solution and mixing it with disodium hydrogen phosphate solution to obtain a fourth solution; adjusting the pH of the fourth solution to 7 to 7.5, for example, 7.1, 7.2, 7.3, 7.4, 7.5 or any combination thereof, using hydrochloric acid solution; and then adding water to make up to a final volume to obtain the substrate solution containing casein.

[0028] Specifically, dried casein can be placed in a beaker, moistened with sodium hydroxide solution, and then disodium hydrogen phosphate solution can be added to obtain the fourth solution. The fourth solution can be heated in boiling water for 30 to 50 minutes, for example, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes or any combination thereof, and then magnetically stirred at 1200 to 1800 rpm, for example, 1200 rpm, 1300 rpm, 1400 rpm, 1500 rpm, 1600 rpm, 1700 rpm, 1800 rpm or any combination thereof, to accelerate the dissolution of casein. After the fourth solution cools to room temperature, the pH of the fourth solution can be adjusted to 7 to 7.5 with hydrochloric acid solution, and then water can be added to make up the volume to obtain a substrate solution containing casein. The volume of water added during the volume adjustment process can be 20mL to 50mL, for example, 20mL, 25mL, 30mL, 35mL, 40mL, 45mL, 50mL or any combination thereof.

[0029] The volume ratio of the fourth solution to the substrate solution containing casein is 70% to 90%, for example, a range of 70%, 75%, 80%, 85%, 90%, or any two of these.

[0030] Specifically, the concentration of the hydrochloric acid solution used to adjust the pH of the fourth solution can be 0.5 to 1.5 mol / L, for example, a range of 0.5 mol / L, 0.8 mol / L, 1 mol / L, 1.2 mol / L, 1.5 mol / L, or any combination thereof.

[0031] In addition, the concentration of the sodium hydroxide solution used to moisten casein can be 0.5 to 1.5 mol / L, for example, 0.5 mol / L, 0.8 mol / L, 1 mol / L, 1.2 mol / L, 1.5 mol / L or any combination thereof.

[0032] Specifically, the concentration of the disodium hydrogen phosphate solution can be 0.05–0.08 mol / L, for example, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L, 0.07 mol / L, 0.08 mol / L, or any combination thereof, and the volume of the disodium hydrogen phosphate solution can be 50–80 mL, for example, 50 mL, 60 mL, 70 mL, 80 mL, 100 mL, or any combination thereof.

[0033] The concentration of casein in the substrate solution containing casein can be (0.3 to 0.5) g / 100 mL, for example, 0.3 g / 100 mL, 0.4 g / 100 mL, 0.5 g / 100 mL or any combination thereof.

[0034] In practice, the substrate solution containing casein can be prepared fresh before use.

[0035] In some embodiments, after adding the sample solution to be tested to a substrate solution containing casein, the voltage value of the sample solution is tested over time under the condition that casein can be decomposed by papain. Specifically, this may include: adding the substrate solution containing casein to a first detection tube, placing the first detection tube in the detection channel of a biochemical process monitor, then adjusting the operating conditions of the biochemical process monitor to the condition that casein can be decomposed by papain, then adding the sample solution to be tested to the first detection tube, and testing the voltage value of the sample solution over time.

[0036] In practice, after adjusting the operating conditions of the biochemical process monitor to allow casein to be broken down by papain, the substrate solution containing casein can be added to the first detection tube after maintaining this condition for 20–40 minutes (e.g., 30 minutes). After adding the substrate solution containing casein to the first detection tube, the condition should be maintained for 5–15 minutes (e.g., 10 minutes) before adding the sample solution to be tested. This makes the conditions in the biochemical process monitor that allow casein to be broken down by papain more stable, which helps improve the accuracy of enzyme activity assay results.

[0037] In some embodiments, after adjusting the operating conditions of the biochemical process monitor to conditions where casein can be decomposed by papain, the sample solution to be tested is added to the first detection tube and the detection tube is placed inside the biochemical process monitor. The test tube can be maintained for 50 to 70 seconds (i.e., equilibrium time), for example, 50 seconds, 55 seconds, 60 seconds, 65 seconds, 70 seconds or any combination thereof, and the voltage value of the sample solution to be tested changes over time.

