A method for determining pectolytic activity

Abstract

The present application belongs to the field of enzyme activity determination method, aiming at the problems of existing pectinase activity determination technology, such as complicated operation, low sensitivity, and the problems of existing MBTH method for determining pectinase activity, such as a large amount of precipitate in the chromogenic product solution and the high background value of the chromogenic product, the present application provides a kind of determination method of pectinase activity: a series of concentration gradient galacturonic acid standard solution is prepared by using substrate solution, the precipitate is provided by using alkaline reagent to provide high concentration alkaline environment, and the precipitation of precipitated material is accelerated by using low temperature condition, then the supernatant is obtained by centrifugation, MBTH reaction is carried out, the absorbance value is determined, and the standard corresponding relationship curve is drawn; Pectinase enzymolysis reaction; preparation of enzymolysis blank; the initial speed of the concentration of reducing end group produced by the enzymolysis of the substrate per unit time is determined to calculate the enzyme activity of pectinase. The method is accurate, reliable, good in repeatability, high in sensitivity and stable in chromogenic product.

Description

Technical Field

[0001] This invention belongs to the field of enzyme activity assay technology, specifically relating to a method for measuring pectinase activity. Background Technology

[0002] Pectinase is a general term for a series of enzymes that can degrade pectin into oligogalacturonic acid and / or galacturonic acid. Enzyme activity is an important indicator for measuring enzyme activity, and it is generally determined by measuring the initial rate of the enzyme-catalyzed reaction. The activity unit of pectinase can be defined as the amount of 1 μmol of galacturonic acid produced per minute per milliliter of reaction solution under certain enzymatic hydrolysis conditions. Currently, the main methods for determining pectinase activity are: (1) The national food safety standard in my country adopts the hypoiodite titration method (GB1886.174—2016), which is cumbersome and lengthy and not convenient for large-scale determination. (2) The DNS method (Wang Xiaomin, Wu Wenlong, Lü Lianfei, Li Weilin, Qu Lewen. Study on the method of determining pectinase activity by spectrophotometer [J]. Food Industry Technology, 2007(05):227-229.) calculates pectinase activity by measuring the rate of increase of reducing sugar concentration during pectinase hydrolysis. The data in this article show that the dilution factor of pectinase has a significant impact on the determination of pectinase activity. For example, the enzyme activities measured by dilution of 100, 500, 1000, and 2000 times are 657.08, 2164.81, 2321.08, and 3980.81, respectively. The accuracy of enzyme activity determination will be affected by the different dilution factors of the enzyme solution. Due to the low sensitivity of DNS, it is difficult to obtain the hydrolysis rate of the 0th order reaction for pectinase with a large Km value, so it is impossible to use the hydrolysis time period (such as 30 min) to calculate the initial hydrolysis rate. In addition, the problem that the DNS method does not conform to chemometrics has been reported before (Mccleary BV, McGeough PA Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase[J]. Applied Biochemistry & Biotechnology, 2015, 177(5): 1152-1163.).

[0003] The MBTH method is a method for determining lysozyme activity. This method is far more sensitive than the DNS method, accurately measuring the rate of increase in reducing sugar concentration even when the concentration of reducing sugar in the enzymatic hydrolysis product is low, thus enabling more accurate determination of enzyme activity. During the research process of this invention, the inventors discovered that the MBTH method is difficult to apply to the determination of pectinase activity because precipitation occurs during the color development process. Even after centrifugation to remove the precipitation, it still affects the absorbance value, making it impossible to accurately determine pectinase activity. Furthermore, commercially available pectin often contains a large amount of pectin degradation products, resulting in excessively high background values. Some pectin products may even cause the absorbance value of the measured colorimetric product to exceed the instrument's measurement limit. Therefore, when applying the MBTH method to determine pectinase activity, the problems of precipitation during the color development process and excessively high background values ​​must be addressed. Summary of the Invention

[0004] To address the problems of cumbersome operation, low sensitivity, and excessive precipitation and high background values ​​in the chromogenic product solution when using the existing MBTH method for pectinase activity determination, this invention provides a method for determining pectinase activity. This method uses a modified 3-methyl-2-benzothiazolinone hydrazone (MBTH) method to determine pectinase activity. A high concentration of alkaline reagent is added to induce precipitation in the enzymatic hydrolysate, and low temperature conditions are used to accelerate the precipitation, thus removing high molecular weight pectin. The 3-methyl-2-benzothiazolinone hydrazone method is then used for pectinase activity determination. This method completely eliminates precipitates in the chromogenic product and simultaneously reduces the background value of the reaction. Furthermore, by improving the preparation method and detection wavelength of the galacturonic acid standard curve, the obtained standard curve more closely reflects the actual enzymatic hydrolysis process, making the intercept of the standard curve closer to the origin of the coordinate axis, and improving the stability of the chromogenic product, thereby improving the sensitivity, accuracy, and repeatability of the experiment.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for determining pectinase activity includes the following steps:

[0007] (1) Preparation of substrate solution and pectinase solution: Prepare substrate solution with pectin as substrate: Dissolve pectin in buffer or water, adjust the pH value to the pH value to be tested for pectinase, and then make up the volume with buffer solution of the pH value to be tested.

[0008] Alternatively, a substrate solution can be prepared using polygalacturonic acid as a substrate: dissolve polygalacturonic acid in an alkaline solution, adjust the pH to the desired pH value for pectinase, and then bring the solution to volume with a buffer solution at the desired pH value. The alkaline solution contains OH-... - The concentration is 0.02-0.05 mol / L;

[0009] Prepare the pectinase solution using a buffer solution with the same pH value as the one to be tested. Specifically, the buffer solution used to prepare the pectinase solution must be exactly the same as the buffer solution used to prepare the substrate solution in terms of type, concentration, and pH value. It is best to choose a buffer solution with good stability and minimize the operation time to ensure that the type, concentration, and pH value of the buffer solution are exactly the same when preparing the pectinase solution and the substrate solution, so as to eliminate any possible influence on the test results.

[0010] (2) Plot the standard correspondence curve between galacturonic acid absorbance value and galacturonic acid concentration:

[0011] a. Preparation of galacturonic acid standard solutions of different concentrations: Using the substrate solution prepared in step (1), a series of galacturonic acid standard solutions of different concentration gradients are prepared, wherein the concentration of the substrate in the series of galacturonic acid standard solutions of different concentration gradients is the same, and the concentration of galacturonic acid is in the range of 0-0.3 μmol / mL.

[0012] b. Preparation of alkaline mixtures: Alkaline reagents are added to a series of galacturonic acid standard solutions of varying concentrations to obtain alkaline mixtures. All alkaline mixtures contain the same concentration of alkaline reagent, and the OH- concentration is... - The concentration is in the range of 0.25-0.5 mol / L;

[0013] c. Precipitation: One method involves placing the alkaline mixture obtained in step b at 0-15°C to precipitate the precipitate, then removing it, mixing it thoroughly, and allowing it to stand until the precipitate separates into layers. This method is suitable for alkaline mixtures with an OH content of [missing value]. - The concentration is 0.25-0.5 mol / L; another method is to let the alkaline mixture stand at 15-35℃ until precipitation and separation occur. This method is suitable for alkaline mixtures with an OH concentration of 0.25-0.5 mol / L. - The concentration is 0.325-0.5 mol / L; the former requires a lower alkali concentration and a shorter precipitation time, while the latter requires a higher alkali concentration and a longer precipitation time.

[0014] d. Centrifugation: The alkaline mixture from step c is centrifuged to obtain the supernatant;

[0015] e. MBTH reaction: Add freshly prepared MBTH reagent to the supernatant from step d. The initial concentration of MBTH is 0.3-4.3 mmol / L, and the initial concentration of DTT is 0-1.6 mmol / L. React at 50-100℃ for 5-180 min. Then add acidic iron reagent, and the reaction system becomes acidic. The H+ in the system... + The concentration range is 0.03-0.3 mol / L, Fe 3+The initial concentration range is 3-10 mmol / L. A colorimetric reaction occurs, and after the colorimetric reaction stabilizes, the absorbance value is measured in the wavelength range of 580-680 nm.

