Method for fluorescence quantitative determination of enzyme activity of endoprotease

Abstract

The invention relates to a method for fluorescence quantitative determination of enzyme activity of endoprotease, the method takes casein as a substrate, adopts a dual-wavelength scheme, shields fluorescence interference brought by protease, can accurately determine absolute enzyme activity of endoprotease, and can avoid use of toxic protein coloring agents, so that the method is simple and convenient to operate. And the detection method is relatively green and healthy.

Description

Technical Field

[0001] This invention belongs to the field of proteases, and specifically relates to a method for quantitative fluorescence determination of endopeptide enzyme activity. Background Technology

[0002] Proteases are classified as endopeptidases and exopeptidases. Endopeptidases hydrolyze proteins or polypeptides from within, forming many smaller peptides; representative examples include glutamine proteases and serine proteases. Exopeptidases continuously detach amino acids from the ends of proteins or polypeptides; representative examples include aminopeptidases and carboxypeptidases. Most literature focuses on exopeptidases, detecting protease activity based on the amount of amino acids produced. For example, the literature [MCDONALD CE, CHEN L L. The lowry modification of the folin reagent for determination of proteinase activity[J]. Analytical Biochemistry, 1965, 10(1):175-177] uses the folin-phenol method to quantify protease activity based on the amount of free tyrosine and tryptophan released after hydrolysis of the protein substrate. However, there is currently less attention paid to endopeptidases in enzyme activity detection.

[0003] The Coomassie Brilliant Blue method is the most classic method for detecting protein content. Coomassie Brilliant Blue is a protein dye that binds to proteins to form a blue conjugate. Its absorbance (OD595) has a certain mathematical relationship with the protein content in the sample (linear relationship within a suitable range). It can be used to detect endopeptides. For example, the literature [An Can, Wang Xin, Chen Meiling, et al. Study on the salty taste produced by protease hydrolysis of lizardfish [J]. China Food Additives, 2017, (01): 135-140] uses the Coomassie Brilliant Blue method to detect the changing trend of papain hydrolysis of lizardfish protein. However, the preparation of Coomassie Brilliant Blue reagent is relatively complicated and it is toxic to the skin, respiratory tract and digestive tract. Therefore, it is necessary to explore a green and healthy detection method.

[0004] Proteins with stable spatial structures exhibit autofluorescence, but proteases, being proteins themselves, can cause significant interference during enzyme activity assays. Therefore, there are very few reports in the published literature on fluorescence methods for detecting endopeptides. The homogeneous assay is the only method that uses fluorescence to measure proteases. This method detects fluorescence at an excitation wavelength of 500 nm and an emission wavelength of 530 nm, using substrate and enzyme solutions as controls. Protease activity is expressed as relative fluorescence intensity (RFU), calculated using the formula: RFU = F C -(F A +F B ), where: F C F represents the sample fluorescence value.A F represents the substrate fluorescence value. B The value represents the fluorescence value of the enzyme solution. Homogeneous methods for measuring protease activity can only detect relative enzyme activity, not absolute enzyme activity. For example, the literature [Cheng Lei, Wang Zhengxiang. Biochemical characteristics analysis of alkaline protease CpyC from Aspergillus niger [J]. Food and Fermentation Industries, 2024, 50(23):1-6] used the homogeneous method to detect the relative enzyme activity of alkaline protease from Aspergillus niger. In the experiment on the effect of metal ions on enzyme activity, the relative fluorescence value of the control group was taken as 100% of the relative enzyme activity to explore the changes in the relative fluorescence value of different metals and calculate the relative enzyme activity of each group. To detect absolute enzyme activity, this method requires prior knowledge of the nominal enzyme activity of the commercial enzyme. The commercial enzyme is then used as a standard enzyme to plot an enzyme activity standard curve before quantification can be performed. For example, the literature [Song Peng, Cheng Lei, Tian Kangming, et al. Establishment and preliminary application of rapid detection method for industrial proteolytic enzymes [J]. Food and Fermentation Industries, 2019, 45(17):216-220] established multiple protease standard curves for trypsin, Aspergillus oryzae acidic protease, papain, etc. However, the literature also pointed out that the standard curves for different proteases are inconsistent. The relative fluorescence intensity of homogeneous method corresponding to the same Folin method enzyme activity is different, which is reflected in the difference in the slope and linear range of the regression equation. For unknown proteases, it is impossible to select an effective target. Without an accurate standard curve, it is impossible to quantitatively detect unknown proteases.

[0005] In summary, there is an urgent need in this field for a green and healthy method to quantitatively detect the absolute enzyme activity of endopeptides. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for quantitative fluorescence detection of endopeptide activity. This method uses casein as a substrate and employs a dual-wavelength scheme of "EM (emission wavelength) = 355nm - Y nm, EX (excitation wavelength) = 280nm" (the fluorescence response value of the endopeptide at an emission wavelength of 355nm is equal to its fluorescence response value at an emission wavelength of Y nm), thus shielding the fluorescence interference caused by the protease. This method can accurately determine the absolute enzyme activity of the endopeptide. Furthermore, the detection method of this invention avoids the use of toxic protein staining agents, making the detection method relatively green and healthy.

[0007] Specifically, in one aspect of the present invention, a method for quantitative fluorescence detection of endopeptide protease activity is provided, comprising:

[0008] (1) Establish a standard curve of the concentration C of casein aqueous solution and the difference X of fluorescence response value, wherein the difference X of fluorescence response value is the difference obtained by subtracting fluorescence response value B from fluorescence response value A, wherein fluorescence response value A is the fluorescence response value of casein aqueous solution under the conditions of excitation wavelength of 280±5nm (preferably 280nm) and emission wavelength of 355±5nm (preferably 355nm), wherein fluorescence response value B is the fluorescence response value of casein aqueous solution under the conditions of excitation wavelength of 280±5nm (preferably 280nm) and emission wavelength of Y±5nm (preferably Y nm), wherein the fluorescence response value of the endopeptide under the condition of emission wavelength of 355nm is equal to its fluorescence response value under the condition of emission wavelength of Y nm;

[0009] (2) After the casein and the endopeptide to be tested undergo an enzymatic reaction, the enzyme is inactivated to obtain a reaction mixture;