[0038] In some embodiments, the conditions under which casein can be decomposed by papain may include a temperature of 20 to 80°C, such as a range of 20°C, 37°C, 47°C, 57°C, 67°C, 77°C, 80°C, or any combination thereof.

[0039] In some embodiments, the process of testing the voltage value of the sample solution under the condition that casein can be decomposed by papain may include: collecting voltage values ​​at different times to obtain the voltage value change curve over time; wherein, the voltage value acquisition period can be 0.5 to 2 s, for example, a range of 0.5 s, 0.8 s, 1 s, 1.2 s, 1.4 s, 1.6 s, 1.8 s, 2 s or any two of these. For example, when the voltage value acquisition period is 1 s, a voltage value is acquired every 1 s.

[0040] In some embodiments, the number of voltage value acquisitions can be 500 to 700 times, for example, a range of 500, 550, 600, 650, 700 times or any combination thereof.

[0041] Specifically, the voltage value corresponds to the conductivity value, the voltage value acquisition period can also be called the conductivity value acquisition period, and the number of voltage value acquisitions can also be called the conductivity value acquisition number.

[0042] In practice, when using a biochemical process monitor to test the voltage value of the sample solution over time, the excitation frequency of the biochemical process monitor can be 200 kHz and the excitation level can be 90%.

[0043] In some embodiments, the voltage values ​​of the standard solution (i.e., the mixture of the standard solution and the substrate solution containing casein) over time can also be tested, and / or the voltage values ​​of the L-cysteine ​​hydrochloride buffer (i.e., the mixture of the L-cysteine ​​hydrochloride buffer and the substrate solution containing casein) over time can also be tested.

[0044] The testing process for the voltage change curve of the standard solution over time includes: mixing papain (or papain standard) and L-cysteine ​​hydrochloride buffer to obtain a standard solution; adding the standard solution to a substrate solution containing casein; and testing the voltage change curve (or enzyme catalytic reaction curve) of the standard solution over time under the condition that casein can be decomposed by papain.

[0045] Specifically, the L-cysteine ​​hydrochloride buffer used in preparing the standard solution is the same as the L-cysteine ​​hydrochloride buffer used in preparing the test sample solution. When testing the voltage value of the standard solution over time, the substrate solution containing casein, the conditions under which casein can be decomposed by papain (conditions for enzyme-catalyzed reaction), and other operating conditions of the biochemical process monitor are the same as those when testing the voltage value of the test sample over time.

[0046] The test procedure for the voltage change curve of L-cysteine ​​hydrochloride buffer over time includes: adding L-cysteine ​​hydrochloride buffer to a substrate solution containing casein, and then testing the voltage change curve of L-cysteine ​​hydrochloride buffer over time under the condition that casein can be decomposed by papain.

[0047] Specifically, the L-cysteine ​​hydrochloride buffer used is the same as the L-cysteine ​​hydrochloride buffer used when preparing the test sample solution. When testing the voltage value of the L-cysteine ​​hydrochloride buffer over time, the substrate solution containing casein, the conditions under which casein can be decomposed by papain (conditions for enzyme-catalyzed reaction), and other operating conditions of the biochemical process monitor are the same as those when testing the voltage value of the test sample over time.

[0048] In some embodiments, the process of determining the papain enzyme activity in the hypersensitive protein complex enzyme by the voltage value change curve of the test sample solution over time includes: determining the papain enzyme activity in the hypersensitive protein complex enzyme based on the voltage value change curve of the test sample solution over time, and referring to the voltage value change curve of the standard solution over time and / or the voltage value change curve of the L-cysteine ​​hydrochloride buffer over time.

[0049] The standard solution contains papain. During the measurement of the voltage change curve of the standard solution over time, under conditions where casein can be decomposed by papain, papain catalyzes the decomposition of casein, i.e., an enzymatic reaction occurs. As the enzymatic reaction proceeds (i.e., as the reaction time increases), casein is gradually decomposed, causing a change in the conductivity of the standard solution, which in turn causes a change in the voltage value of the standard solution. When the casein is basically completely decomposed, the voltage value of the standard solution reaches a stable state (i.e., as the reaction time increases, the voltage value of the standard solution essentially no longer changes). Therefore, the voltage change curve of the standard solution over time (enzyme-catalyzed reaction time) can be measured. This curve is a single parabola (i.e., in a coordinate system with time as the abscissa and voltage as the ordinate, the curve initially shows an upward trend until it reaches a stable state). In other words, the voltage change curve of the standard solution over time has a peak value (i.e., the voltage value at which the curve reaches a stable state).