[0016] The MBTH reagent is an aqueous solution prepared from 3-methyl-2-benzothiazolinone hydrazone (MBTH) and dithiothreitol (DTT), or an aqueous solution prepared from MBTH alone. The acidic iron reagent is composed of soluble Fe... 3+ An aqueous solution prepared from salt and strong acid;

[0017] "The initial concentration range of MBTH is 0.3-4.3 mmol / L, and the initial concentration range of DTT is 0-1.6 mmol / L." This means that the concentration of MBTH reagent described here refers to the initial concentration at the moment of addition of MBTH reagent before the reaction has started.

[0018] Fe 3+ The initial concentration range is 3-10 mmol / L. This can be understood as Fe... 3+ The concentration describes the initial concentration at the moment the acidic iron reagent is added, before any reaction has occurred.

[0019] Unreacted MBTH reagent with Fe 3+ The reaction needs to occur in an acidic environment, as mentioned above, H + The concentration describes the H+ after acid-base neutralization following the addition of acidic ferric reagent. + concentration.

[0020] f. Plot the standard correspondence curve: Plot the standard correspondence curve based on the relationship between the galacturonic acid concentration and the measured absorbance value for each group;

[0021] (3) Enzymatic hydrolysis of pectinase: Add the substrate solution prepared in step (1) to the pectinase solution to ensure that the concentration of the substrate in the resulting solution is the same as the concentration of the substrate in the galacturonic acid standard solution in step (2), and then carry out the enzymatic hydrolysis reaction. Then add an alkaline reagent to terminate the enzymatic hydrolysis reaction and obtain an alkaline enzymatic hydrolysate. The concentration of the alkaline reagent in the alkaline enzymatic hydrolysate is the same as the concentration of the alkaline reagent in the alkaline mixture in step b.

[0022] (4) Prepare an alkaline enzymatic hydrolysis blank solution;

[0023] (5) Prepare the supernatant of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysate blank solution: Obtain the supernatant of alkaline enzymatic hydrolysate in step (3) and the supernatant of alkaline enzymatic hydrolysate blank solution in step (4) by referring to the methods in steps c and d in step (2).

[0024] (6) Determine the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysate blank solution: Perform the MBTH reaction according to the method in step e of step (2), and determine the supernatant of the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysate blank solution respectively, so as to obtain the concentration of the reducing end group generated by enzymatic hydrolysis, and then obtain the pectinase activity.

[0025] Prior Art MBTH method (Lv The measurement wavelength used in Chemistry, 2021, 343:128532.) is 590 nm, and the intercept of the linear equation of the standard curve is close to 0. The optimal measurement wavelength for galacturonic acid is also 590 nm. However, since the enzyme digest contains substrate, the intercept of the linear equation of the standard curve obtained by preparing a series of galacturonic acid standard solutions with different concentration gradients using substrate solution deviates from 0 at 590 nm. The intercept of the linear equation of the standard curve measured at 630-655 nm is closer to 0. The accuracy of pectinase activity measurement will not be affected by different dilution factors of pectinase. Therefore, we choose the standard curve equation obtained by preparing a series of galacturonic acid standard solutions with different concentration gradients using substrate solution at 630-655 nm for calculating enzyme activity.

[0026] The pectinase described in this invention is a collective term for a series of enzymes that degrade pectin, and its cleavage site is the α-1,4 glycosidic bond in the pectin molecule.

[0027] Preferably, in step (1), the solvents used to prepare the substrate solution and pectinase solution are the same type, concentration, and pH value of the same buffer solution. The buffer pH range can be acidic or alkaline. Preferably, the buffer solution is an acetate buffer, glycine buffer, borate buffer, succinate buffer, or phosphate buffer. More preferably, it is an acetate-sodium acetate buffer, glycine-NaOH buffer, borate-sodium borate buffer, succinate-sodium succinate buffer, or phosphate-sodium phosphate buffer.

[0028] Preferably, the concentration of the substrate solution obtained in step (1) is 1-10 mg / mL, and the substrate solution needs to be prepared on the same day.

[0029] Preferably, the alkaline solution used in step (1) is a NaOH solution or a KOH solution.

[0030] Preferably, in steps (2), (3), and (4), the alkaline reagent used is NaOH solution or KOH solution, preferably NaOH solution, alkaline mixture, alkaline enzymatic hydrolysate, or alkaline enzymatic hydrolysate blank solution containing OH. - The concentration is 0.25-0.5 mol / L. The main structural component of the substrate solution, polygalacturonic acid, reacts under alkaline conditions (e.g., the OH- in the above solution). - At a concentration of 0.25 mol / L, it can be converted into polygalacturonate, thus accelerating its dissolution. However, at this time, the colorimetric product of the MBTH reaction produces insoluble substances, and the background value is very high. However, this invention has found that high concentrations of alkali (e.g., OH- in the above solution) can also accelerate its dissolution. - At a concentration of 0.375 mol / L, pectinase promotes the enzymatic hydrolysis blank and the precipitation of the hydrolysate. After removing these insoluble substances by centrifugation, the MBTH reaction was performed. The resulting solution not only showed no precipitation but also a significant decrease in absorbance, avoiding interference from large-molecule pectin in the assay; and at the same OH concentration... - At the same precipitation time at different concentrations, pectin concentration and absorbance values ​​showed a linear increase, indicating that the molecular weight of the precipitate was independent of pectin concentration. Furthermore, if the precipitate was fully precipitated, the molecular weight of the precipitate was affected by OH... - Concentration has a relatively small effect, but the precipitation rate will be affected by OH. - The rate increases with increasing concentration;

[0031] Preferably, in steps (2), (3) and (4), the precipitation and standing time of the alkaline mixture, alkaline enzymatic hydrolysate, and alkaline enzymatic hydrolysate blank solution is 30 min to 180 min; the precipitation time of step c in step (2) is consistent with the precipitation time in step (5).

[0032] Preferably, in steps (2) c and (5), there are two methods for treating the alkaline mixture, alkaline enzymatic hydrolysate, and alkaline enzymatic blank solution: one method is to first precipitate the alkaline mixture, alkaline enzymatic hydrolysate, or alkaline enzymatic blank solution in an ice-water bath at 0-8℃ for 10-30 minutes, then remove it, mix it well, and then let it stand at 20-30℃ for 30-150 minutes until it separates into layers. At this time, the OH- in the alkaline mixture, alkaline enzymatic hydrolysate, or alkaline enzymatic blank solution will be reduced. - The concentration is 0.3-0.35 mol / L; another method is to directly let the alkaline mixture, alkaline enzymatic hydrolysate, or alkaline enzymatic hydrolysis blank solution stand at 20-30℃ for 30-180 min until precipitation and separation. At this time, the OH in the alkaline mixture, alkaline enzymatic hydrolysate, or alkaline enzymatic hydrolysis blank solution is reduced. -The concentration is 0.35-0.425 mol / L. Ice bath conditions can promote precipitate formation, such as in OH... - At a concentration of 0.25 mol / L, a small amount of precipitate formed after 30 minutes in an ice bath, but no precipitation occurred at room temperature (25°C), even after standing for 24 hours. Treatment at room temperature should be adapted to higher alkali concentrations and longer precipitation times, such as in OH... - At a concentration of 0.375 mol / L, the precipitate precipitates and separates into layers after 90 min at room temperature, and the mixture is basically stable. If the alkali concentration is too high, the viscosity of the alkaline mixture will increase. Ice bath operation can also promote the increase in viscosity. However, the increase in viscosity will affect the MBTH reaction efficiency and make it difficult to transfer the supernatant, which will increase the operation error.