[0010] (3) Obtain the fluorescence response value difference X1 of the reaction mixture, wherein the fluorescence response value difference X1 is the difference obtained by subtracting the fluorescence response value B1 from the fluorescence response value A1, wherein the fluorescence response value A1 is the fluorescence response value of the reaction mixture under the conditions of excitation wavelength of 280±5nm (preferably 280nm) and emission wavelength of 355±5nm (preferably 355nm), and the fluorescence response value B1 is the fluorescence response value of the reaction mixture under the conditions of excitation wavelength of 280±5nm (preferably 280nm) and emission wavelength of Y±5nm (preferably Y nm);

[0011] (4) Substitute the difference in fluorescence response value X1 of the reaction mixture into the standard curve of the difference in fluorescence response value X between the concentration C and the concentration C to obtain the concentration of casein in the reaction mixture;

[0012] (5) Calculate the amount of casein consumed in the enzymatic reaction and calculate the enzyme activity of the endopeptide to be tested according to the definition of enzyme activity.

[0013] In some embodiments, the enzymatic reaction is carried out in the presence of a buffer solution whose matrix comprises tris(hydroxymethyl)aminomethane, glycine, boric acid, L-tartaric acid, or citric acid.

[0014] In some embodiments, the matrix of the buffer solution contains boric acid.

[0015] In some embodiments, the buffer solution is a borate buffer solution.

[0016] In some embodiments, the enzyme inactivation is performed using an enzyme-inactivating reagent.

[0017] In some embodiments, the enzyme-inactivating reagent is an acidic solution.

[0018] In some embodiments, the acidic solution is an aqueous solution of hydrochloric acid, an aqueous solution of acetic acid, an aqueous solution of lactic acid, or an aqueous solution of citric acid.

[0019] In some embodiments, the acidic solution is an aqueous solution of lactic acid.

[0020] In some embodiments, the endopeptidase is selected from chymotrypsin, Bacillus subtilis protease, and Bacillus licheniformis protease.

[0021] In some embodiments, when the endopeptide is chymotrypsin, Y is 332.

[0022] In some embodiments, when the endopeptide is Bacillus subtilis protease, Y is 369.

[0023] In some embodiments, when the endopeptide is Bacillus licheniformis protease, Y is 381.

[0024] In some embodiments, the casein is provided in the form of an aqueous casein solution, wherein the concentration of casein in the aqueous casein solution is 2-3 mg / mL, preferably 2.5 mg / mL.

[0025] In some embodiments, the volume ratio of the casein aqueous solution to the buffer solution is 1:1.

[0026] In some embodiments, the endopeptide to be tested is provided in the form of an aqueous solution of the endopeptide, wherein the concentration of the endopeptide in the aqueous solution is 4-6 mg / mL, preferably 5 mg / mL.

[0027] In some embodiments, the volume ratio of the casein aqueous solution to the endopeptide aqueous solution is 5:1.

[0028] In some embodiments, the volume ratio of the casein aqueous solution to the enzyme inactivating reagent is 1:1.

[0029] In some embodiments, the enzymatic reaction is carried out at a temperature of 20–45°C (e.g., 20–30°C or 35–45°C, preferably 25°C or 40°C).

[0030] In some embodiments, when the endopeptide is chymotrypsin, the enzymatic reaction is carried out at a temperature of 20–30°C (preferably 25°C).

[0031] In some embodiments, when the endopeptide is Bacillus subtilis protease, the enzymatic reaction is carried out at a temperature of 35–45°C (preferably 40°C).

[0032] In some embodiments, when the endopeptide is Bacillus licheniformis protease, the enzymatic reaction is carried out at a temperature of 35–45°C (preferably 40°C).

[0033] In some embodiments, the enzymatic reaction takes 20 to 40 minutes, preferably 30 minutes.

[0034] In some embodiments, the concentration of the buffer solution is 0.04–0.06 mol / L, preferably 0.05 mol / L.

[0035] In some embodiments, the pH of the buffer solution is 6.5 to 11 (e.g., 6.5 to 7.5, 7.0 to 8.0, or 10 to 11), preferably 7.0, 7.5, or 10.5.

[0036] In some embodiments, when the endopeptide is chymotrypsin, the pH of the buffer solution is 6.5 to 7.5, preferably 7.0.

[0037] In some embodiments, when the endopeptide is Bacillus subtilis protease, the pH of the buffer solution is 7.0 to 8.0, preferably 7.5.

[0038] In some embodiments, when the endopeptide is Bacillus licheniformis protease, the pH of the buffer solution is 10-11, preferably 10.5.

[0039] In some embodiments, the concentration of the solute in the acidic solution is 0.4 to 0.6 mol / L, preferably 0.5 mol / L.

[0040] In some embodiments, the pH of the acidic solution is 0.2 to 0.4, preferably 0.3.

[0041] In some implementations, step (2) includes:

[0042] Experimental group: In the presence of buffer solution, casein and the target endopeptide protease were subjected to an enzymatic reaction, and the enzyme was then inactivated to obtain a reaction mixture;

[0043] Control group: Under the same buffer conditions as the experimental group, a mixture of casein and enzyme inactivation reagent was mixed with the endopeptide to be tested, and the same experimental operation as the enzymatic reaction in the experimental group was performed to obtain a control mixture.

[0044] Furthermore, the casein concentration and mass in the control group are the same as those in the experimental group; the concentration and volume of the enzyme-inactivating reagent in the control group are the same as those used in the experimental group; the concentration and mass of the endonuclease to be tested in the control group are the same as those in the experimental group; and the volume and pH of the buffer solution in the control group are the same as those in the experimental group.

[0045] Step (3) further includes: obtaining the fluorescence response value difference X2 of the control mixture, wherein the fluorescence response value difference X2 is the difference obtained by subtracting the fluorescence response value B2 from the fluorescence response value A2, wherein the fluorescence response value A2 is the fluorescence response value of the control mixture under the conditions of excitation wavelength of 280±5nm (preferably 280nm) and emission wavelength of 355±5nm (preferably 355nm), and the fluorescence response value B2 is the fluorescence response value of the control mixture under the conditions of excitation wavelength of 280±5nm (preferably 280nm) and emission wavelength of Y±5nm (preferably Y nm);

[0046] Step (4) further includes: substituting the fluorescence response value difference X2 of the control mixture into the standard curve of the concentration C and the fluorescence response value difference X to obtain the concentration of casein in the control mixture.