[0050] L-cysteine ​​hydrochloride buffer does not contain papain and cannot break down casein. Therefore, the voltage value of L-cysteine ​​hydrochloride buffer changes over time in a basically linear manner.

[0051] Therefore, when the voltage change curve of the test sample solution over time is basically consistent with the voltage change curve of the standard solution over time (i.e., the voltage change curve of the test sample solution over time is also a single parabola), it indicates that the papain in the hypersensitive protein complex enzyme is active. However, when the voltage change curve of the test sample solution over time is basically consistent with the voltage change curve of the L-cysteine ​​hydrochloride buffer over time, it indicates that the hypersensitive protein complex enzyme does not contain active papain (i.e., it has no activity).

[0052] Furthermore, the voltage change curve of L-cysteine ​​hydrochloride buffer over time also serves a calibration function. Specifically, if the measured voltage change curve of L-cysteine ​​hydrochloride buffer over time has a peak (e.g., a parabolic or other peak-shaped curve) rather than being nearly linear, it indicates an error in the testing conditions (e.g., reagent preparation, experimental parameter / condition settings, operating procedures, etc.). Therefore, calibration can be performed by testing the voltage change curve of L-cysteine ​​hydrochloride buffer over time.

[0053] In some embodiments, the mass-to-volume ratio of papain and L-cysteine ​​hydrochloride buffer in the standard solution can be 0.05 g: (4-6) mL, for example, a range of 0.05: 4 mL, 0.05: 4.5 mL, 0.05: 5 mL, 0.05: 5.5 mL, 0.05: 6 mL, or any combination thereof.

[0054] In some embodiments, the process of determining the papain enzyme activity in the hypersensitive protein complex enzyme based on the voltage value change curve of the sample solution over time includes: determining the papain enzyme activity value in the hypersensitive protein complex enzyme by using papain as a standard, based on the voltage value when the voltage value change curve over time reaches a stable state, and using papain as a standard.

[0055] Specifically, as the enzyme-catalyzed reaction proceeds, the voltage value gradually increases. Once the casein is completely decomposed, the voltage value basically stops changing, and the curve reaches a stable state. The voltage value at which the voltage-time curve reaches a stable state is the maximum voltage value.

[0056] Specifically, the equation for the voltage value at which the enzyme activity and voltage change curve of standard papain over time reach a steady state is obtained using the external standard method. The equation can be X = 3715e 139.9C X represents the enzyme activity of papain, and C represents the voltage value when the voltage-time curve reaches a steady state. Based on this equation and the voltage value when the voltage-time curve of the test sample solution reaches a steady state, the enzyme activity of papain in the hypersensitive protein complex enzyme can be calculated.

[0057] Specifically, papain and the L-cysteine ​​hydrochloride buffer can be used to prepare standard solutions with different concentrations of papain. These standard solutions have different concentrations of papain, and correspondingly, different enzyme activities (X) of the papain in these standard solutions. Then, referring to the test procedure of the voltage value change curve of the aforementioned standard solutions over time, the voltage value change curve of each standard solution is tested, and the voltage value (C) at which the voltage value change curve reaches a stable state is obtained. Based on the enzyme activity (X) of the papain in these standard solutions and the voltage values ​​(C) obtained through these standard solutions, the relationship (equation) between the voltage value (C) and X is determined.

[0058] Specifically, the above equation can be expressed as X = 3715e 139.9C X represents the enzyme activity of papain, and C represents the voltage value when the voltage-time curve reaches a steady state.

[0059] In specific implementation of this invention, to further improve accuracy, when testing any test solution (such as the above-mentioned test sample solution or standard solution), multiple tests can be performed, or multiple test samples can be taken and tested simultaneously to obtain the voltage value C when the voltage value change curve over time reaches a stable state, for example, three. Then, the average value of these voltage values ​​C is calculated as the final voltage value C when the voltage value change curve over time reaches a stable state.

