Method for fluorescence quantitative determination of activity of endo-pectin lyase

Abstract

The invention provides the method for fluorescence quantitative determination of the activity of the endo-pectinase, the enzyme activity of the endo-pectinase can be accurately detected by constructing a proper endo-pectinase enzyme activity detection system, the method is green, safe, convenient to operate and low in cost, the defects of the existing detection method can be avoided, and the method can be widely applied to detection of the endo-pectinase activity of the endo-pectinase activity of the endo-pectinase activity of the endo-pectinase activity of the endo-pectinase activity of the endo-pectinase. The application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pectinase, and particularly relates to a method for fluorescent quantitative determination of endo-type pectin lyase activity. BACKGROUND

[0002] Pectinases are divided into two categories, one of which can catalyze the depolymerization of pectin, and the other of which can catalyze the hydrolysis of ester in the pectin molecule. Among them, the enzyme catalyzing the depolymerization of pectin substance (hereinafter referred to as "pectin lyase") is widely used in the food industry, such as crushing fruits, increasing juice yield, clarifying fruit juice, etc., and is also used in flue-cured tobacco processing to reduce the pectin content in flue-cured tobacco and improve the quality of tobacco leaves. Pectin lyase is divided into endo-type and exo-type, endo-type enzyme randomly hydrolyzes α-1, 4 bond from the inside of the molecule, which rapidly reduces the viscosity of pectin, but cannot generate reducing sugar; exo-type enzyme strips reducing sugar from the end, but the overall structure of pectin remains, and the viscosity does not decrease significantly.

[0003] DNS method is the most commonly used method for detecting pectin lyase, but this method can only detect exo-type pectin lyase, and its principle is that DNS reagent can color with reducing sugar in the sample to represent how much reducing sugar is stripped from pectin by pectinase, so as to calculate the enzyme activity. For example, the literature

Zhou Pan, Lei Dandan, Li Haoxin, et al. Extraction and characterization of pectinase from potato cell wall [J]. Chinese Journal of Food Science, 2024, 24 (12): 215-223

[0004] Endo-type pectin lyase is difficult to characterize because its enzymatic product is small fragments of pectin or oligosaccharides. Viscosity method is a commonly used method before, but it is abandoned because of large error and easy to be affected by environmental temperature. The commonly used detection method today is ultraviolet absorption method, which detects C4, C5 unsaturated bond produced in the pectin lyase process, the literature

Xing Mingxia, Wang Pengbo, Xu Wenting, et al. Optimization of determination method of specific activity of alkaline pectinase by kinetic method [J]. Food Industry Science and Technology, 2020, 41 (23): 223-228, 235

Zhang Ying, Han Xiaojing, Cai Yitan, et al. Application research on screening of endogenous promoter of bacillus amyloliquefaciens and expression of alkaline pectinase [J]. Acta Microbiologica Sinica, 2023, 63 (4): 1575-1586

[0005] In view of the problems in the prior art, the application provides a method for quantitatively determining the activity of endo-type pectin lyase. The method can accurately detect the enzyme activity of endo-type pectin lyase by constructing a suitable endo-type pectin lyase enzyme activity detection system. The method is green and safe, convenient to operate, low in cost, can avoid the defects of the existing detection methods, and has a wide application prospect.

[0006] Specifically, the application provides a method for detecting the enzyme activity of endo-type pectin lyase, which comprises the following steps:

[0007] (1) establishing a standard curve of the concentration C of a pectin aqueous solution and a fluorescence response value A, wherein the fluorescence response value A is the fluorescence response value of the pectin aqueous solution under the condition that the excitation wavelength is 285 nm and the emission wavelength is 463 nm;

[0008] (2) performing parallel experimental group experiments and control group experiments;

[0009] The experimental group experiments comprise: under the condition that a buffer solution exists, performing an enzymatic hydrolysis reaction of pectin and the endo-type pectin lyase to be detected, and then inactivating the enzyme to obtain an enzymatic hydrolysis mixture, wherein the buffer solution is an L-tartaric acid buffer solution, the enzyme inactivation is achieved by adding an acidic solution, and the acidic solution is a lactic acid aqueous solution;

[0010] The control group experiments comprise: under the condition that a buffer solution exists, mixing pectin and an acidic solution, then mixing the mixture with the endo-type pectin lyase to be detected and performing the same experimental operation as the enzymatic hydrolysis reaction of the experimental group to obtain a control mixture, wherein the buffer solution is an L-tartaric acid buffer solution, and the acidic solution is a lactic acid aqueous solution;