[0047] In some embodiments, when the endopeptide is chymotrypsin, in step (1), when the concentration of casein in the casein aqueous solution is in the range of 0.05–0.6 mg / mL, the established standard curve is y = 55757626x + 4371913(R 2 =1), when the concentration of casein in the casein aqueous solution is in the range of 0.6 to 2.0 mg / mL, the established standard curve is y = 17509569ln(x) + 47369891(R). 2 =1);

[0048] When the endopeptide is Bacillus subtilis protease, in step (1), the concentration of casein in the casein aqueous solution is in the range of 0.1–1.8 mg / mL, and the established standard curve is y = 7,223,846x + 962,556(R). 2 =0.9996);

[0049] When the endopeptide is Bacillus licheniformis protease, in step (1), the concentration of casein in the casein aqueous solution is in the range of 0.1 to 1.8 mg / mL, and the established standard curve is y = 17,123,471x + 1,885,497(R). 2 =0.9991);

[0050] Where y represents the difference in fluorescence response values, and x represents the casein concentration (unit: mg / mL).

[0051] In some embodiments, the enzyme activity is defined as the amount of enzyme required to degrade 1 μg of casein in 1 min per 1 mL of endopeptide aqueous solution, with the enzyme activity unit being U / mL.

[0052] In some embodiments, the enzyme activity of the endopeptide to be tested is calculated using the following formula: enzyme activity of the endopeptide to be tested (U / mL) = [casein content of control group (μg) - casein content of experimental group (μg)] / [volume of endopeptide aqueous solution (mL) × reaction time (min)], wherein the casein content of the control group is calculated using the concentration of casein in the control mixture, and the casein content of the experimental group is calculated using the concentration of casein in the reaction mixture.

[0053] Beneficial effects

[0054] Currently, there are few methods for detecting endopeptides. The detection method constructed in this invention can accurately determine the enzyme activity of endopeptides. Compared with the existing Coomassie Brilliant Blue method, the method of this invention can avoid the use of protein staining agents that are toxic to humans, making the detection method greener and healthier. Compared with the existing homogeneous method, the method of this invention can subtract the influence of protease fluorescence on substrate fluorescence by using dual wavelengths. The homogeneous method can only detect relative enzyme activity, while the method of this invention can detect absolute enzyme activity. Attached Figure Description

[0055] Figure 1 The fluorescence emission spectra of casein, protease (chymotrypsin), and L-tyrosine are shown.

[0056] Figure 2 The curves showing the relationship between casein concentration (0.05–8 mg / mL) and fluorescence response value when the protease is chymotrypsin;

[0057] Figure 3 The curves showing the relationship between casein concentration (0.05–0.6 mg / mL) and fluorescence response values ​​when the protease is chymotrypsin;

[0058] Figure 4 The curves showing the relationship between casein concentration (0.6–2 mg / mL) and fluorescence response value when the protease is chymotrypsin;

[0059] Figure 5 The fluorescence emission spectra of casein and Bacillus subtilis protease are shown.

[0060] Figure 6 The curve showing the relationship between casein concentration and fluorescence response value when the protease is Bacillus subtilis protease;

[0061] Figure 7 The fluorescence emission spectra of casein and Bacillus licheniformis protease;

[0062] Figure 8 The curve showing the relationship between casein concentration and fluorescence response value when the protease is Bacillus licheniformis protease;

[0063] Figure 9 The effect of different buffer matrices on the fluorescence response value of casein;

[0064] Figure 10 The effect of different acidic solutions on the fluorescence response value of casein;

[0065] Figure 11 The curve showing the relationship between casein concentration (0–8 mg / mL) and Coomassie Brilliant Blue absorbance is shown.

[0066] Figure 12 The curve showing the relationship between casein concentration (0–0.2 mg / mL) and Coomassie Brilliant Blue absorbance is shown.

[0067] Figure 13 The curve showing the relationship between casein concentration (0.2–2 mg / mL) and Coomassie Brilliant Blue absorbance is shown.

[0068] Figure 14 The results show the full-wavelength scan of fluorescence emission wavelengths of bovine serum albumin. Detailed Implementation

[0069] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments.

[0070] To achieve the objectives of this invention, the following technical solution is adopted:

[0071] Add 0.5 mL of 2.5 mg / mL casein and 0.5 mL of 0.05 mol / L pH 7.0 buffer solution to a 2 mL centrifuge tube and mix well. Then add 0.1 mL of protease solution and react at 40 °C for 30 min. After the reaction, add 0.5 mL of 0.5 mol / L acidic solution to inactivate the enzyme. Use the dual-wavelength method to compare the standard curve of casein concentration and fluorescence response value to calculate the amount of casein consumed, and calculate the enzyme activity according to the definition of enzyme activity.

[0072] In some embodiments, the matrix of the buffer solution may be one of tris(hydroxymethyl)aminomethane, glycine, boric acid, L-tartaric acid, and citric acid, preferably boric acid.

[0073] In some embodiments, the acidic solution may be one of hydrochloric acid, acetic acid, lactic acid, or citric acid, and the pH is adjusted to 0.3 with concentrated hydrochloric acid before use.

[0074] In some implementations, the dual-wavelength method uses fluorescence spectroscopy for detection, with an excitation wavelength of 280 nm and the fluorescence response value at the emission wavelength obtained by subtracting the fluorescence value at 332 nm from the fluorescence value at 355 nm.

[0075] In some implementations, the standard curve needs to be plotted in segments: a linear standard curve is plotted when the casein concentration is low, and a logarithmic standard curve is plotted when the casein concentration is high. For example, when the casein concentration is in the range of 0.05 to 0.6 mg / mL, the standard curve is y = 55757626x + 4371913; when the casein concentration is in the range of 0.6 to 2.0 mg / mL, the standard curve is y = 17509569ln(x) + 47369891.