[0033] Preferably, the centrifugation step d is performed by centrifuging at 1500-6500 RCF for 5-15 minutes, and the system is mixed before centrifugation to ensure uniformity.

[0034] Preferably, the soluble Fe in the acidic iron reagent 3+ The salt is at least one of ferric ammonium sulfate, ferric chloride, and ferric nitrate.

[0035] Preferably, the acidic iron reagent may contain at least one of hydrochloric acid, aminosulfonic acid, and sulfuric acid as the strong acid. More preferably, the acidic iron reagent is prepared from ferric ammonium sulfate, aminosulfonic acid, and hydrochloric acid.

[0036] Preferably, in step e, after adding MBTH reagent to the supernatant from step d, the initial concentration of MBTH in the system is in the range of 0.7-3.6 mmol / L, more preferably 1.5-2.5 mmol / L; the initial concentration of DTT is in the range of 0.5-1.1 mmol / L, more preferably 0.5-0.7 mmol / L; then, acidic iron reagent is added, and the Fe concentration in the system is... 3+ The initial concentration range is 5-7 mmol / L, more preferably 5.5-6.5 mmol / L; H + The concentration range is 0.07-0.2 mol / L, more preferably 0.12-0.18 mol / L.

[0037] Preferably, in step e, acidic iron reagent is added while hot, cooled to room temperature, and the absorbance value is measured after the color development is stable.

[0038] MBTH reagent should be prepared fresh and is effective within one day. In some embodiments of the present invention, the concentration of MBTH in the prepared MBTH reagent is 2.8-10.7 mmol / L, and the concentration of DTT is 1.6-3.2 mmol / L; the concentration of ferric ammonium sulfate in the prepared acidic iron reagent is 7.5-20 mmol / L, the concentration of aminosulfonic acid is 0.175-0.3 mol / L, and the concentration of hydrochloric acid is 0.5-0.6 mol / L.

[0039] Preferably, the volume ratio of the supernatant, MBTH reagent, and acidic iron reagent in step e is 1:(0.3-0.7):(0.8-1.2).

[0040] Preferably, in step (2), the reaction temperature of the supernatant in step d with MBTH reagent is 75-85℃ and the reaction time is 9-17 min.

[0041] Preferably, in step (2), the color development time is 50-190 min, more preferably 80-180 min, and the absorbance value is measured within this color development time.

[0042] Preferably, the wavelength for measuring the absorbance value in step (2) is 590-670 nm, more preferably 630-655 nm.

[0043] Preferably, in step (3), pectinase solution is added to the substrate solution. The enzymatic hydrolysis temperature is preferably 30-60℃, and the enzymatic hydrolysis time is preferably 1-60 min, more preferably 3-40 min, and even more preferably 10-30 min. As long as the enzymatic hydrolysis reaction occurs, the enzyme activity can be determined using the method of the present invention. A longer enzymatic hydrolysis time is beneficial for large-scale determination, but it should not be too long to avoid a decrease in enzyme activity during the hydrolysis process. The enzymatic hydrolysis temperature should not be too high to avoid non-enzymatic degradation of the substrate. Preferably, the enzymatic hydrolysis reaction is carried out at 37℃ for 10-30 min.

[0044] Preferably, the absorbance value is measured using an enzyme-linked immunosorbent assay (ELISA) reader or a UV-Vis spectrophotometer.

[0045] The method for preparing the alkaline enzymatic hydrolysis blank solution in step (4) is specifically the same as step (3), except that the alkaline reagent is added first, and then the pectinase solution is added to obtain the alkaline enzymatic hydrolysis blank solution.

[0046] Preferably, in steps (3) and (4), the pectinase is subjected to enzymatic hydrolysis at 37°C. Taking a hydrolysis reaction time of 30 min as an example, the concentration of pectinase in the hydrolysis reaction system is in the range of 0-14 mU / mL.

[0047] The present invention has the following beneficial effects:

[0048] 1. The method of the present invention is simple to operate and rapid to measure, and is suitable for high-throughput analysis of large-scale determination, which is far superior to the hypoiodite method; the method of the present invention has high sensitivity and can accurately measure the initial rate of pectinase hydrolysis, which is far superior to the DNS method.

[0049] 2. This invention solves the problems of excessively high background values ​​and precipitation of colorimetric products in the MBTH method for determining lysozyme activity in the prior art.

[0050] 3. Compared with the prior art, the intercept of the galacturonic acid standard curve obtained by the MBTH method of the present invention is closer to 0, which is more in line with Beer-Lambert law. It can more accurately detect the concentration of reducing end groups generated by enzymatic hydrolysis, and will not affect the accuracy of enzyme activity determination due to different dilution factors of enzyme solution.

[0051] 4. The method of this invention can be used for product quality control in enzyme preparation manufacturing enterprises. Attached Figure Description

[0052] Figure 1 The diagram shows a comparison of the galacturonic acid standard curves (cuvettes) of the method of the present invention and the prior art DNS method. The intercept of the standard curve of the prior art DNS method deviates from 0, making it difficult to accurately measure when the galacturonic acid concentration is low. The intercept of the standard curve of the method of the present invention is close to 0, and it can still accurately measure when the galacturonic acid concentration is low. Moreover, the method of the present invention has a higher slope and is more sensitive.

[0053] Figure 2 The graph shows a comparison of the precipitation and background values ​​of the colorimetric products under acidic enzymatic hydrolysis conditions (Figure a) and alkaline enzymatic hydrolysis conditions (Figure b) in the existing MBTH method and the method of the present invention. The existing MBTH method produces precipitation of colorimetric products under both acidic and alkaline enzymatic hydrolysis conditions, and the background value is high. The method of the present invention produces no precipitation of colorimetric products under both acidic and alkaline enzymatic hydrolysis conditions, and the background value is low.

[0054] Figure 3This is a comparison of the galacturonic acid standard curves of the method of the present invention at different measurement wavelengths (the measurement wavelengths in the upper, middle, and lower graphs are 590 nm, 630 nm, and 655 nm, respectively, measured using cuvettes; the graphs with and without a substrate indicate the presence and absence of the substrate in the galacturonic acid solution, respectively). When the galacturonic acid solution does not contain the substrate, the intercept of the linear equation of the standard curve deviates from 0 at 630 nm and 655 nm, while it is closer to 0 at 590 nm. When the galacturonic acid solution contains the substrate, the intercept of the linear equation of the standard curve obtained deviates from 0 at 590 nm, while the intercepts of the linear equation of the standard curves measured at 630 nm and 655 nm are closer to 0. Therefore, when the galacturonic acid solution does not contain the substrate, measurement at 590 nm is better, and when the galacturonic acid solution contains the substrate, measurement at 630 nm or 655 nm is better.

[0055] Figure 4 The images show the results of precipitating pectinase ① hydrolysate with different NaOH concentrations for 180 min at room temperature (25℃). (In the images, 0, 1.5, and 3 represent the concentrations of pectinase ① in the hydrolysis system, in μg / mL, where the concentration of pectin ① in the hydrolysis system is 2.5 mg / mL.) In the image, a represents the alkaline hydrolysate of the existing MBTH method with an alkaline concentration of 0.25 mol / L, in which no precipitation occurred. In the image, b represents the alkaline hydrolysate with an alkaline concentration of 0.375 mol / L, in which obvious precipitation occurred. This indicates that precipitation is difficult to observe at the alkaline concentrations used in the existing technology. However, under high alkaline concentrations, pectin will precipitate insoluble substances with the alkaline reagent.

[0056] Figure 5 The graph shows the relationship between pectin concentration and absorbance. Under the same NaOH concentration and for the same precipitation time, the pectin concentration and absorbance increase linearly, indicating that the molecular weight of the precipitate is independent of the pectin concentration.