[0060] For example, when testing the voltage value C at which the voltage change curve of any test sample solution reaches a steady state over time, multiple first detection tubes can be used. Substrate solution is added to each first detection tube, and then multiple portions of the test sample solution are taken and added to each detection tube. The voltage change curve of each test sample solution is tested, and the voltage value C at which the change curve reaches a steady state is obtained, thus obtaining multiple voltage values ​​C. Then, the average value of these voltage values ​​C is calculated as the voltage value C at which the voltage change curve of the test sample solution reaches a steady state over time. This average value is then substituted into the above equation to calculate the papain enzyme activity in the hypersensitive protein complex enzyme.

[0061] Generally, biochemical process monitors have multiple detection channels. In specific implementation, the same biochemical process monitor can be used to simultaneously test the voltage changes of the above-mentioned sample solution, standard solution, and L-cysteine ​​hydrochloride buffer over time. Specifically, a substrate solution containing casein can be added to the first, second, and third detection tubes, respectively. These detection tubes are then placed in the detection channels of the biochemical process monitor. The operating conditions of the biochemical process monitor are then adjusted to conditions that allow casein to be decomposed by papain. The sample solution to be tested is then added to the first detection tube, the second sample solution to be tested is added to the second detection tube, and the L-cysteine ​​hydrochloride buffer is added to the third detection tube. The voltage changes of these solutions over time are then tested.

[0062] In practice, the biochemical process monitor used can be the BER816 model from Qingdao Elf Analytical Instruments Co., Ltd. The biochemical process monitor has a dedicated software CellStatz main interface window, through which parameters such as temperature, equilibrium time, voltage value acquisition cycle, excitation frequency, excitation level, and conductivity value acquisition number can be set.

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below through specific embodiments. In the following embodiments, unless otherwise specified, the papain used is from Shanghai Yuanye Biotechnology Co., Ltd., the hypersensitive protein complex enzyme used is from Qingdao Huashengrong Biotechnology Co., Ltd., the biochemical process monitoring instrument used is from Qingdao Jingling Analytical Instrument Co., Ltd., and all reagents used are analytical grade reagents or biological reagents conforming to national standards.

[0064] Unless otherwise specified, the instruments and equipment used in the following examples are as follows: a biochemical process monitor (purchased from Qingdao Elf Analytical Instruments Co., Ltd., model BER816); a magnetic stirrer heater; disposable glass test tubes (hereinafter referred to as test tubes); micropipettes and matching pipette tips: 200μL scale (accuracy 1μL), 1mL scale (accuracy 0.01mL), and 10mL (0.1mL scale) can be selected according to the operation; a balance (sensitivity 0.001g, 6.6 pH meter: ±0.01); centrifuge tubes: 2mL and 10mL capacity can be selected according to the operation; volumetric flasks: 100mL, 500mL, and 1000mL capacity can be selected according to the operation; beakers: 200mL and 500mL capacity can be selected according to the operation; graduated cylinder (specification 100mL); reagent bottle (capacity 500mL).

[0065] In the following examples, unless otherwise specified, the reagents and materials used are as follows:

[0066] (1) Water (GB / T 6682, Grade I).

[0067] (2) Sodium hydroxide solution (1 mol / L): Prepare and standardize according to the method specified in GB / T 601. Weigh 20 g of sodium hydroxide solid using a balance, dissolve it in water, and transfer it to a 500 mL volumetric flask by means of a glass rod. Wash the beaker and glass rod 2-3 times, and transfer the washing solution to the volumetric flask by means of a glass rod. Then add water to the volumetric flask until it is 1-2 cm away from the mark, and then add it dropwise with a dropper until the liquid level is the same as the mark. Stopper the volumetric flask, shake well, and transfer it to a reagent bottle.

[0068] (3) Hydrochloric acid solution (1 mol / L): Prepare and standardize according to the method specified in GB / T 601. Measure 43 mL of 36% hydrochloric acid with a graduated cylinder and pour it into a beaker. Dilute with water and transfer it to a 500 mL volumetric flask by means of a glass rod. Wash the beaker and glass rod 2-3 times. Transfer the washing solution to the volumetric flask by means of a glass rod. Then add water to the volumetric flask until it is 1-2 cm away from the graduation mark. Use a dropper to add water dropwise until the liquid level is the same as the graduation mark. Stopper the volumetric flask, shake well, and transfer it to a reagent bottle.