[0076] In some implementations, the enzyme activity definition can be customized according to reaction conditions such as enzyme reaction temperature, pH, and reaction time. For example, the amount of enzyme required to degrade 1 μg of casein in 1 min per 1 mL of enzyme solution is 1 U, and the enzyme activity unit is U / mL.

[0077] The present invention will be further explained and described below with reference to specific embodiments.

[0078] In the following examples, casein was purchased from Sinopharm Group, catalog number 69006237; L-tyrosine was purchased from Sinopharm Group, catalog number 62023536; chymotrypsin was purchased from Shanghai Yuanye Biotechnology, catalog number S10001-1g; Bacillus subtilis protease was purchased from Beijing Hongrun Baoshun Technology Co., Ltd., catalog number M008; and Bacillus licheniformis protease was purchased from Beijing Hongrun Baoshun Technology Co., Ltd., catalog number M007.

[0079] In the following examples, Tris buffer (0.05 mol / L, pH = 7.0) was purchased from Shanghai Yuanye Biotechnology, catalog number: R27666-100ml. Other buffers (L-tartaric acid buffer, glycine buffer, borate buffer, citrate buffer) could be prepared from commercially available reagents. The source of the reagents and the specific preparation methods are as follows:

[0080] Glycine buffer (0.05 mol / L, pH=7.0): prepared by diluting commercially available glycine buffer (0.1 mol / L, pH=7.0, purchased from Shanghai Yuanye, product number R26382-100ml) with deionized water by 2 times;

[0081] Citrate buffer (0.05 mol / L, pH=7.0): prepared by diluting commercially available citrate-sodium citrate buffer (0.1 mol / L, pH=7.0, purchased from Shangbao Biotechnology, product number T17395) with deionized water by 2 times;

[0082] NaOH (1mol / L): Weigh 40g of NaOH (purchased from Sinopharm Group, item number 10019718) and add deionized water to a final volume of 1L;

[0083] L-Tartrate Buffer (0.05mol / L, pH=7.0): Weigh 0.75g of L-tartaric acid (purchased from Sinopharm Group, catalog number 10022018), dissolve it in 80mL of deionized water, adjust the pH to 7.0 with 1mol / L NaOH, and make up the volume to 100mL.

[0084] Boric acid buffer (0.05 mol / L, pH=7.0): Weigh 0.309 g of boric acid (purchased from Sinopharm Group, catalog number 10004808), dissolve it in 80 mL of deionized water, adjust the pH to 7.0 with 1 mol / L NaOH, and make up the volume to 100 mL.

[0085] In the following examples, the fluorescent microplate reader used was the SpectraMax i3x multi-functional microplate reader (American Valley Molecular Company, USA).

[0086] Preparation of casein solutions of different concentrations: Weigh 1.00 g of casein, add 90 mL of ultrapure water, heat to boiling to dissolve, then immediately cool in an ice bath and bring the volume to 100 mL to prepare a 10 mg / mL casein solution. Dilute with ultrapure water to prepare casein solutions of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.2, 1.6, 2, 4, 6, and 8 mg / mL, respectively.

[0087] Preparation of L-tyrosine solutions of different concentrations: Weigh 0.0200 g of L-tyrosine, add 100 mL of ultrapure water to dissolve, and prepare an L-tyrosine solution of 200 μg / mL. Then dilute with ultrapure water to prepare L-tyrosine solutions of 40, 80, 120, and 160 μg / mL respectively.

[0088] Preparation of protease solution: Weigh 0.10g of protease, add 20mL of ultrapure water to dissolve, and prepare a 5mg / mL protease solution.

[0089] Example 1: Fluorescence emission spectra of casein, protease, and L-tyrosine, and plotting of the casein standard curve.

[0090] Fluorescence emission spectra of casein, protease, and L-tyrosine were scanned: 200 μL of each of the following solutions were placed in a fluorescent microplate: 2 mg / mL casein solution, 200 μg / mL L-tyrosine solution, and 5 mg / mL protease solution. The excitation wavelength was 280 nm (the maximum absorption wavelength of proteins and aromatic amino acids), and the emission wavelength was in full-wavelength scan mode, with a scan range of 320 nm to 700 nm. Fluorescence spectra of these three substances were plotted.

[0091] Using chymotrypsin as an example, the fluorescence emission wavelength was determined and a standard curve was plotted. Figure 1 It is known that casein has a maximum emission wavelength of 355 nm, chymotrypsin has a maximum emission wavelength of 340 nm, and L-tyrosine does not have a complete emission peak, with a maximum emission wavelength at 320 nm. Based on the fluorescence spectra characteristics of these three substances, it is known that they are suitable for detecting endopeptides (enzymes that hydrolyze proteins or peptides from the inside to form small peptides; because the protein structure is severely damaged, the fluorescence response value will be significantly reduced), but not suitable for detecting exopeptides (which cleave amino acids from the ends of proteins or peptides; their impact on the overall protein structure is not as great as that of endopeptides). Because the fluorescence response value of protein (casein) is high at the excitation wavelength of 355 nm, while the fluorescence response value of L-tyrosine (amino acid) is extremely low, the inventors believe that the change in protein can be reflected by the fluorescence response value at 355 nm, which is not affected by the change in amino acid; while the changes in both protein and amino acid will affect the fluorescence response value at 320 nm.

[0092] However, the inventors considered that the fluorescence response value at 355 nm alone might not be sufficient to accurately describe the changes in casein, as the maximum emission wavelength of proteases is close to 340 nm, and they also exhibit a large fluorescence response value at 355 nm. Based on this, after extensive experimental exploration, the inventors of this application were pleasantly surprised to discover that by employing a dual-emission wavelength method to characterize the changes in casein, interference from proteases could be shielded, as detailed below.

[0093] After analysis, the inventors found that the fluorescence response value of chymotrypsin at 332nm was the same as that at 355nm. Therefore, they speculated that a dual-wavelength scheme of "EM (emission wavelength) = 355nm-332nm, EX (excitation wavelength) = 280nm" could be used to shield the interference caused by the protease.