[0011] The concentration and volume of the acidic solution of the control group are the same as those of the experimental group, the volume and pH of the buffer solution of the control group are the same as those of the experimental group, the concentration and mass of the substrate pectin of the control group are the same as those of the experimental group, and the concentration and mass of the endo-type pectin lyase to be detected of the control group are the same as those of the experimental group;

[0012] (3) obtaining the fluorescence response value A of the enzymatic hydrolysis mixture under the condition that the excitation wavelength is 285 nm and the emission wavelength is 463 nm 实验 ; and simultaneously, obtaining the fluorescence response value A of the control mixture under the condition that the excitation wavelength is 285 nm and the emission wavelength is 463 nm 对照 ;

[0013] (4) substituting the fluorescence response value A of the enzymatic hydrolysis mixture into the standard curve of the concentration C and the fluorescence response value A to obtain the concentration C of the pectin in the enzymatic hydrolysis mixture 实验 实验 ​; at the same time, the fluorescence response value A of the control mixture is measured 对照 The concentration C of pectin in the control mixture is obtained by substituting the standard curve of the concentration C and the fluorescence response value A 对 ;

[0014] (5) The concentration C of pectin in the enzyme hydrolysis mixture is obtained 实验 , and the mass M of pectin in the enzyme hydrolysis mixture is obtained 实验 ; at the same time, the concentration C of pectin in the control mixture is obtained 对照 , and the mass M of pectin in the control mixture is obtained 对照 ;

[0015] (6) The enzyme activity of the endo-pectinase to be tested is obtained based on the mass M of pectin in the enzyme hydrolysis mixture 实验 and the mass M of pectin in the control mixture 对照 .

[0016] In some embodiments, in step (2):

[0017] The experimental group experiment includes: after the enzyme hydrolysis reaction of pectin and the endo-pectinase to be tested in the presence of a buffer and an active factor, the enzyme is inactivated to obtain an enzyme hydrolysis mixture, wherein the buffer is an L-tartaric acid buffer, the enzyme inactivation is achieved by adding an acidic solution, and the acidic solution is a lactic acid aqueous solution;

[0018] The control group experiment includes: after the pectin is mixed with an acidic solution, the mixture is mixed with the endo-pectinase to be tested and the same experimental operation as the enzyme hydrolysis reaction of the experimental group is performed to obtain a control mixture, wherein the buffer is an L-tartaric acid buffer, and the acidic solution is a lactic acid aqueous solution;

[0019] The concentration and volume of the acidic solution of the control group are the same as those of the experimental group, the volume and pH of the buffer of the control group are the same as those of the experimental group, the concentration and mass of the active factor of the control group are the same as those of the experimental group, the concentration and mass of the substrate pectin of the control group are the same as those of the experimental group, and the concentration and mass of the endo-pectinase to be tested of the control group are the same as those of the experimental group.

[0020] It should be noted that in the control group experiment, "the same experimental operation as the enzyme hydrolysis reaction of the experimental group is performed", which means that if the conditions of the enzyme hydrolysis reaction in the experimental group are what, the same operation is also performed in the control group. For example, the enzyme hydrolysis reaction is performed at a temperature of 50°C for 30 min, and the mixture in the control group is also placed at a temperature of 50°C for 30 min.

[0021] In the present application, unless otherwise specified, "a solution of a certain substance" or "a solution of a certain substance" means an aqueous solution of the substance by default.

[0022] In some embodiments, the active factor is selected from salicin, esculin, stevioside, mogroside.

[0023] In some embodiments, the active factor is selected from salicin, stevioside, mogroside.

[0024] In some embodiments, the active factor is salicin or mogroside.

[0025] In some embodiments, the active factor is salicin.

[0026] In some embodiments, the active factor is provided in the form of an aqueous solution, and the concentration of the active factor is 100-500 μg / mL, such as 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL or 500 μg / mL.

[0027] In some embodiments, the concentration of the active factor is 300 μg / mL.

[0028] In some embodiments, the pH of the buffer is 6.8-7.2.

[0029] In some embodiments, the pH of the buffer is 7.0.

[0030] In some embodiments, in the acidic solution, the concentration of lactic acid is 0.4-0.6 mol / L.

[0031] In some embodiments, in the acidic solution, the concentration of lactic acid is 0.5 mol / L.

[0032] In some embodiments, when the concentration of pectin is in the range of 0-1.0 mg / mL, the standard curve is: y=1608056x+101919 (R 2 =1); when the concentration of pectin is in the range of 1.0-10 mg / mL, the standard curve is: y=1094596ln(x)+1643514 (R 2 =1), wherein x represents the concentration of pectin (unit: mg / mL), and y represents the fluorescence response value.