[0057] Figure 6 The graph shows the relationship between NaOH concentration and absorbance under different pectinase ① enzyme concentrations (0, 1.5, 3, 4.5, and 5 in the graph represent the enzyme concentration in the enzymatic hydrolysis system, in μg / mL; the concentration of pectin ① in the enzymatic hydrolysis system is 2.5 mg / mL; and the NaOH concentration is the concentration in the alkaline enzymatic hydrolysate). Under the condition that the precipitate is fully separated, the absorbance value changes little with the NaOH concentration, indicating that the molecular weight of the precipitate is less affected by the NaOH concentration.

[0058] Figure 7The precipitation results of pectinase ① hydrolysate (enzyme concentration of 0 μg / mL and pectin ① concentration of 2.5 mg / mL) under different NaOH concentrations for 60 min. The higher the NaOH concentration, the more precipitate appears in the same time period, that is, the faster the precipitation rate. The NaOH concentration is the concentration in the alkaline hydrolysate.

[0059] Figure 8 The images show the precipitation of pectin ① solution (2.5 mg / mL) in the alkaline mixed solution of the existing MBTH method with a NaOH concentration of 0.25 mol / L for 30 minutes at room temperature (Figure a) and in an ice bath (Figure b). No precipitate was observed in Figure a, while a small amount of precipitate was observed in Figure b, indicating that the ice bath condition can promote precipitation.

[0060] Figure 9 The images show the precipitation conditions and precipitation effects of the method of the present invention on different substrates. From left to right, they are pectin ① (Sigma-Aldrich Trading Co., Ltd.), pectin ② (Shanghai Aladdin Biochemical Technology Co., Ltd.), pectin ③ (Shanghai Maclean Biochemical Technology Co., Ltd.), and polygalacturonic acid (Sigma-Aldrich Trading Co., Ltd.). Under the precipitation conditions of the method of the present invention, precipitation and stratification are produced in all of them.

[0061] Figure 10 This invention presents standard curves of galacturonic acid under different types of soluble iron salts, along with their slopes, intercepts, and R values. 2 The graphs show the relationship between galacturonic acid and time. The top left graph shows the standard curves for galacturonic acid under different types of soluble iron salts. All three types of soluble iron salts can be used for pectinase activity determination. The top right graph shows the slope of the galacturonic acid standard curves for different types of soluble iron salts as a function of time. The bottom left graph shows the intercept of the galacturonic acid standard curves for different types of soluble iron salts as a function of time. The bottom right graph shows the R-squared value of the galacturonic acid standard curves for different types of soluble iron salts. 2 Over time, the standard curve intercept obtained when the soluble iron salt is ferric ammonium sulfate is closer to 0, and the slope, intercept, and R0 are all more consistent. 2 It has good time stability.

[0062] Figure 11 The figures above show the reaction kinetics curve of pectinase ① using the MBTH method of this invention (top figure, representing the zero-order reaction) and the reaction rate curve of enzyme mass concentration (bottom figure, showing a linear curve). In the bottom figure, "pectinase concentration" refers to the concentration of pectinase in the enzymatic hydrolysis reaction system.

[0063] Figure 12 The figures show the reaction kinetics curve of pectinase ① in the DNS method (top figure, which is a non-zero order reaction) and the reaction rate curve of enzyme mass concentration (bottom figure, which is not linear). In the figure below, "pectinase concentration" refers to the concentration of pectinase in the enzymatic hydrolysis reaction system. Detailed Implementation

[0064] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0065] The main instruments and reagents used in the following examples are as follows:

[0066] Main instruments: Water bath constant temperature shaker, SHZ-82 type, Changzhou Zhiborui Co., Ltd.; FE2 pH meter, Mettler Toledo Instruments Co., Ltd.; Bio-rad 680 microplate reader, Bio-Rad Biomedical Products (Shanghai) Co., Ltd.; UV-2600 UV-Vis spectrophotometer, SHIMADZU; AL204 electronic balance, Mettler Toledo Instruments Co., Ltd.; CT14RD centrifuge, Shanghai Tianmei Biochemical Instrument Equipment Engineering Co., Ltd.

[0067] Main reagents: Galacturonic acid standard was purchased from Sigma-Aldrich Trading Co., Ltd.; Pectin ① was purchased from Sigma-Aldrich Trading Co., Ltd., Pectin ② from Shanghai Aladdin Biochemical Technology Co., Ltd., and Pectin ③ from Shanghai Maclean Biochemical Technology Co., Ltd.; Polygalacturonic acid was purchased from Sigma-Aldrich Trading Co., Ltd.; Pectinase ① (acidic pectinase) was purchased from Sigma-Aldrich Trading Co., Ltd., Pectinase ② (alkaline pectinase) was purchased from Wuhan Xinhua Yang Biotechnology Co., Ltd., and Pectinase ③ was purchased from Qingdao Weilan Biotechnology Co., Ltd. Alkaline pectinase was obtained from Sinopharm Chemical Reagent Co., Ltd.; 3-methyl-2-benzothiazolinone hydrochloride monohydrate (MBTH) was obtained from Sigma-Aldrich Trading Co., Ltd.; DL-dithiothreitol (DTT) was obtained from Sigma-Aldrich Trading Co., Ltd.; aminosulfonic acid, (FeNH4(SO4)2)·12H2O, FeCl3, Fe(NO3)3·9H2O, and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0068] The enzyme activity calculation formula in the following examples is as follows:

[0069]

[0070] In the formula, U-pectinase activity is expressed in μmol / mL / min;

[0071] Absorbance value of A-enzyme hydrolysate;

[0072] A0 - Absorbance value of the enzymatically digested blank solution;

[0073] Intercept of the β-galacturonic acid standard curve;

[0074] The slope of the k-galacturonic acid standard curve, in mL / μmol;

[0075] t-Enzymatic hydrolysis time (min).

[0076] Example 1:

[0077] Step (1) Prepare a 5 mg / mL substrate solution: First, wet the accurately weighed pectin ① with a small amount of 0.1 mol / L pH 4.0 acetate-sodium acetate buffer, then slowly add the buffer to make the pectin swell and dissolve into a paste without any lumps or insoluble matter. Adjust the pH value to 4.0 with sodium acetate, and then make up the volume with 0.1 mol / L pH 4.0 acetate-sodium acetate buffer.

[0078] Step (2) Plot the standard correlation curve between galacturonic acid absorbance and galacturonic acid concentration: Take a portion of the substrate solution prepared in step (1) and mix it 1:1 with galacturonic acid solutions of different concentrations to prepare a series of galacturonic acid standard solutions with a concentration range of 0-60 μg / mL (equivalent to 0-0.28 μmol / mL). Add 800 μL of 0.65 mol / L NaOH solution to 800 μL of the above galacturonic acid standard solutions of different concentrations respectively. The resulting alkaline mixture contains OH... - The concentration was 0.325 mol / L. After rapid mixing, it was placed in an ice bath at 5°C for 20 min, then removed and mixed again. After precipitation, it was allowed to stand at room temperature (25°C) for 40 min, and centrifuged at 6000 rpm (3440 RCF) for 15 min (mixed before centrifugation). 800 μL of the supernatant was collected. 400 μL of freshly prepared MBTH reagent, which is made by mixing equal volumes of 3 mg / mL (12.8 mmol / L) MBTH solution and 0.5 mg / mL (3.2 mmol / L) DTT solution, was added to the supernatant. At this point, the initial concentration of MBTH in the reaction system was 2.13 mmol / L, and the initial concentration of DTT was 0.5 mmol / L. The mixture was heated in a water bath at 80°C for 13 minutes. Then, while still hot, 800 μL of an acidic iron reagent consisting of 15 mmol / L (FeNH4(SO4)2)·12H2O, 0.2 mol / L aminosulfonic acid, and 0.5 mol / L hydrochloric acid was added. At this point, the H2O concentration in the reaction system was... + The concentration is 0.15 mol / L, Fe 3+The initial concentration was 6 mmol / L. After cooling to room temperature, the solution was developed for 90 min, thoroughly mixed, and 200 μL was added to the wells of the microplate. After shaking the plate, the absorbance of galacturonic acid was measured at 655 nm using a microplate reader. Three replicates were prepared for each concentration. A standard correlation curve was plotted based on the relationship between the concentration of galacturonic acid solution and the corresponding absorbance value.