[0094] Furthermore, the inventors used a dual-wavelength scheme of "EM (emission wavelength) = 355nm-332nm, EX (excitation wavelength) = 280nm" to plot the relationship curve (standard curve) between casein concentration and fluorescence response value. Specifically, with an excitation wavelength of 280nm, fluorescence response values ​​A at an emission wavelength of 355nm for casein solutions of different concentrations were collected, and fluorescence response values ​​B at an emission wavelength of 332nm for casein solutions of different concentrations were also collected. The difference AB between the fluorescence response values ​​of casein solutions of different concentrations at the two emission wavelengths was calculated. Finally, the relationship curve between casein concentration and this difference in fluorescence response value was plotted, and the results are as follows: Figure 2 As shown.

[0095] Depend on Figure 2 It can be seen that when the casein concentration is below 0.6 mg / mL, the casein concentration and the fluorescence response value have a linear relationship; when the casein concentration is between 0.6 and 2.0 mg / mL, the casein concentration and the fluorescence response value have a logarithmic relationship; and when it is above 2 mg / mL, it exceeds the detection range of the instrument. Based on this, a standard curve was further plotted segmentally, and the results are as follows. Figure 3 and Figure 4 As shown.

[0096] Depend on Figure 3 It can be seen that when the casein concentration is in the range of 0.05–0.6 mg / mL, the standard curve is y = 55757626x + 4371913(R). 2 =1); by Figure 4 It can be seen that when the casein concentration is in the range of 0.6–2.0 mg / mL, its standard curve is y = 17509569ln(x) + 47369891(R). 2 =1). It is evident that the aforementioned standard curve exhibits excellent linearity and extremely high reliability.

[0097] Following the experimental steps described above, using 280 nm as the excitation wavelength, a full-wavelength fluorescence emission scan was performed on Bacillus subtilis protease and Bacillus licheniformis protease, and the full-wavelength scan curves of casein were plotted. Figure 5 and Figure 7 .

[0098] Depend on Figure 5 It is known that the fluorescence emission wavelength of Bacillus subtilis protease has a maximum absorption peak at 361 nm. Following the above dual-wavelength method, a standard curve (EM (emission wavelength) = 355 nm - 369 nm, EX (excitation wavelength) = 280 nm) is plotted to show the relationship between casein concentration and fluorescence response value. Figure 6 It can be seen that within the range of 0.1–1.8 mg / mL, the standard curve is y = 7,223,846x + 962,556(R). 2 =0.9996).

[0099] Depend on Figure 7 It is known that the fluorescence emission wavelength of Bacillus licheniformis protease has a maximum absorption peak at 369 nm. Following the above dual-wavelength method, a standard curve (EM (emission wavelength) = 355 nm - 381 nm, EX (excitation wavelength) = 280 nm) is plotted to show the relationship between casein concentration and fluorescence response value. Figure 8 It can be seen that within the range of 0.1–1.8 mg / mL, the standard curve is y = 17,123,471x + 1,885,497 (R²). 2 =0.9991).

[0100] Example 2 Construction of a protease detection system

[0101] Taking chymotrypsin as an example, a preliminary enzyme activity detection system for the experimental group was constructed: 0.5 mL of 2.5 mg / mL casein (aqueous solution) and 0.5 mL of buffer (pH 7.0) were added to a 2 mL centrifuge tube and mixed well. Then, 0.1 mL of protease solution (5 mg / mL) was added and reacted at 25 °C for 30 min. After the reaction, 0.5 mL of 0.5 mol / L acidic solution was added to inactivate the enzyme. The fluorescence response values ​​of casein in the system at emission wavelengths of 355 nm and 332 nm were collected and the difference was calculated. The concentration of casein in the system after the reaction was obtained by using the standard curve of the dual-wavelength scheme "EM (emission wavelength) = 355 nm - 332 nm, EX (excitation wavelength) = 280 nm" in Example 1.

[0102] Calculate enzyme activity: Calculate the amount of casein consumed and calculate the enzyme activity according to the definition of enzyme activity.

[0103] In this embodiment, enzyme activity is defined as follows: the amount of enzyme required to degrade 1 μg of casein in 1 min is 1 U. Protease activity (U / mL) = [casein content of control group (μg) - casein content of experimental group (μg)] / [enzyme solution volume (mL) × reaction time (min)].

[0104] Compared to the experimental group, the control group's enzyme activity detection system differed only in that the step "adding 0.1 mL of protease solution (5 mg / mL) and reacting at 25°C for 30 min, followed by adding 0.5 mL of 0.5 mol / L acidic solution to inactivate the enzyme" was changed to "adding 0.5 mL of 0.5 mol / L acidic solution, then adding 0.1 mL of protease solution (5 mg / mL) and mixing at 25°C for 30 min". In other words, the acidic solution was added first, followed by the enzyme solution, preventing the enzyme from undergoing an enzymatic reaction. The remaining steps were the same as those in the experimental group.

[0105] Based on the enzyme activity detection system constructed above, the inventors further screened the optimal buffer solution and enzyme-inactivating acidic solution in the enzyme-catalyzed reaction.

[0106] To compare the effects of different buffer matrices on the fluorescence response of casein, the concentration of all buffer matrices was 0.05 mol / L (aqueous solution). The enzymatic reaction system used was the same as that of the control group. The enzyme inactivation solution was a lactic acid aqueous solution (0.5 mol / L, adjusted to pH 0.3 with concentrated hydrochloric acid before use). The blank group used an equal volume of deionized water instead of the buffer. Figure 9 It is known that Tris (tris(hydroxymethyl)aminomethane) and borate buffer can significantly increase the fluorescence response value of casein, while glycine can significantly decrease the fluorescence response value of casein.

[0107] Furthermore, using the aforementioned constructed enzyme activity detection system (the enzyme inactivation solution was a lactic acid aqueous solution, 0.5 mol / L, adjusted to pH 0.3 with concentrated hydrochloric acid before use), the enzyme activity of the protease solution (Bacillus subtilis protease, purchased from Shanghai Yuanye, catalog number S10051-250mg; the definition and calculation method of enzyme activity here refer to its instructions) was detected using deionized water (i.e., the buffer solution was replaced with deionized water), Tris buffer, and borate buffer, respectively. The enzyme activity values ​​were 151±2 U / mg, 135±3 U / mg, and 152±3 U / mg, respectively, indicating that Tris may inhibit protease activity. Therefore, borate buffer was used as the optimal buffer for the enzyme-catalyzed reaction system.