[0033] In some embodiments, the temperature of the enzymatic reaction is 48-52°C, preferably 50°C.

[0034] In some embodiments, the time of the enzymatic reaction is 25-35 min, preferably 30 min.

[0035] In some embodiments, the enzyme activity is defined as: 1 U of enzyme is the amount of enzyme required to degrade 1 μg of pectin in 1 min per 1 mL of pectinase aqueous solution.

[0036] In some embodiments, step (6) comprises: based on the mass M 实验 of pectin in the enzyme hydrolysis mixture and the mass M 对照 of pectin in the control mixture, the enzyme activity of the endo-type pectinase to be tested is calculated using the following formula,

[0037] Enzyme activity (U / mL) = [M 对照 (μg) - M 实验 (μg)] / [volume of pectinase aqueous solution (mL) x enzyme hydrolysis reaction time (min)].

[0038] In some embodiments, the substrate pectin is provided in the form of an aqueous solution, and the concentration of the pectin is 8-12 mg / mL (such as 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL or 12 mg / mL), preferably 10 mg / mL.

[0039] In some embodiments, the pectinase is provided in the form of an aqueous solution, and the concentration of the pectinase is 0.2-0.3 mg / mL, preferably 0.25 mg / mL.

[0040] In some embodiments, the ratio of the volume of the buffer solution to the volume of the pectin aqueous solution is (0.5-1):1, such as 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or 1:1, preferably 0.8:1.

[0041] In some embodiments, the ratio of the volume of the active factor aqueous solution to the volume of the pectin aqueous solution is (0.1-0.3):1, preferably 0.2:1.

[0042] In some embodiments, the ratio of the volume of the pectinase aqueous solution to the volume of the pectin aqueous solution is (0.1-0.3):1, preferably 0.2:1.

[0043] In some embodiments, the ratio of the volume of the acid solution to the volume of the pectin aqueous solution is (0.5-1):1, such as 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1 or 1:1, preferably 0.8:1.

[0044] In some embodiments, the endo-type pectinase is a polygalacturonase.

[0045] Beneficial effects

[0046] The current endo-type pectinase detection method is less, and the constructed detection method can accurately determine the enzyme activity of endo-type pectinase; compared with the existing ultraviolet absorption method, the sensitivity is higher, and the stability is better. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 It is a full wavelength scanning graph of pectin solution;

[0048] Figure 2 It is a fluorescence spectrum of pectin, galacturonic acid and pectinase;

[0049] Figure 3 It is a relationship curve between pectin concentration (0-10 mg / mL) and fluorescence response value;

[0050] Figure 4 It is a relationship curve between pectin concentration (0-1.0 mg / mL) and fluorescence response value;

[0051] Figure 5 It is a relationship curve between pectin concentration (1.0-10 mg / mL) and fluorescence response value;

[0052] Figure 6 It is the influence of different acid solutions on the fluorescence response value of pectin;

[0053] Figure 7 It is the influence of different buffer matrixes on the fluorescence response value of pectin;

[0054] Figure 8 It is the influence of different active factors on the fluorescence spectrum of pectin;

[0055] Figure 9 It is the influence of different active factors on the fluorescence value of pectin;

[0056] Figure 10 It is the standard curve of the existing ultraviolet quantitative method. DETAILED DESCRIPTION

[0057] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not limited to the scope of the present application. According to the following detailed description of the preferred embodiments and the drawings, the various purposes and advantages of the present application will become apparent to those skilled in the art.

[0058] In the following examples, unless otherwise specified, pectin was purchased from Shanghai Yuan Ye Bio, item number S11083-100g; galacturonic acid was purchased from Sinopharm Group, item number 63004582; standard pectinase (polygalacturonase) was purchased from Shanghai Yuan Ye Bio, item number S10007-25g.

[0059] Preparation of standard pectinase solution: 0.025 g of standard pectinase was weighed and dissolved in 100 mL of ultrapure water to prepare a 0.25 mg / mL standard pectinase solution.

[0060] Preparation of galacturonic acid solution: 0.100 g of galacturonic acid was weighed and dissolved in 100 mL of deionized water to prepare a 1 mg / mL galacturonic acid solution.

[0061] In addition, in the following examples, salicin, aescin, stevioside, mogroside, and Tris buffer can be purchased from the market, and exemplary purchase sources are shown as follows.

[0062]

[0063] Other buffers (L-tartaric acid buffer, glycine buffer, boric acid buffer, citric acid buffer) involved in the present application can be prepared from commercially available reagents, and the sources of reagents and specific preparation methods are as follows.