[0079] Step (3) Determine pectinase activity: Add 400 μL of the substrate solution from step (1) to a test tube and preheat in a water bath shaker at 37°C for 5 min. Then add 400 μL of pectinase ① solution (2 μg / mL, prepared with 0.1 mol / L pH 4.0 acetate-sodium acetate buffer) preheated to the same temperature, mix quickly to start the enzymatic hydrolysis reaction, and after 30 min, quickly add 800 μL of 0.65 mol / L NaOH solution and mix thoroughly to terminate the enzyme reaction and obtain an alkaline enzymatic hydrolysate.

[0080] Add 400 μL of the substrate solution from step (1) into a test tube and preheat it in a water bath shaker at 37°C for 5 min. Then continue to keep it warm for 30 min. Then quickly add 800 μL of 0.65 mol / L NaOH solution and mix thoroughly immediately. Add 400 μL of the same pectinase solution and mix thoroughly to obtain an alkaline enzymatic hydrolysis blank solution.

[0081] Step (4) Determine the absorbance values ​​of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution: According to the method in step (2), the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are subjected to precipitation and centrifugation and MBTH reaction, and the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are measured to obtain the concentration of the reducing end group generated by enzymatic hydrolysis, and then obtain the pectinase activity.

[0082] Table 1 Results of pectinase activity assay in Example 1

[0083]

[0084] Example 2:

[0085] Step (1) Prepare a 5 mg / mL substrate solution: Dissolve the accurately weighed pectin ① in a small amount of 0.1 mol / L pH 4.0 acetate-sodium acetate buffer, adjust the pH to 4.0 with sodium acetate, and then make up the volume with 0.1 mol / L pH 4.0 acetate-sodium acetate buffer.

[0086] Step (2) Plot the standard correlation curve between galacturonic acid absorbance and galacturonic acid concentration: Take a portion of the substrate solution prepared in step (1) and mix it 1:1 with galacturonic acid solutions of different concentrations to prepare a series of galacturonic acid standard solutions with a concentration range of 0-60 μg / mL (equivalent to 0-0.28 μmol / mL). Add 800 μL of 0.7 mol / L NaOH solution to 800 μL of the above galacturonic acid standard solutions of different concentrations respectively. The resulting alkaline mixture contains OH... - The concentration was 0.35 mol / L. After rapid mixing, the mixture was allowed to stand at room temperature (25°C) for 1.5 h to precipitate. It was then centrifuged at 6000 rpm (3440 RCF) for 15 min (mixed before centrifugation), and 800 μL of the supernatant was collected. 400 μL of freshly prepared MBTH reagent, consisting of equal volumes of 3 mg / mL (12.8 mmol / L) MBTH solution and 0.5 mg / mL (3.2 mmol / L) DTT solution, was added to the supernatant. At this point, the initial concentration of MBTH in the reaction system was 2.13 mmol / L, and the initial concentration of DTT was 0.5 mmol / L. The mixture was heated in an 80°C water bath for 13 min, and then 800 μL of acidic iron reagent, consisting of 12.5 mmol / L (FeNH4(SO4)2)·12H2O, 0.225 mol / L aminosulfonic acid, and 0.5 mol / L hydrochloric acid, was added while hot. At this point, the H+ concentration in the reaction system was... + The concentration is 0.15 mol / L, Fe 3+ The initial concentration was 5 mmol / L. After cooling to room temperature, the solution was developed for 90 min, thoroughly mixed, and 200 μL was added to the wells of the microplate. After shaking the plate, the absorbance of galacturonic acid was measured at 655 nm using a microplate reader. Three replicates were prepared for each concentration. A standard correlation curve was plotted based on the relationship between the concentration of galacturonic acid solution and the corresponding absorbance value.

[0087] Step (3) Determine pectinase activity: Add 400 μL of the substrate solution from step (1) to a test tube and preheat in a water bath shaker at 37°C for 5 min. Then add 400 μL of pectinase ① solution (2 μg / mL, prepared with 0.1 mol / L pH 4.0 acetate-sodium acetate buffer) preheated to the same temperature, mix quickly to start the enzymatic hydrolysis reaction, and after 30 min, quickly add 800 μL of 0.7 mol / L NaOH solution and mix thoroughly to terminate the enzyme reaction and obtain an alkaline enzymatic hydrolysate.

[0088] Add 400 μL of the substrate solution from step (1) into a test tube and preheat it in a water bath shaker at 37°C for 5 min. Then continue to keep it warm for 30 min. Then quickly add 800 μL of 0.7 mol / L NaOH solution and mix thoroughly immediately. Add 400 μL of the same pectinase solution and mix thoroughly to obtain an alkaline enzymatic hydrolysis blank solution.

[0089] Step (4) Determine the absorbance values ​​of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution: According to the method in step (2), the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are subjected to precipitation and centrifugation and MBTH reaction, and the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are measured to obtain the concentration of the reducing end group generated by enzymatic hydrolysis, and then obtain the pectinase activity.

[0090] Table 2 Results of pectinase activity assay in Example 2

[0091]

[0092] Example 3:

[0093] Step (1) Prepare a 5 mg / mL substrate solution: Weigh the pectin ① accurately, first soak it in a small amount of 0.1 mol / L pH 4.0 acetate-sodium acetate buffer, then slowly add the buffer to make the pectin swell and dissolve into a paste without any lumps or insoluble matter. Adjust the pH value to 4.0 with sodium acetate, and then make up the volume with 0.1 mol / L pH 4.0 acetate-sodium acetate buffer.

[0094] Step (2) Plot the standard correlation curve between galacturonic acid absorbance and galacturonic acid concentration: Take a portion of the substrate solution prepared in step (1) and mix it 1:1 with galacturonic acid solutions of different concentrations to prepare a series of galacturonic acid standard solutions with a concentration range of 0-60 μg / mL (equivalent to 0-0.28 μmol / mL). Add 800 μL of 0.6 mol / L NaOH solution to 800 μL of the above galacturonic acid standard solutions of different concentrations respectively. The resulting alkaline mixture contains OH... -The concentration was 0.3 mol / L. After rapid mixing, it was placed in an ice bath at 5℃ for 20 min, then removed and mixed again. The mixture was allowed to stand at room temperature (25℃) for 70 min to precipitate. It was then centrifuged at 6000 rpm (3440 RCF) for 15 min (mixed before centrifugation), and 800 μL of the supernatant was collected. 400 μL of freshly prepared MBTH reagent, consisting of equal volumes of 3 mg / mL (12.8 mmol / L) MBTH solution and 0.5 mg / mL (3.2 mmol / L) DTT solution, was added to the supernatant. At this point, the initial concentration of MBTH in the reaction system was 2.13 mmol / L, and the initial concentration of DTT was 0.5 mmol / L. The mixture was heated in an 80℃ water bath for 13 min, and then 800 μL of acidic iron reagent, consisting of 15 mmol / L FeCl3, 0.175 mol / L aminosulfonic acid, and 0.5 mol / L hydrochloric acid, was added while hot. At this point, the H+ concentration in the reaction system was... + The concentration is 0.15 mol / L, Fe 3+ The initial concentration was 6 mmol / L. After cooling to room temperature, the solution was developed for 90 min, thoroughly mixed, and 200 μL was added to the wells of the microplate. After shaking the plate, the absorbance of galacturonic acid was measured at 630 nm using a microplate reader. Three replicates were prepared for each concentration. A standard correlation curve was plotted based on the relationship between the concentration of galacturonic acid solution and the corresponding absorbance value.