[0108] In addition, using the aforementioned constructed control group enzyme activity detection system, borate buffer was selected as the buffer for the enzyme-catalyzed reaction system to compare the effects of different acidic solutions (enzyme inactivating reagents) on the fluorescence response value of casein. The concentration of the matrix in the acidic solutions (all solvents were water) was 0.5 mol / L (the pH of 0.5 mol / L HCl was 0.3, and the other acids were weak acids, adjusted to pH 0.3 with concentrated hydrochloric acid before use). The results are as follows. Figure 10 As shown. By Figure 10 It can be seen that inactivating enzymes with lactic acid solution can increase the fluorescence response value of the system, and acidic solution is the most suitable as enzyme inactivation reagent.

[0109] Example 3 Comparison with existing protease detection methods

[0110] The protease detection method of this invention is applicable to the detection of endopeptides. The Coomassie Brilliant Blue method is currently the most commonly used method for detecting endopeptides. Coomassie Brilliant Blue is a protein staining agent that exhibits maximum light absorption at 595 nm after binding to proteins. Its absorbance value is directly proportional to the protein content; when the protein content in the reaction system decreases, the absorbance value after staining also decreases. The Coomassie Brilliant Blue method for protease assay consists of two steps: ① Enzymatic reaction: To ensure comparability between the two methods, the enzymatic reaction steps are identical for both methods. Taking chymotrypsin as an example, add 0.5 mL of 2.5 mg / mL casein (aqueous solution) and 0.5 mL of 0.05 mol / L borate buffer (pH 7.0) to a 2 mL centrifuge tube, mix well, then add 0.1 mL of protease solution (5 mg / mL) and react at 25°C for 30 min. After the reaction, add 0.5 mL of 0.5 mol / L lactate solution to inactivate the enzyme. ② Colorimetric detection: The colorimetric detection steps for the Coomassie Brilliant Blue method are as follows: Add 1 mL of deionized water and 0.5 mL of Coomassie Brilliant Blue chromogenic solution (100 mg Coomassie Brilliant Blue G-250 dissolved in 50 mL of water) to 0.1 mL of the reaction solution. Add 100 mL of 85% phosphoric acid to 95% ethanol, then add deionized water to make up to 200 mL. Mix well and let stand for 5 min before measuring the absorbance at 595 nm.

[0111] The standard curve for the Coomassie brilliant blue method was plotted using casein solutions of varying concentrations. Figure 11 It can be seen that when the casein concentration is below 0.2 mg / mL, the casein concentration has a linear relationship with OD595; when the casein concentration is between 0.2 and 2.0 mg / mL, the casein concentration has a logarithmic relationship with OD595; and above 2 mg / mL, it exceeds the detection range of Coomassie Brilliant Blue. Based on this, a standard curve was plotted piecewise. Figure 12 It can be seen that when the casein concentration is in the range of 0–0.2 mg / mL, its standard curve is y = 0.9949x + 0.2542(R). 2 =0.9869); by Figure 13 It can be seen that when the casein concentration is in the range of 0.2–2.0 mg / mL, its standard curve is y = 0.0416ln(x) + 0.5288(R). 2 =0.9515).

[0112] Compared with the fluorescence method for casein determination of the present invention in Examples 1 and 2, the Coomassie Brilliant Blue method has a narrower linear range, reaching only 0.2 mg / mL. Furthermore, the slope of this colorimetric method is too low; the difference between the OD595 of high-concentration casein and low-concentration casein is small, resulting in greater fluctuations in detection. The fitting degree of both the linear and logarithmic standard curves is also poor (R0). 2 (All values ​​are below 0.99). Therefore, from the perspective of standard curve plotting, the fluorescence method for protein detection of this invention is superior to the Coomassie brilliant blue method for protein detection.

[0113] Furthermore, the enzyme activity of the proteases was detected using different methods. The proteases used were chymotrypsin (purchased from Shanghai Yuanye, catalog number S10001-1g), Bacillus subtilis protease (purchased from Beijing Hongrun Baoshun Technology Co., Ltd., catalog number M008), and Bacillus licheniformis protease (purchased from Beijing Hongrun Baoshun Technology Co., Ltd., catalog number M007). According to their instructions, the optimal temperature and optimal pH of the above three enzymes are different: the optimal reaction temperature of chymotrypsin is 25℃ and the optimal pH is 7.0; the optimal reaction temperature of Bacillus subtilis protease is 40℃ and the optimal pH is 7.5; and the optimal reaction temperature of Bacillus licheniformis protease is 40℃ and the optimal pH is 10.5. Here, the enzyme activity detection system of the experimental group constructed above was still used, but the reaction temperature and pH were adjusted to their respective optimal temperatures and pH. The pH was adjusted to 7.0, 7.5, and 10.5 respectively by using 1 mol / L NaOH during the preparation of 0.05 mol / L borate buffer.

[0114] The fluorescence quantitative method is the optimal enzyme activity detection method described in Example 2 of this invention;

[0115] The Coomassie brilliant blue assay is the method for detecting the enzyme activity described above.

[0116] The homogeneous method for protease assay involves two steps: ① Enzyme-catalyzed reaction, the steps of which are the same as those for quantitative fluorescence assay and Coomassie brilliant blue assay; ② Fluorescence detection, where fluorescence is detected under excitation wavelength of 500 nm and emission wavelength of 530 nm, with substrate and enzyme solution used as controls. The protease activity is expressed as relative fluorescence intensity (RFU), calculated using the formula: RFU = F C -(F A +F B ), where: F C F represents the sample fluorescence value. A F represents the substrate fluorescence value. B This represents the fluorescence value of the enzyme solution.

[0117] The results are shown in Table 1. Table 1 shows that there was no significant difference in the protease activity measured by the quantitative fluorescence method and the Coomassie brilliant blue method, and both were close to the nominal activity of the commercial enzyme. However, the standard deviation of the Coomassie brilliant blue method was significantly larger. This indicates that although both detection methods are accurate, the quantitative fluorescence method of this invention is significantly more stable (with smaller deviation) than the Coomassie brilliant blue method. The homogeneous method, lacking the construction of a standard curve, cannot yield specific enzyme activity values; it only provides the relative fluorescence value of the sample, which is meaningless for the quantitative detection of protease activity.