[0064] Glycine buffer (0.05 mol / L, pH = 7.0): commercially available glycine buffer (0.1 mol / L, pH = 7.0, purchased from Shanghai Yuan Ye, item number R26382-100ml) was diluted 2 times with deionized water;

[0065] Citric acid buffer (0.05 mol / L, pH = 7.0): commercially available citric acid-sodium citrate buffer (0.1 mol / L, pH = 7.0, purchased from Shangbao Biology, item number T17395) was diluted 2 times with deionized water;

[0066] NaOH (1 mol / L): 40 g of NaOH (purchased from China National Pharmaceutical Group, item number 10019718) was weighed and dissolved in deionized water to a final volume of 1 L;

[0067] L-tartaric acid buffer (0.05 mol / L, pH = 7.0): 0.75 g of L-tartaric acid (purchased from China National Pharmaceutical Group, item number 10022018) was dissolved in 80 mL of deionized water, and then 1 mol / L NaOH was used to adjust the pH to 7.0, and the volume was made up to 100 mL with water.

[0068] Boric acid buffer (0.05 mol / L, pH = 7.0): 0.309 g of boric acid (purchased from China National Pharmaceutical Group, item number 10004808) was dissolved in 80 mL of deionized water, and then 1 mol / L NaOH was used to adjust the pH to 7.0, and the volume was made up to 100 mL with water.

[0069] Example 1: Fluorescence emission spectrum of pectin, standard pectinase, and galacturonic acid

[0070] Preparation of pectin solution at room temperature: 1 g of pectin was dissolved in 100 mL of deionized water (10 mg / mL) at room temperature, and diluted with deionized water to 0.2, 0.4, 0.5, 0.8, 1, 1.2, 1.6, 2, 4, 6, 8 mg / mL, respectively.

[0071] The 10 mg / mL pectin solution was taken in 200 μL and subjected to full wavelength scanning from 230 to 600 nm in a UV enzyme-coated plate, and the results are shown in Figure 1 It can be seen from Figure 1 that pectin has a special absorption peak at 285 nm, and thus the excitation wavelength of the fluorescence spectrum is selected as 285 nm. The 10 mg / mL pectin solution, 0.25 mg / mL standard pectinase solution and 1 mg / mL galacturonic acid solution were taken in 200 μL, respectively, and subjected to fluorescence spectrum scanning in a fluorescence enzyme-coated plate, with the excitation wavelength being 285 nm and the emission wavelength being 325-700 nm, and the results are shown in Figure 2 It can be seen from Figure 2 that the fluorescence spectrum of pectin has fluorescence response peak values at 355 nm and 463 nm, respectively, but the fluorescence response at 355 nm is easily interfered by the standard pectinase (one is that the fluorescence response peak value of the standard pectinase is 338 nm, and the other is that the fluorescence response value of the standard pectinase is much higher than that of pectin within 400 nm), and thus 463 nm is selected as the emission wavelength of pectin, at which the interference of galacturonic acid and standard pectinase is smaller.

[0072] Preparation of pectin standard curve: the 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.6, 2, 4, 6, 8, 10 mg / mL pectin solutions were taken in 200 μL, respectively, and subjected to fluorescence spectrum scanning in a fluorescence enzyme-coated plate, with the excitation wavelength being 285 nm and the emission wavelength being 463 nm, and the standard curve was plotted according to the fluorescence response value, and the results are shown in Figure 3 It can be seen from Figure 3 that the pectin concentration within the range of 0-10 mg / mL is not a straight line, but a curve, and thus the standard curve is plotted in sections, as shown in Figure 4 and Figure 5 It can be seen from Figure 4 that within the range of 0-1.0 mg / mL, the standard curve is y=1608056x+101919 (R 2 =1); and it can be seen from Figure 5 that within the range of 1.0-10 mg / mL, the standard curve is y=1,094,596ln(x)+1,643,514 (R 2 =1).