[0069] (4) Hydrochloric acid solution (0.1 mol / L): Prepare and calibrate according to the method specified in GB / T 601. Measure 4.3 mL of 36% hydrochloric acid into a beaker using a graduated cylinder, dilute with water, and transfer to a 500 mL volumetric flask by means of a glass rod. Wash the beaker and glass rod 2-3 times, and transfer the washing solution to the volumetric flask by means of a glass rod. Then add water to the volumetric flask until it is 1-2 cm away from the graduation mark, and then add water dropwise with a dropper until the liquid level is the same as the graduation mark. Stopper the volumetric flask, shake well, and transfer to a reagent bottle.

[0070] (5) Disodium hydrogen phosphate solution (0.05 mol / L): Prepare and standardize according to the method specified in GB / T 601. Weigh 1.7745 g of disodium hydrogen phosphate solid, dissolve it in water, and transfer it to a 500 mL volumetric flask by means of a glass rod. Wash the beaker and glass rod 2-3 times, and transfer the washing solution to the volumetric flask by means of a glass rod. Then add water to the volumetric flask to 1-2 cm away from the mark, and then use a dropper to add water dropwise until the liquid level is the same as the mark. Stopper the volumetric flask, shake well, and transfer it to a reagent bottle.

[0071] (6) L-cysteine ​​hydrochloride buffer: Weigh 5.27 g of cysteine ​​hydrochloride and 23.4 g of sodium chloride, accurate to 0.001 g, and dissolve in 500 mL of water; weigh 2.23 g of disodium ethylenediaminetetraacetate, accurate to 0.001 g, and dissolve in 200 mL of water; mix the solutions prepared in 1.6.1 and 1.6.2, adjust the pH to 7-7.5 with 1 mol / L sodium hydroxide solution, transfer to a 1000 mL volumetric flask, add water to the mark and shake well.

[0072] (7) Casein solution (substrate solution): Prepare fresh before use. Weigh 0.4 g of dried casein (NICPBP National Drug Standard Material), accurate to 0.001 g, and place it in a beaker. Moisten with a small amount of 1 mol / L sodium hydroxide solution, then add 80 mL of 0.05 mol / L disodium hydrogen phosphate solution. Stir magnetically at 1500 rpm and 100 °C for 40 min using a magnetic stirrer. After cooling to room temperature, adjust the pH to 7–7.5 with 1 mol / L hydrochloric acid solution, then transfer to a 100 mL volumetric flask, add water to the mark, and mix well.

[0073] In the following examples, unless otherwise specified, the sample preparation and determination are as follows:

[0074] (1) Standard solution: Prepare fresh before use. Weigh 0.05 g of papain standard, accurate to 0.001 g, place it in a beaker, dissolve it thoroughly with about 5 mL of L-cysteine ​​hydrochloride buffer, and then transfer it to a 10 mL centrifuge tube. The activity unit of the papain standard used is 800 U / mg.

[0075] (2) Hypersensitive protein complex enzyme sample solution (sample solution to be tested): Prepare fresh before use. Take 100 μL of the hypersensitive protein complex enzyme sample and add it to a 2 mL centrifuge tube containing 900 μL of L-cysteine ​​hydrochloride buffer. Shake gently to mix.

[0076] Example 1: Detection process of papain enzyme activity in hypersensitive protein complex enzyme

[0077] Prepare the instrument: Turn on the biochemical process monitor. In the CellStatz software's main interface window, set the temperature to 37℃, the equilibration time to 60s, the voltage acquisition cycle to 1s, the excitation frequency to 200kHz, the excitation level to 90%, and the voltage acquisition count to 600. Preheat for 30 minutes.

[0078] Tube loading and instrument preheating: The experiment consisted of three parallel experiments. 2 mL of casein solution was added to each of the nine test tubes using a micropipette. The test tubes were then inserted into the detection channels of the biochemical process monitor and preheated for 10 minutes to reach a temperature of 37°C. This temperature was maintained during subsequent testing of the voltage values ​​of each solution over time (i.e., the voltage value over time was tested at this temperature).

[0079] Sample loading and detection: Using a micropipette, quickly add 200 μL of L-cysteine ​​hydrochloride buffer as a negative control solution to three test tubes (i.e., the third test tube); quickly add 200 μL of high-sensitivity protein complex enzyme sample solution to three other test tubes (i.e., the first test tube); quickly add 200 μL of standard solution as a positive control solution to the remaining three test tubes (i.e., the second test tube). Immediately click the "Start" button on the CellStatz interface window to measure the enzyme catalytic reaction curve. It will automatically stop after 600 seconds; click the "Save" button.