[0118] Table 1. Enzyme activity (U / mg) of standard enzymes measured by different detection methods.

[0119]

[0120] Comparative Example 1:

[0121] The detection effects of the single-wavelength method (Comparative Example 1) and the dual-wavelength method (the method of this invention) on different proteases were compared.

[0122] The enzyme activity assay steps (including the reaction temperature and pH for each protease) were the same as in Example 3. The wavelength selection and standard curves for the dual-wavelength method for different proteases were the same as in Example 1. However, compared to Example 1, a single-wavelength scheme of "EM (emission wavelength) = 355 nm, EX (excitation wavelength) = 280 nm" was used to plot the curve (standard curve) relating casein concentration to fluorescence response. The resulting standard curve was: y = 40,318,094x + 5,477,549 (R0). 2 =0.9974), where the concentration of casein is ≤2mg / mL, and when the concentration is higher than 2mg / mL, it is beyond the detection range of the instrument.

[0123] The enzyme activities of different proteases are shown in Table 2 below.

[0124] Table 2. Enzyme activity (U / mg) of standard enzymes measured by different detection methods.

[0125]

[0126]

[0127] The results showed that for proteases with low enzyme activity (Bacillus subtilis protease and Bacillus licheniformis protease), there was no significant difference between the dual-wavelength method and the single-wavelength method. However, for proteases with high enzyme activity (chymotrypsin), there was a significant difference between the two methods. Obviously, the detection results of the dual-wavelength method of the present invention are closer to the nominal enzyme activity, while the enzyme activity measured by the single-wavelength method is distorted.

[0128] Comparative Example 2:

[0129] To investigate whether bovine serum albumin is suitable as a substrate for the method of this invention, Comparative Example 2 was constructed.

[0130] A full-wavelength scan of the fluorescence emission wavelength of bovine serum albumin was performed using 280 nm as the excitation wavelength, such as... Figure 14 As shown, its fluorescence emission wavelength has a maximum absorption peak at 346 nm.

[0131] The enzyme activity assay steps (including the reaction temperature and pH for each protease) were the same as in Example 3. Using the dual-wavelength method approach from Example 1, standard curves were plotted for each protease. For chymotrypsin, EM (emission wavelength) = 346nm-337nm, EX (excitation wavelength) = 280nm. At concentrations below 2mg / mL, the standard curve was y = -1,709,570x. 2 +6,762,458x-109,404(R 2 =0.9975); For Bacillus subtilis protease, EM (emission wavelength) = 346nm-377nm, EX (excitation wavelength) = 280nm, at concentrations below 2mg / mL, the standard curve is y = -13,231,243x 2 +58,210,740x+1,284,360(R 2 =0.9971); For Bacillus licheniformis protease, EM (emission wavelength) = 346nm-414nm, EX (excitation wavelength) = 280nm, at concentrations below 2mg / mL, the standard curve is y = -26,806,186x 2 +120,226,892x+2,310,208(R 2 =0.9975).

[0132] The enzyme activities of different proteases are shown in Table 3 below.

[0133] Table 3. Enzyme activity (U / mg) of standard enzymes measured by different detection methods.

[0134]

[0135]

[0136] The results showed that the enzyme activities of the proteases measured by the fluorescence quantitative method in Comparative Example 2 were all lower than the nominal enzyme activities, and the differences were significant. The fluorescence quantitative method of the present invention, which uses casein as a substrate, has significantly better accuracy.

[0137] It should be understood that the invention described herein is not limited to specific methodologies, experimental protocols, or reagents, as these can vary. The discussions and examples provided herein are for illustrative purposes only and are not intended to limit the scope of the invention, which is defined solely by the claims.

Claims

1. A method for quantitative fluorescence detection of endopeptide protease activity, comprising: (1) Establish a standard curve between the concentration C of casein aqueous solution and the difference X of fluorescence response value, wherein the difference X of fluorescence response value is the difference obtained by subtracting fluorescence response value B from fluorescence response value A, wherein fluorescence response value A is the fluorescence response value of casein aqueous solution under the conditions of excitation wavelength of 280±5nm and emission wavelength of 355±5nm, wherein fluorescence response value B is the fluorescence response value of casein aqueous solution under the conditions of excitation wavelength of 280±5nm and emission wavelength of Y±5nm, wherein the fluorescence response value of the endopeptide under the condition of emission wavelength of 355nm is equal to its fluorescence response value under the condition of emission wavelength of Y nm; (2) After the casein and the endopeptide to be tested undergo an enzymatic reaction, the enzyme is inactivated to obtain a reaction mixture; (3) Obtain the fluorescence response value difference X1 of the reaction mixture, wherein the fluorescence response value difference X1 is the difference obtained by subtracting the fluorescence response value B1 from the fluorescence response value A1, wherein the fluorescence response value A1 is the fluorescence response value of the reaction mixture under the conditions of excitation wavelength of 280±5nm and emission wavelength of 355±5nm, and the fluorescence response value B1 is the fluorescence response value of the reaction mixture under the conditions of excitation wavelength of 280±5nm and emission wavelength of Y±5nm; (4) Substitute the difference in fluorescence response value X1 of the reaction mixture into the standard curve of the difference in fluorescence response value X between the concentration C and the concentration C to obtain the concentration of casein in the reaction mixture; (5) Calculate the amount of casein consumed in the enzymatic reaction and calculate the enzyme activity of the endopeptide to be tested according to the definition of enzyme activity.

2. The method according to claim 1, wherein, The enzymatic reaction is carried out in the presence of a buffer solution whose matrix contains tris(hydroxymethyl)aminomethane, glycine, boric acid, L-tartaric acid, or citric acid. Preferably, the matrix of the buffer solution comprises boric acid; Preferably, the buffer solution is a borate buffer solution.