[0073] Example 2: Construction of pectinase detection system

[0074] Preliminary construction of enzyme activity detection system: in 2 mL centrifuge tube, 0.5 mL of 10 mg / mL pectin solution and 0.5 mL buffer (pH 7.0) were mixed, then 0.1 mL of 0.25 mg / mL pectinase solution was added and reacted at 50°C for 30 min, after the reaction, 0.4 mL of 0.5 mol / L acid solution was added for enzyme inactivation, then 200 μL of the reaction solution was taken for fluorescence detection, excitation wavelength 285 nm, emission wavelength 463 nm, the standard curve of pectin concentration and fluorescence response value was used to calculate the consumption of pectin, and the enzyme activity was calculated according to the definition of enzyme activity. The definition of enzyme activity is: under the conditions of 50°C and pH=7.0, the amount of enzyme required for degrading 1 μg of pectin per 1 mL of enzyme solution in 1 min is 1 U. Pectinase enzyme activity (U / mL) = [control group pectin mass (μg) - experimental group pectin mass (μg)] / [enzyme solution volume (mL) x reaction time (min)]. The enzyme activity of the control group was added with acid solution first and then with enzyme solution, so that the enzyme could not occur enzymatic reaction, and the rest of the steps were consistent with the experimental group.

[0075] 1. Effect of enzyme inactivation reagent on fluorescence response of substrate pectin

[0076] Deionized water was used instead of buffer, and except for the type of acid solution, the rest of the steps were the same as the above-mentioned preliminary construction of enzyme activity detection system, so as to compare the effects of different acid solutions (enzyme inactivation reagents) on the fluorescence response value of pectin.

[0077] The concentration of the matrix of the acid solution was 0.5 mol / L (the pH of 0.5 mol / L HCl was 0.3, and other acids were weak acids, and the pH was adjusted to 0.3 with concentrated hydrochloric acid before use, so as to keep the pH of each acid solution consistent). Figure 6 It can be seen that acetic acid solution and lactic acid solution can improve the fluorescence response value of the system, but acetic acid is easy to volatilize, so the preparation of the reagent needs to be operated in a fume hood, which is relatively inconvenient, therefore, lactic acid solution is the most suitable as the acid solution of enzyme inactivation reagent.

[0078] 2. Effect of buffer on fluorescence response of substrate pectin and pectinase activity

[0079] (1) Comparison of the effects of different buffer matrices on the fluorescence response value of pectin. The concentration of the buffer matrix was 0.05 mol / L, the enzyme reaction system used was the enzyme reaction system of the control group in the above-mentioned preliminary construction of enzyme activity detection system, the blank group used the same volume of deionized water instead of buffer, and the enzyme inactivation reagent used was lactic acid solution. From Figure 7 It can be seen that boric acid buffer, L-tartaric acid buffer and citric acid buffer can improve the fluorescence response value of pectin, and other buffer matrices have little effect on the fluorescence response value of pectin.

[0080] (2) Further, except for the different buffer types, the rest of the steps remain the same as the above-mentioned preliminary construction of enzyme activity detection system (the enzyme inactivating reagent used is lactic acid solution), so as to compare the effects of different buffers on pectinase activity. Deionized water (i.e. the buffer is replaced by deionized water), boric acid, L-tartaric acid and citric acid buffer are used to detect the enzyme activity of the pectinase sample (purchased from Shanghai Yuan Ye Biology, product number S10007-25g), and the enzyme activity values are 31.2±0.6 U / mL, 25.8±3.1 U / mL, 31.6±0.6 U / mL and 28.5±1.0 U / mL respectively, indicating that the boric acid buffer and the citric acid buffer may inhibit the pectin lyase enzyme activity, and therefore the L-tartaric acid buffer is used as the optimal buffer for the enzymatic reaction system.

[0081] 3. Effects of active factors on pectinase activity and fluorescence response of substrate pectin

[0082] (1) Effects of active factors on pectinase activity. The reaction system is slightly modified: 0.5 mL of 10 mg / mL pectin solution, 0.4 mL of L-tartaric acid buffer (pH 7.0), 0.1 mL of active factor solution with a concentration of 100 μg / mL (0.1 mL of deionized water is added to the blank group) are mixed, and then 0.1 mL of 0.25 mg / mL pectinase solution is added for reaction at 50°C for 30 min. After the reaction, 0.4 mL of 0.5 mol / L lactic acid solution is added for enzyme inactivation. After enzyme inactivation, 200 μL of the reaction solution is taken for fluorescence detection, with an excitation wavelength of 285 nm and an emission wavelength of 463 nm. The consumption of pectin is calculated by comparing with the standard curve of pectin concentration and fluorescence response value, and the enzyme activity is calculated according to the definition of enzyme activity. The enzyme activity of the control group is added with an acidic solution first and then with an enzyme solution, so that the enzyme cannot occur enzymatic reaction, and the rest of the steps are consistent with the experimental group. Enzyme activity improvement rate = (enzyme activity of experimental group - enzyme activity of blank group) / enzyme activity of blank group x 100%. As shown in Table 1, the four kinds of active factors in the reaction system can all improve the pectinase enzyme activity.