[0095] Step (3) Determine pectinase activity: Add 400 μL of the substrate solution from step (1) to a test tube and preheat in a water bath shaker at 37°C for 5 min. Then add 400 μL of pectinase ① solution (2 μg / mL, prepared with 0.1 mol / L pH 4.0 acetate-sodium acetate buffer) preheated to the same temperature, mix quickly to start the enzymatic hydrolysis reaction, and after 30 min, quickly add 800 μL of 0.6 mol / L NaOH solution and mix thoroughly to terminate the enzyme reaction and obtain an alkaline enzymatic hydrolysate.

[0096] Add 400 μL of the substrate solution from step (1) into a test tube and preheat it in a water bath shaker at 37°C for 5 min. Then continue to keep it warm for 30 min. Then quickly add 800 μL of 0.6 mol / L NaOH solution and mix thoroughly immediately. Add 400 μL of the same pectinase solution and mix thoroughly to obtain an alkaline enzymatic blank solution.

[0097] Step (4) Determine the absorbance values ​​of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution: According to the method in step (2), the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are subjected to precipitation and centrifugation and MBTH reaction, and the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are measured to obtain the concentration of the reducing end group generated by enzymatic hydrolysis, and then obtain the pectinase activity.

[0098] Table 3 Results of pectinase activity assay in Example 3

[0099]

[0100]

[0101] Example 4:

[0102] Step (1) Prepare a 5 mg / mL substrate solution: Weigh the pectin ① accurately, first soak it in a small amount of 0.1 mol / L pH 4.0 acetate-sodium acetate buffer, then slowly add the buffer to make the pectin swell and dissolve into a paste without any lumps or insoluble matter. Adjust the pH value to 4.0 with sodium acetate, and then make up the volume with 0.1 mol / L pH 4.0 acetate-sodium acetate buffer.

[0103] Step (2) Plot the standard correlation curve between galacturonic acid absorbance and galacturonic acid concentration: Take a portion of the substrate solution prepared in step (1) and mix it 1:1 with galacturonic acid solutions of different concentrations to prepare a series of galacturonic acid standard solutions with a concentration range of 0-60 μg / mL (equivalent to 0-0.28 μmol / mL). Add 800 μL of 0.85 mol / L NaOH solution to 800 μL of the above galacturonic acid standard solutions of different concentrations respectively. The resulting alkaline mixture contains OH... - The concentration was 0.425 mol / L. After rapid mixing, the mixture was allowed to stand at room temperature (25°C) for 1 hour to precipitate. It was then centrifuged at 6000 rpm (3440 RCF) for 15 minutes (mixed before centrifugation), and 800 μL of the supernatant was collected. 400 μL of freshly prepared MBTH reagent, consisting of equal volumes of 3 mg / mL (12.8 mmol / L) MBTH solution and 0.5 mg / mL (3.2 mmol / L) DTT solution, was added to the supernatant. At this point, the initial concentration of MBTH in the reaction system was 2.13 mmol / L, and the initial concentration of DTT was 0.5 mmol / L. The mixture was heated in an 80°C water bath for 13 minutes, and then 800 μL of acidic iron reagent, consisting of 15 mmol / L Fe(NO3)3·9H2O, 0.3 mol / L aminosulfonic acid, and 0.5 mol / L hydrochloric acid, was added while hot. At this point, the H+ concentration in the reaction system was... + The concentration is 0.15 mol / L, Fe 3+ The initial concentration was 6 mmol / L. After cooling to room temperature, the solution was developed for 90 min, thoroughly mixed, and 200 μL was added to the wells of the microplate. After shaking the plate, the absorbance of galacturonic acid was measured at 590 nm using a microplate reader. Three replicates were prepared for each concentration. A standard correlation curve was plotted based on the relationship between the concentration of galacturonic acid solution and the corresponding absorbance value of galacturonic acid.

[0104] Step (3) Determine pectinase activity: Add 400 μL of the substrate solution from step (1) to a test tube and preheat in a water bath shaker at 37°C for 5 min. Then add 400 μL of pectinase ① solution (2 μg / mL, prepared with 0.1 mol / L pH 4.0 acetate-sodium acetate buffer) preheated to the same temperature, mix quickly to start the enzymatic hydrolysis reaction, and after 30 min, quickly add 800 μL of 0.85 mol / L NaOH solution and mix thoroughly to terminate the enzyme reaction and obtain an alkaline enzymatic hydrolysate.

[0105] Add 400 μL of the substrate solution from step (1) into a test tube and preheat it in a water bath shaker at 37°C for 5 min. Then continue to keep it warm for 30 min. Then quickly add 800 μL of 0.85 mol / L NaOH solution and mix thoroughly immediately. Add 400 μL of the same pectinase solution and mix thoroughly to obtain an alkaline enzymatic blank solution.

[0106] Step (4) Determine the absorbance values ​​of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution: According to the method in step (2), the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are subjected to precipitation and centrifugation and MBTH reaction, and the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are measured to obtain the concentration of the reducing end group generated by enzymatic hydrolysis, and then obtain the pectinase activity.

[0107] Table 4 Results of pectinase activity assay in Example 4

[0108]

[0109] Example 5:

[0110] Step (1) Prepare a 5 mg / mL substrate solution: First, wet the accurately weighed pectin ② with a small amount of 0.1 mol / L pH 9.8 glycine-NaOH buffer solution, then slowly add the buffer solution to make the pectin swell and dissolve into a paste without any lumps or insoluble matter. Adjust the pH value to 9.8 with NaOH, and then make up the volume with 0.1 mol / L pH 9.8 glycine-NaOH buffer solution.

[0111] Step (2) Plot the standard correlation curve between galacturonic acid absorbance and galacturonic acid concentration: Take a portion of the substrate solution prepared in step (1) and mix it 1:1 with galacturonic acid solutions of different concentrations to prepare a series of galacturonic acid standard solutions with a concentration range of 0-60 μg / mL (equivalent to 0-0.28 μmol / mL). Add 800 μL of 0.65 mol / L NaOH solution to 800 μL of the above galacturonic acid standard solutions of different concentrations respectively. The resulting alkaline mixture contains OH... -The concentration was 0.325 mol / L. After rapid mixing, it was placed in an ice bath at 5°C for 20 min, then removed and mixed again. After precipitation, it was allowed to stand at room temperature (25°C) for 70 min, and centrifuged at 7000 rpm (4700 RCF) for 10 min (mixed before centrifugation). 800 μL of the supernatant was collected. 400 μL of freshly prepared MBTH reagent, which is a mixture of equal volumes of 3 mg / mL (12.8 mmol / L) MBTH solution and 0.5 mg / mL (3.2 mmol / L) DTT solution, was added to the supernatant. At this point, the initial concentration of MBTH in the reaction system was 2.13 mmol / L, and the initial concentration of DTT was 0.5 mmol / L. The mixture was heated in a water bath at 80°C for 13 minutes. Then, while still hot, 800 μL of an acidic iron reagent consisting of 15 mmol / L (FeNH4(SO4)2)·12H2O, 0.2 mol / L aminosulfonic acid, and 0.5 mol / L hydrochloric acid was added. At this point, the H2O concentration in the reaction system was... + The concentration is 0.15 mol / L, Fe 3+ The initial concentration was 6 mmol / L. After cooling to room temperature, the solution was developed for 90 min, thoroughly mixed, and 200 μL was added to the wells of the microplate. After shaking the plate, the absorbance of galacturonic acid was measured at 655 nm using a microplate reader. Three replicates were prepared for each concentration. A standard correlation curve was plotted based on the relationship between the concentration of galacturonic acid solution and the corresponding absorbance value.