[0080] Result reading and calculation:

[0081] After the assay is completed, if the detection channel corresponding to the standard papain solution shows a parabolic enzyme catalytic reaction curve and the detection channel corresponding to the L-cysteine ​​hydrochloride buffer shows a nearly horizontal straight line, the test results are reliable. Otherwise, the test should be repeated after checking the reagent preparation, instrument parameter settings, experimental operation, and other experimental procedures.

[0082] The appearance of a parabolic enzyme catalytic reaction curve in the detection channel corresponding to the hypersensitive protein complex enzyme sample solution indicates the presence of active papain in the tested product (i.e., the hypersensitive protein complex enzyme). If a horizontal straight line appears, similar to the curve of the L-cysteine ​​hydrochloride buffer, it indicates the absence of papain in the hypersensitive protein complex enzyme or that the present papain lacks papain activity.

[0083] In the CellStatz interface window, under the "Data Processing" function module, select any channel corresponding to a negative control solution as the "Reference Channel," and click the "Calibration Mode" button to obtain three calibration enzyme catalytic reaction curves corresponding to the high-sensitivity protein complex enzyme test sample solution. Read the endpoint ordinate values ​​(i.e., the voltage values ​​when the enzyme catalytic reaction curve reaches a stable state) C1, C2, and C3 of the three curves respectively, calculate the average value C, and substitute it into the following formula to calculate the papain activity in the high-sensitivity protein complex enzyme sample: X = 3715e 139.9C In the formula, X represents the papain activity in the sample (i.e., the enzyme activity test value), in U / L, and C represents the endpoint ordinate value of the enzyme catalytic reaction curve of the sample. Wherein, the formula (X=3715e 139.9C The voltage value was determined by the external standard method (in the process of determining the formula by the external standard method, refer to the test process of the voltage value change curve of the test standard solution over time in Example 1).

[0084] Example 2

[0085] Experiments 1 through 10 were conducted according to the following procedures: a standard solution with an enzyme activity of 5200 U / L (i.e., a standard solution with known enzyme activity) was prepared. The standard solution with known enzyme activity was tested according to the detection procedure of papain enzyme in hypersensitive protein complex enzyme in Example 1. The voltage value and enzyme activity test value at steady state are shown in Table 1.

[0086] Table 1

[0087]

[0088] From Table 1, the accuracy of the test results can be calculated as A = EMC / TC = 97%, where EMC is the average value of the enzyme activity test values ​​of Test 1 to Test 10, and TC is the average value of the enzyme activity of the standard solutions of Test 1 to Test 10 in Table 1.

[0089] As can be seen from Table 1, the enzyme activity test values ​​of the standard solution with known enzyme activity measured in Example 2 are close to those of the standard solution with known enzyme activity, indicating that the detection method of the present invention can accurately detect the papain enzyme activity in the hypersensitive protein complex enzyme.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting papain enzyme activity in a hypersensitive protein complex enzyme, characterized in that, Includes the following steps: The high-sensitivity protein complex enzyme and L-cysteine ​​hydrochloride buffer were mixed to obtain the sample solution to be tested. After adding the test sample solution to a substrate solution containing casein, the voltage value of the test sample solution over time is tested under the condition that the casein can be decomposed by papain. The papain activity in the hypersensitive protein complex enzyme was determined based on the voltage value of the test sample solution over time.

2. The method for detecting papain enzyme activity in the hypersensitive protein complex enzyme according to claim 1, characterized in that, The volume ratio of the hypersensitive protein complex enzyme to the L-cysteine ​​hydrochloride buffer is 100 μl: (800-1000) μL; And / or, the preparation process of the L-cysteine ​​hydrochloride buffer includes: L-cysteine ​​hydrochloride, sodium chloride, disodium ethylenediaminetetraacetate, and water are mixed to obtain a third solution; wherein the mass ratio of L-cysteine ​​hydrochloride to sodium chloride is 1:(4-5); and the mass ratio of L-cysteine ​​hydrochloride to disodium ethylenediaminetetraacetate is 1:(0.35-0.5). The pH of the third solution was adjusted to 7-7.5 using sodium hydroxide solution, and then water was added to make up the volume to obtain the L-cysteine ​​hydrochloride buffer solution; wherein the volume ratio of the third solution to the L-cysteine ​​hydrochloride buffer solution was 60%-80%.