[0083] Table 1 Effects of different types of active factors on pectinase enzyme activity

[0084]

[0085] Note: In the table, a, b, c and d in the upper right corner represent the degree of significant difference. In the same comparison data, the groups marked with the same letter have no significant difference (P value≥0.05), and the groups marked with different letters have significant difference (P value<0.05). The larger the mean value is, the earlier the order of the letter marked is.

[0086] (2) The effect of active factors on the fluorescence response of substrate pectin. The detection system was as follows: 0.5 mL of 10 mg / mL pectin solution, 0.4 mL of L-tartaric acid buffer (pH 7.0), 0.1 mL of active factor solution with a concentration of 100 μg / mL (0.1 mL of deionized water was added to the blank group), 0.1 mL of water, and 0.4 mL of 0.5 mol / L lactic acid solution were added to a 2 mL centrifuge tube, and then 200 μL of the reaction solution was taken for fluorescence scanning at an excitation wavelength of 285 nm. It was found that Figure 8 The results showed that esculin itself had a strong fluorescence color development effect, which covered the fluorescence of pectin itself. The fluorescence intensity at an emission wavelength of 463 nm was compared for the groups without esculin, and it was found that Figure 9 The results showed that salicin and mogroside could improve the fluorescence response value of pectin, while stevioside reduced the fluorescence response value of pectin.

[0087] Based on the effects of active factors on enzyme activity and substrate fluorescence response, salicin and mogroside could be used as additives in the reaction system, which could promote enzyme activity and improve the sensitivity of detection, and salicin was the best.

[0088] (3) The effect of the concentration of active factors on pectinase activity or the fluorescence response of substrate pectin

[0089] The reaction system of (1) or the detection system of (2) was used to study the effect of different concentrations of salicin on pectinase activity or the fluorescence response of substrate pectin, so as to further determine the appropriate addition concentration of salicin.

[0090] Among them, the enzyme activity improvement rate = (experimental group enzyme activity - blank group enzyme activity) / blank group enzyme activity × 100%; the fluorescence value improvement rate = (experimental group fluorescence value - blank group fluorescence value) / blank group fluorescence value × 100%.

[0091] As shown in Table 2, the optimal addition concentration of salicin was 300 μg / mL, which had the strongest ability to promote pectinase activity and the strongest ability to improve the fluorescence response of pectin.

[0092] Table 2 Effect of salicin addition concentration on pectinase activity and pectin fluorescence response

[0093]

[0094] Note: In the table, a, b, and c on the right top represent the degree of significant difference. In the same comparison data, groups with the same letter have no significant difference (P value ≥ 0.05), and groups with different letters have significant difference (P value < 0.05). The larger the mean value is, the earlier the order of the letter marked is.

[0095] 0.05), and the larger the mean value is, the earlier the order of the letter marked is.

[0096] Based on the above researches, the enzyme activity detection system is finally determined as follows:

[0097] In a 2 mL centrifuge tube, 0.5 mL of 10 mg / mL pectin solution, 0.4 mL of L-tartaric acid buffer (pH 7.0), 0.1 mL of 300 μg / mL salicin solution are mixed, 0.1 mL of 0.25 mg / mL pectinase solution is added, and the mixture is reacted at 50°C for 30 min. After the reaction is completed, 0.4 mL of 0.5 mol / L lactic acid solution is added for enzyme inactivation. After enzyme inactivation, 200 μL of the reaction solution is taken for fluorescence detection, the excitation wavelength is 285 nm, and the emission wavelength is 463 nm. The standard curve of pectin concentration and fluorescence response value is used to calculate the consumption of pectin, and the enzyme activity is calculated according to the definition of enzyme activity. The definition of enzyme activity is: under the conditions of 50°C and pH=7.0, the amount of enzyme required for 1 μg of pectin to be degraded in 1 min per 1 mL of enzyme solution is 1 U. The pectinase enzyme activity (U / mL) = [control group pectin mass (μg) - experimental group pectin mass (μg)] / [enzyme solution volume (mL) x reaction time (min)]. The enzyme activity system of the control group is added with an acidic solution first, and then with an enzyme solution, so that the enzyme cannot occur enzymatic reaction, and the remaining steps are consistent with the experimental group.