[0112] Step (3) Determine pectinase activity: Add 400 μL of the substrate solution from step (1) to a test tube and preheat in a water bath shaker at 37°C for 5 min. Then add 400 μL of pectinase ③ solution (0.2 μL / mL, prepared with 0.1 mol / L pH 9.8 glycine-NaOH buffer) preheated to the same temperature, mix quickly to start the enzymatic hydrolysis reaction, and after 30 min, quickly add 800 μL of 0.65 mol / L NaOH solution and mix thoroughly to terminate the enzyme reaction and obtain an alkaline enzymatic hydrolysate.

[0113] Add 400 μL of the substrate solution from step (1) into a test tube and preheat it in a water bath shaker at 37°C for 5 min. Then continue to keep it warm for 30 min. Immediately add 800 μL of 0.65 mol / L NaOH solution and mix thoroughly. Add 400 μL of the same pectinase solution and mix thoroughly to obtain an alkaline enzymatic hydrolysis blank solution.

[0114] Step (4) Determine the absorbance values ​​of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution: According to the method in step (2), the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are subjected to precipitation and centrifugation and MBTH reaction, and the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are measured to obtain the concentration of the reducing end group generated by enzymatic hydrolysis, and then obtain the pectinase activity.

[0115] Table 5 Results of pectinase activity assay in Example 5

[0116]

[0117] Example 6:

[0118] Step (1) Prepare a 5 mg / mL substrate solution: Accurately weigh pectin ③, first soak it in a small amount of 0.1 mol / L pH 9.8 glycine-NaOH buffer, then slowly add buffer to make the pectin swell and dissolve into a paste without any lumps or insoluble matter. Adjust the pH value to 9.8 with NaOH, and then make up the volume with 0.1 mol / L pH 9.8 glycine-NaOH buffer.

[0119] Step (2) Plot the standard correlation curve between galacturonic acid absorbance and galacturonic acid concentration: Take a portion of the substrate solution prepared in step (1) and mix it 1:1 with galacturonic acid solutions of different concentrations to prepare a series of galacturonic acid standard solutions with a concentration range of 0-60 μg / mL (equivalent to 0-0.28 μmol / mL). Add 800 μL of 0.7 mol / L NaOH solution to 800 μL of the above galacturonic acid standard solutions of different concentrations respectively. The resulting alkaline mixture contains OH... - The concentration was 0.35 mol / L. After rapid mixing, the solution was placed in an ice bath at 5°C for 20 min. After mixing again, the solution was allowed to stand at room temperature (25°C) for 130 min to precipitate. The solution was then centrifuged at 8000 rpm (6140 RCF) for 15 min (mixed before centrifugation). 800 μL of the supernatant was collected. 400 μL of freshly prepared MBTH reagent, prepared by mixing equal volumes of 3 mg / mL (12.8 mmol / L) MBTH solution and 0.5 mg / mL (3.2 mmol / L) DTT solution, was added to the supernatant. At this point, the initial concentration of MBTH in the reaction system was 2.13 mmol / L, and the initial concentration of DTT was 0.5 mmol / L. The mixture was heated in a water bath at 80°C for 13 minutes. Then, while still hot, 800 μL of an acidic iron reagent consisting of 15 mmol / L (FeNH4(SO4)2)·12H2O, 0.225 mol / L aminosulfonic acid, and 0.5 mol / L hydrochloric acid was added. At this point, the H2O concentration in the reaction system was... + The concentration is 0.15 mol / L, Fe 3+ The initial concentration was 6 mmol / L. After cooling to room temperature, the solution was developed for 90 min, thoroughly mixed, and 200 μL was added to the wells of the microplate. After shaking the plate, the absorbance of galacturonic acid was measured at 655 nm using a microplate reader. Three replicates were prepared for each concentration. A standard correlation curve was plotted based on the relationship between the concentration of galacturonic acid solution and the corresponding absorbance value.

[0120] Step (3) Determine pectinase activity: Add 400 μL of the substrate solution from step (1) to a test tube and preheat in a water bath shaker at 37°C for 5 min. Then add 400 μL of pectinase ② solution (0.01 μL / mL, prepared with 0.1 mol / L pH 9.8 glycine-NaOH buffer) preheated to the same temperature, mix quickly to start the enzymatic hydrolysis reaction, and after 30 min, quickly add 800 μL of 0.7 mol / L NaOH solution and mix thoroughly to terminate the enzyme reaction and obtain an alkaline enzymatic hydrolysate.

[0121] Add 400 μL of the substrate solution from step (1) into a test tube and preheat it in a water bath shaker at 37°C for 5 min. Then continue to keep it warm for 30 min. Then quickly add 800 μL of 0.7 mol / L NaOH solution and mix thoroughly immediately. Add 400 μL of the same pectinase solution and mix thoroughly to obtain an alkaline enzymatic hydrolysis blank solution.

[0122] Step (4) Determine the absorbance values ​​of alkaline enzymatic hydrolysate and alkaline enzymatic blank: According to the method in step (2), the alkaline enzymatic hydrolysate and alkaline enzymatic blank solution are subjected to precipitation and centrifugation and MBTH reaction, and the alkaline enzymatic hydrolysate and alkaline enzymatic blank solution are measured to obtain the concentration of the reducing end group generated by enzymatic hydrolysis, and then the pectinase activity is obtained.

[0123] Table 6 Results of pectinase activity assay in Example 6

[0124]

[0125]

[0126] Example 7:

[0127] Step (1) Prepare a 5 mg / mL substrate solution: Dissolve the accurately weighed polygalacturonic acid in a small amount of 0.025 mol / L NaOH, adjust the pH to 4.0 with acetic acid, and then bring the volume up with 0.1 mol / L pH 4.0 acetic acid-sodium acetate buffer.

[0128] Step (2) Plot the standard correlation curve between galacturonic acid absorbance and galacturonic acid concentration: Take a portion of the substrate solution prepared in step (1) and mix it 1:1 with galacturonic acid solutions of different concentrations to prepare a series of galacturonic acid standard solutions with a concentration range of 0-60 μg / mL (equivalent to 0-0.28 μmol / mL). Add 800 μL of 0.65 mol / L NaOH solution to 800 μL of the above galacturonic acid standard solutions of different concentrations respectively. The resulting alkaline mixture contains OH... -The concentration was 0.325 mol / L. After rapid mixing, it was placed in an ice bath at 5°C for 20 min, then removed and mixed again. After precipitation, it was allowed to stand at room temperature (25°C) for 40 min, and centrifuged at 6000 rpm (3440 RCF) for 15 min (mixed before centrifugation). 800 μL of the supernatant was collected. 400 μL of freshly prepared MBTH reagent, which is made by mixing equal volumes of 3 mg / mL (12.8 mmol / L) MBTH solution and 0.5 mg / mL (3.2 mmol / L) DTT solution, was added to the supernatant. At this point, the initial concentration of MBTH in the reaction system was 2.13 mmol / L, and the initial concentration of DTT was 0.5 mmol / L. The mixture was heated in a water bath at 80°C for 13 minutes. Then, while still hot, 800 μL of an acidic iron reagent consisting of 15 mmol / L (FeNH4(SO4)2)·12H2O, 0.2 mol / L aminosulfonic acid, and 0.5 mol / L hydrochloric acid was added. At this point, the H2O concentration in the reaction system was... + The concentration is 0.15 mol / L, Fe 3+ The initial concentration was 6 mmol / L. After cooling to room temperature, the solution was developed for 90 min, thoroughly mixed, and 200 μL was added to the wells of the microplate. After shaking the plate, the absorbance of galacturonic acid was measured at 655 nm using a microplate reader. Three replicates were prepared for each concentration. A standard correlation curve was plotted based on the relationship between the concentration of galacturonic acid solution and the corresponding absorbance value.