3. The method according to claim 2, wherein, The enzyme inactivation is achieved by using an enzyme-inactivating reagent. Preferably, the enzyme-inactivating reagent is an acidic solution; Preferably, the acidic solution is an aqueous solution of hydrochloric acid, an aqueous solution of acetic acid, an aqueous solution of lactic acid, or an aqueous solution of citric acid; Preferably, the acidic solution is an aqueous solution of lactic acid.

4. The method according to any one of claims 1-3, wherein, The endopeptidase is selected from chymotrypsin, Bacillus subtilis protease, and Bacillus licheniformis protease. Preferably, when the endopeptide is chymotrypsin, Y is 332; Preferably, when the endopeptide is Bacillus subtilis protease, Y is 369; Preferably, when the endopeptide is Bacillus licheniformis protease, Y is 381.

5. The method according to claim 4, wherein, Step (2) further includes one or more technical features selected from the following (i)-(xi): (i) The casein is provided in the form of an aqueous casein solution, wherein the concentration of casein in the aqueous casein solution is 2-3 mg / mL, preferably 2.5 mg / mL; (ii) The volume ratio of the casein aqueous solution to the buffer solution is 1:1; (iii) The endopeptide to be tested is provided in the form of an aqueous solution of the endopeptide, wherein the concentration of the endopeptide in the aqueous solution of the endopeptide is 4-6 mg / mL, preferably 5 mg / mL; (iv) The volume ratio of the casein aqueous solution to the endopeptide aqueous solution is 5:1; (v) The volume ratio of the casein aqueous solution to the enzyme inactivating reagent is 1:1; (vi) The enzymatic reaction is carried out at a temperature of 20–45°C (preferably 25°C or 40°C); Preferably, when the endopeptide is chymotrypsin, the enzymatic reaction is carried out at a temperature of 20-30°C (preferably 25°C); Preferably, when the endopeptide is Bacillus subtilis protease, the enzymatic reaction is carried out at a temperature of 35-45°C (preferably 40°C). Preferably, when the endopeptide is Bacillus licheniformis protease, the enzymatic reaction is carried out at a temperature of 35-45°C (preferably 40°C); (vii) The time for the enzymatic reaction is 20 to 40 minutes, preferably 30 minutes; (viii) The concentration of the buffer solution is 0.04–0.06 mol / L, preferably 0.05 mol / L; (ix) The pH of the buffer solution is 6.5 to 11, preferably 7.0, 7.5 or 10.5; Preferably, when the endopeptide is chymotrypsin, the pH of the buffer solution is 6.5 to 7.5, preferably 7.0; Preferably, when the endopeptide is Bacillus subtilis protease, the pH of the buffer solution is 7.0 to 8.0, preferably 7.5; Preferably, when the endopeptide is Bacillus licheniformis protease, the pH of the buffer solution is 10-11, preferably 10.5; (x) In the acidic solution, the concentration of the solute is 0.4 to 0.6 mol / L, preferably 0.5 mol / L; (xi) The pH of the acidic solution is 0.2 to 0.4, preferably 0.

3.

6. The method according to any one of claims 1-5, wherein, Step (2) includes: Experimental group: In the presence of buffer solution, casein and the target endopeptide protease were subjected to an enzymatic reaction, and the enzyme was then inactivated to obtain a reaction mixture; Control group: Under the same buffer conditions as the experimental group, a mixture of casein and enzyme inactivation reagent was mixed with the endopeptide to be tested, and the same experimental operation as the enzymatic reaction in the experimental group was performed to obtain a control mixture. Furthermore, the casein concentration and mass in the control group are the same as those in the experimental group; the concentration and volume of the enzyme-inactivating reagent in the control group are the same as those used in the experimental group; the concentration and mass of the endonuclease to be tested in the control group are the same as those in the experimental group; and the volume and pH of the buffer solution in the control group are the same as those in the experimental group. Step (3) further includes: obtaining the fluorescence response value difference X2 of the control mixture, wherein the fluorescence response value difference X2 is the difference obtained by subtracting the fluorescence response value B2 from the fluorescence response value A2, wherein the fluorescence response value A2 is the fluorescence response value of the control mixture under the conditions of excitation wavelength of 280±5nm and emission wavelength of 355±5nm, and the fluorescence response value B2 is the fluorescence response value of the control mixture under the conditions of excitation wavelength of 280±5nm and emission wavelength of Y±5nm; Step (4) further includes: substituting the fluorescence response value difference X2 of the control mixture into the standard curve of the concentration C and the fluorescence response value difference X to obtain the concentration of casein in the control mixture.

7. The method according to any one of claims 4-6, wherein: When the endopeptide is chymotrypsin, in step (1), when the concentration of casein in the casein aqueous solution is in the range of 0.05–0.6 mg / mL, the established standard curve is y = 55757626x + 4371913(R 2 =1), when the concentration of casein in the casein aqueous solution is in the range of 0.6 to 2.0 mg / mL, the established standard curve is y = 17509569ln(x) + 47369891(R). 2 =1); When the endopeptide is Bacillus subtilis protease, in step (1), the concentration of casein in the casein aqueous solution is in the range of 0.1–1.8 mg / mL, and the established standard curve is y = 7,223,846x + 962,556(R). 2 =0.9996); When the endopeptide is Bacillus licheniformis protease, in step (1), the concentration of casein in the casein aqueous solution is in the range of 0.1 to 1.8 mg / mL, and the established standard curve is y = 17,123,471x + 1,885,497(R). 2 =0.9991); Where y represents the difference in fluorescence response values, and x represents the casein concentration (unit: mg / mL).

8. The method according to any one of claims 1-7, wherein, The enzyme activity is defined as the amount of enzyme required to degrade 1 μg of casein in 1 min by 1 mL of endopeptide aqueous solution, and the enzyme activity unit is U / mL.

9. The method according to claim 8, wherein, The enzyme activity of the endopeptide to be tested is calculated using the following formula: enzyme activity of the endopeptide to be tested (U / mL) = [casein content of control group (μg) - casein content of experimental group (μg)] / [volume of endopeptide aqueous solution (mL) × reaction time (min)], wherein the casein content of the control group is calculated using the concentration of casein in the control mixture, and the casein content of the experimental group is calculated using the concentration of casein in the reaction mixture.

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