[0098] Example 3: Comparison with existing endo-type pectin lyase detection methods

[0099] The ultraviolet absorption method is the most commonly used method for detecting endo-type pectin lyase at present. The comparison method is the detection method of the literature

Xing Mingxia, Wang Pengbo, Xu Wenting, et al. Kinetic method for optimizing the determination of specific activity of alkaline pectinase [J]. Food Industry Science and Technology, 2020, 41(23): 223-228, 235

[0100] The method (fluorescence quantitative method) of the present application: after enzyme inactivation, 200 μL of the reaction solution is taken for fluorescence detection, the excitation wavelength is 285 nm, and the emission wavelength is 463 nm. The standard curve of pectin concentration and fluorescence response value is used to calculate the consumption of pectin;

[0101] Reference method (UV quantification): After enzyme inactivation, 200 μL of the reaction solution was collected for UV detection at a wavelength of 232 nm. The standard curve was constructed using the absorbance values ​​measured at 232 nm for all concentrations of pectin solutions prepared in Example 1. The reference pectinase standards included pectinase I (described in Example 2); pectinase II (purchased from Shifeng Biotechnology, catalog number A0627-25g); and pectinase III (purchased from Hongrun Baoshun, catalog number M012).

[0102] Depend on Figure 10 It can be seen that the standard curve R of the UV quantitative method 2 =0.9992, slightly less than the R of the fluorescence quantitative method standard curve 2 =1, primarily due to the low UV absorbance values. Even small fluctuations in absorbance can lead to assay bias, resulting in data points in the standard curve not being perfectly aligned but fluctuating around the line. Table 3 shows that the means of the two assays are relatively close, but the UV quantification method has a larger standard deviation (larger fluctuations in enzyme activity values).

[0103] Table 3 Enzyme activities of standard enzymes measured by different detection methods (U / mL)

[0104] Method Pectinase I Pectinase II Pectinase III Fluorometric quantification 31.67±0.58 46.33±0.85 12.03±0.15 UV quantification 30.00±1.00 45.50±2.65 11.53±0.86 P value 0.0668 0.6309 0.3786 Is the difference significant No No No

[0105] It should be understood that the invention described herein is not limited to specific methodology, experimental protocols or reagents, as these may vary. The discussion and examples provided herein are presented only to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the claims.

Claims

1. A method for detecting endo-pectinase activity, comprising: (1) establishing a standard curve of the concentration C of the pectin aqueous solution and the fluorescence response value A, wherein the fluorescence response value A is the fluorescence response value of the pectin aqueous solution under the conditions of an excitation wavelength of 285 nm and an emission wavelength of 463 nm; (2) Conducting experimental and control group experiments in parallel; The experimental group experiment includes: in the presence of a buffer, allowing pectin to undergo an enzymatic hydrolysis reaction with the endo-pectinase to be tested, followed by inactivation of the enzyme to obtain an enzymatic hydrolysis mixture, wherein the buffer is L-tartaric acid buffer, and the inactivation of the enzyme is achieved by adding an acidic solution, wherein the acidic solution is a lactic acid aqueous solution; The control group experiment comprises: in the presence of a buffer, mixing pectin with an acidic solution, then mixing with the endo-pectinase to be tested, and performing the same experimental operation as the enzymatic hydrolysis reaction of the experimental group to obtain a control mixed solution, wherein the buffer is L-tartaric acid buffer and the acidic solution is a lactic acid aqueous solution; The acidic solution of the control group has the same concentration and volume as the acidic solution of the experimental group, the buffer of the control group has the same volume and pH as the buffer of the experimental group, the pectin substrate of the control group has the same concentration and mass as the pectin substrate of the experimental group, and the endo-pectinase to be tested in the control group has the same concentration and mass as the endo-pectinase to be tested in the experimental group; (3) Obtain the fluorescence response value A of the enzymatic hydrolysis mixture under the conditions of an excitation wavelength of 285 nm and an emission wavelength of 463 nm 实验 At the same time, the fluorescence response value A of the control mixture under the conditions of excitation wavelength of 285nm and emission wavelength of 463nm was obtained. 对照 ; (4) The fluorescence response value A of the enzymatic mixture 实验 Substitute the concentration C and the fluorescence response value A into the standard curve to obtain the concentration C of pectin in the enzymatic mixture. 实验 At the same time, the fluorescence response value A of the control mixture 对照 Substitute the concentration C and the fluorescence response value A into the standard curve to obtain the concentration C of pectin in the control mixture. 对 According to; (5) Based on the concentration C of pectin in the enzymatic mixture 实验 , obtain the mass M of pectin in the enzymatic hydrolysis mixture 实验 At the same time, based on the concentration of pectin in the control mixture C 对照 , obtain the mass M of pectin in the control mixture 对照 ; (6) Based on the mass M of pectin in the enzymatic mixture 实验 and the mass M of pectin in the control mixture 对照 , and obtain the enzyme activity of the endo-pectinase to be tested.