[0129] Step (3) Determine pectinase activity: Add 400 μL of the substrate solution from step (1) to a test tube and preheat in a water bath shaker at 37°C for 5 min. Then add 400 μL of pectinase ① solution (2 μg / mL, prepared with 0.1 mol / L pH 4.0 acetate-sodium acetate buffer) preheated to the same temperature, mix quickly to start the enzymatic hydrolysis reaction, and after 30 min, quickly add 800 μL of 0.65 mol / L NaOH solution and mix thoroughly to terminate the enzyme reaction and obtain an alkaline enzymatic hydrolysate.

[0130] Add 400 μL of the substrate solution from step (1) into a test tube and preheat it in a water bath shaker at 37°C for 5 min. Then continue to keep it warm for 30 min. Then quickly add 800 μL of 0.65 mol / L NaOH solution and mix thoroughly immediately. Add 400 μL of the same pectinase solution and mix thoroughly to obtain an alkaline enzymatic hydrolysis blank solution.

[0131] Step (4) Determine the absorbance values ​​of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution: According to the method in step (2), the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are subjected to precipitation and centrifugation and MBTH reaction, and the alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysis blank solution are measured to obtain the concentration of the reducing end group generated by enzymatic hydrolysis, and then obtain the pectinase activity.

[0132] Table 7 Results of pectinase activity assay in Example 7

[0133]

[0134] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above operations. All changes, modifications, additions or substitutions made within the scope of the present invention should be protected by the present invention.

Claims

1. A method for determining pectinase activity, characterized in that, Specifically, the following steps are included: (1) Preparation of substrate solution and pectinase solution: Prepare substrate solution with pectin as substrate: Dissolve pectin in buffer or water, adjust the pH value to the pH value to be tested for pectinase, and then make up the volume with buffer solution of the pH value to be tested. Alternatively, a substrate solution can be prepared using polygalacturonic acid as a substrate: dissolve polygalacturonic acid in an alkaline solution, adjust the pH to the desired pH value for pectinase, and then bring the solution to volume with a buffer solution at the desired pH value. The alkaline solution contains OH-... - The concentration is 0.02-0.05 mol / L; Prepare pectinase solutions using the same buffer solution with the same pH value to be tested; (2) Plot the standard correspondence curve between galacturonic acid absorbance value and galacturonic acid concentration: a. Preparation of galacturonic acid standard solutions of different concentrations: Using the substrate solution prepared in step (1), a series of galacturonic acid standard solutions of different concentration gradients are prepared, wherein the concentration of the substrate in the series of galacturonic acid standard solutions of different concentration gradients is the same, and the concentration of galacturonic acid is in the range of 0-0.3 μmol / mL. b. Preparation of alkaline mixtures: Alkaline reagents are added to a series of galacturonic acid standard solutions of varying concentrations to obtain alkaline mixtures. All alkaline mixtures contain the same concentration of alkaline reagent, and the OH- concentration is... - The concentration is in the range of 0.25-0.5 mol / L; c. Precipitation: One method involves placing the alkaline mixture obtained in step b at 0-15°C to precipitate the precipitate, then removing it, mixing it thoroughly, and allowing it to stand until the precipitate separates into layers. This method is suitable for alkaline mixtures with an OH content of [missing value]. - The concentration is 0.25-0.5 mol / L; another method is to let the alkaline mixture stand at 15-35℃ until precipitation and separation occur. This method is suitable for alkaline mixtures with an OH concentration of 0.25-0.5 mol / L. - The concentration is 0.325-0.5 mol / L; d. Centrifugation: The alkaline mixture from step c is centrifuged to obtain the supernatant; e. MBTH reaction: Add freshly prepared MBTH reagent to the supernatant from step d. The initial concentration of MBTH is 0.3-4.3 mmol / L, and the initial concentration of DTT is 0-1.6 mmol / L. React at 50-100℃ for 5-180 min. Then add acidic iron reagent to make the reaction system acidic, with the H+ concentration in the system ranging from 0.03-0.3 mol / L and Fe... 3+ The initial concentration range is 3-10 mmol / L. A colorimetric reaction occurs, and after the colorimetric reaction stabilizes, the absorbance value is measured in the wavelength range of 590-670 nm. The MBTH reagent is an aqueous solution prepared from MBTH and DTT, or an aqueous solution prepared from MBTH alone. The acidic iron reagent is a soluble Fe... 3+ An aqueous solution prepared from salt and strong acid; f. Plot the standard correspondence curve: Plot the standard correspondence curve based on the relationship between the galacturonic acid concentration and the measured absorbance value for each group; (3) Enzymatic hydrolysis of pectinase: Add the substrate solution prepared in step (1) to the pectinase solution to ensure that the concentration of the substrate in the resulting solution is the same as the concentration of the substrate in the galacturonic acid standard solution in step (2), and then carry out the enzymatic hydrolysis reaction. Then add an alkaline reagent to terminate the enzymatic hydrolysis reaction and obtain an alkaline enzymatic hydrolysate. The concentration of the alkaline reagent in the alkaline enzymatic hydrolysate is the same as the concentration of the alkaline reagent in the alkaline mixture in step b. (4) Prepare an alkaline enzymatic hydrolysis blank solution; (5) Preparation of supernatant of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysate blank solution: Obtain the supernatant of alkaline enzymatic hydrolysate in step (3) and the supernatant of alkaline enzymatic hydrolysate blank solution in step (4) by referring to the methods in steps c and d respectively. (6) Determination of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysate blank solution: Perform MBTH reaction according to the method in step e, and determine the supernatant of alkaline enzymatic hydrolysate and alkaline enzymatic hydrolysate blank solution respectively, so as to obtain the concentration of reducing end groups generated by enzymatic hydrolysis, and then obtain the pectinase activity.

2. The method for determining pectinase activity according to claim 1, characterized in that, The buffer solution mentioned in step (1) is an acetate buffer, a glycine buffer, a borate buffer, a succinate buffer, or a phosphate buffer.

3. The method for determining pectinase activity according to claim 1, characterized in that, The alkaline solution used in step (1) and the alkaline reagent used in step (2) are both NaOH solution or KOH solution.

4. The method for determining pectinase activity according to claim 1, characterized in that, In step (2), the soluble Fe in the acidic iron reagent 3+ The salt is at least one of ferric ammonium sulfate, ferric chloride, and ferric nitrate, and the strong acid is at least one of aminosulfonic acid, hydrochloric acid, and sulfuric acid.

5. The method for determining pectinase activity according to claim 1, characterized in that, In step e, after adding MBTH reagent to the supernatant from step d, the initial concentration of MBTH in the system ranges from 0.7 to 3.6 mmol / L, and the initial concentration of DTT ranges from 0.5 to 1.1 mmol / L. Then, acidic iron reagent is added, and the concentration of Fe in the system... 3+ The initial concentration range is 5-7 mmol / L, H + The concentration range is 0.07-0.2 mol / L.

6. The method for determining pectinase activity according to claim 1, characterized in that, The precipitation treatment method in step c includes one approach: first, precipitate the alkaline mixture in an ice-water bath at 0-8°C for 10-30 minutes, then remove and mix thoroughly, and finally allow it to stand at 20-30°C for 30-150 minutes until separation occurs. This method is suitable for alkaline mixtures with an OH content of [missing value]. - The concentration is 0.3-0.35 mol / L; another method is to directly let the alkaline mixture stand at 20-30℃ for 30-180 min until precipitation and separation. This method is suitable for alkaline mixtures with an OH concentration of 0.3-0.35 mol / L. - The concentration is 0.35-0.425 mol / L.

7. The method for determining pectinase activity according to claim 1, characterized in that, In step (2), the reaction temperature of the supernatant in step d with MBTH reagent is 75-85℃ and the reaction time is 9-17 min.

8. The method for determining pectinase activity according to claim 1, characterized in that, The color development time in step (2) is 50-190 min, and the absorbance value is measured within this color development time.

9. The method for determining pectinase activity according to claim 1, characterized in that, The wavelength for measuring the absorbance value in step (2) is 630-655 nm.

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