3. The method for detecting papain enzyme activity in the hypersensitive protein complex enzyme according to claim 1, characterized in that, The volume ratio of the sample solution to the substrate solution containing casein is 200 μL: (1.5–2.5) mL; And / or, the preparation process of the casein-containing substrate solution includes: wetting the casein with sodium hydroxide solution and mixing it with disodium hydrogen phosphate solution to obtain a fourth solution; adjusting the pH of the fourth solution to 7-7.5 with hydrochloric acid solution, and then adding water to make up the volume to obtain the casein-containing substrate solution; wherein the volume ratio of the fourth solution to the casein-containing substrate solution is 70%-90%.

4. The method for detecting papain enzyme activity in the hypersensitive protein complex enzyme according to any one of claims 1-3, characterized in that, The step of adding the test sample solution to a substrate solution containing casein, and then testing the voltage change curve of the test sample solution over time under the condition that casein can be decomposed by papain, specifically includes: The substrate solution containing casein is added to the first detection tube, which is then placed in the detection channel of a biochemical process monitor. The operating conditions of the biochemical process monitor are then adjusted to conditions that allow casein to be decomposed by papain. Subsequently, the sample solution to be tested is added to the first detection tube, and the voltage value of the sample solution to be tested is measured over time.

5. The method for detecting papain enzyme activity in the hypersensitive protein complex enzyme according to claim 4, characterized in that, After adjusting the operating conditions of the biochemical process monitor to the condition that casein can be decomposed by papain, maintain this condition for 50-70 seconds, and then add the test sample solution to the first detection tube to test the voltage value of the test sample solution over time.

6. The method for detecting papain enzyme activity in the hypersensitive protein complex enzyme according to claim 1 or 5, characterized in that, The conditions under which casein can be decomposed by papain include a temperature of 20–80°C.

7. The method for detecting papain enzyme activity in the hypersensitive protein complex enzyme according to any one of claims 1-3, characterized in that, The process of testing the voltage value of the sample solution under the condition that casein can be decomposed by papain includes: collecting voltage values ​​at different times to obtain the voltage value change curve over time; wherein the voltage value acquisition period is 0.5 to 2 seconds, and the number of voltage value acquisitions is 500 to 700 times.

8. The method for detecting papain enzyme activity in the hypersensitive protein complex enzyme according to any one of claims 1-3, characterized in that, It also includes the voltage change curve of the test standard solution over time and / or the voltage change curve of the L-cysteine ​​hydrochloride buffer solution over time; The testing process for the voltage change curve of the standard solution over time includes: mixing papain and the L-cysteine ​​hydrochloride buffer to obtain a standard solution; adding the standard solution to the substrate solution containing casein, and testing the voltage change curve of the standard solution over time under the condition that the casein can be decomposed by papain. The test process for the voltage change curve of the L-cysteine ​​hydrochloride buffer over time includes: adding the L-cysteine ​​hydrochloride buffer to the substrate solution containing casein, and then testing the voltage change curve of the L-cysteine ​​hydrochloride buffer over time under the condition that the casein can be decomposed by papain. The process of determining the papain enzyme activity in the hypersensitive protein complex enzyme based on the voltage value change curve of the test sample solution over time includes: determining the papain enzyme activity in the hypersensitive protein complex enzyme based on the voltage value change curve of the test sample solution over time, and referring to the voltage value change curve of the standard solution over time and / or the voltage value change curve of L-cysteine ​​hydrochloride buffer over time.

9. The method for detecting papain enzyme activity in the hypersensitive protein complex enzyme according to claim 8, characterized in that, In the standard solution, the mass-to-volume ratio of papain to L-cysteine ​​hydrochloride buffer is 0.05 g: (4-6) mL.

10. The method for detecting papain enzyme activity in the hypersensitive protein complex enzyme according to any one of claims 1-3, characterized in that, The process of determining the papain enzyme activity in the hypersensitive protein complex enzyme based on the voltage value change curve of the test sample solution over time includes: based on the voltage value when the voltage value change curve over time reaches a stable state, using papain as a standard, determining the papain enzyme activity value in the hypersensitive protein complex enzyme by external standard method.

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