2. The method according to claim 1, wherein In step (2): The experimental group experiment includes: in the presence of a buffer and an active factor, allowing pectin to undergo an enzymatic hydrolysis reaction with the endo-pectinase to be tested, followed by inactivation of the enzyme to obtain an enzymatic hydrolysis mixture, wherein the buffer is an L-tartaric acid buffer, and the inactivation of the enzyme is achieved by adding an acidic solution, wherein the acidic solution is a lactic acid aqueous solution; The control group experiment comprises: in the presence of a buffer and an active factor, mixing pectin with an acidic solution, then mixing with the endo-pectinase to be tested, and performing the same experimental operation as the enzymatic hydrolysis reaction of the experimental group to obtain a control mixed solution, wherein the buffer is L-tartaric acid buffer and the acidic solution is a lactic acid aqueous solution; The acidic solution of the control group has the same concentration and volume as the acidic solution of the experimental group, the buffer solution of the control group has the same volume and pH as the buffer solution of the experimental group, the active factor of the control group has the same concentration and mass as the active factor of the experimental group, the substrate pectin of the control group has the same concentration and mass as the substrate pectin of the experimental group, and the endo-pectinase to be tested in the control group has the same concentration and mass as the endo-pectinase to be tested in the experimental group.

3. The method according to claim 2, wherein: The active factor is selected from salicin, esculin, stevioside, and mogroside; Preferably, the active factor is selected from salicin, stevioside, and mogroside; More preferably, the active factor is salicin or mogroside; Most preferably, the active factor is salicin.

4. The method according to claim 3, wherein: The active factor is provided in the form of an aqueous solution, and the concentration of the active factor is 100-500 μg / mL, preferably 300 μg / mL.

5. The method according to any one of claims 1 to 4, wherein: The pH of the buffer solution is 6.8 to 7.2, preferably 7.

0.

6. The method according to any one of claims 1 to 5, wherein: In the acidic solution, the concentration of the lactic acid is 0.4-0.6 mol / L, preferably 0.5 mol / L.

7. The method according to any one of claims 1 to 6, wherein: The pectin concentration is in the range of 0 to 1.0 mg / mL, and the standard curve is: y = 1608056x + 101919 (R 2 =1); the pectin concentration is in the range of 1.0 to 10 mg / mL, and the standard curve is: y = 1094596ln(x) + 1643514(R 2 =1), where x represents the pectin concentration (in mg / mL) and y represents the fluorescence response value.

8. The method according to any one of claims 1 to 7, wherein: The temperature of the enzymatic hydrolysis reaction is 48-52° C., preferably 50° C.; and / or, The enzymatic hydrolysis reaction time is 25 to 35 minutes, preferably 30 minutes.

9. The method according to any one of claims 1 to 8, wherein: The method further has one or more technical features selected from the following (i)-(ii): (i) Enzyme activity is defined as the amount of enzyme required to degrade 1 μg of pectin in 1 min per 1 mL of pectinase aqueous solution as 1 U; (ii) Step (6) comprises: based on the mass M of pectin in the enzymatic hydrolysis mixture 实验 and the mass M of pectin in the control mixture 对照 , the enzyme activity of the endo-pectinase to be tested was calculated using the following formula: Enzyme activity (U / mL) = [M 对照 (μg)-M 实验 (μg)] / [volume of pectinase aqueous solution (mL) × enzymatic reaction time (min)].

10. The method according to any one of claims 1 to 9, wherein: The method further has one or more technical features selected from the following (i)-(vii): (i) the substrate pectin is provided in the form of an aqueous solution, and the concentration of the pectin is 8 to 12 mg / mL, preferably 10 mg / mL; (ii) the pectinase is provided in the form of an aqueous solution, and the concentration of the pectinase is 0.2-0.3 mg / mL, preferably 0.25 mg / mL; (iii) the ratio of the volume of the buffer solution to the volume of the pectin aqueous solution is (0.5-1):1, preferably 0.8:1; (iv) the ratio of the volume of the active factor aqueous solution to the volume of the pectin aqueous solution is (0.1-0.3):1, preferably 0.2:1; (v) the ratio of the volume of the pectinase aqueous solution to the volume of the pectin aqueous solution is (0.1-0.3):1, preferably 0.2:1; (vi) the ratio of the volume of the acidic solution to the volume of the pectin aqueous solution is (0.5-1):1, preferably 0.8:1; (vii) The endo-pectinase is polygalacturonase.

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