Activity detection method of arabinofuranosidase and application thereof

Abstract

The invention relates to an activity detection method of arabinofuranosidase and application of the activity detection method. The activity detection method comprises the following steps: mixing p-nitrophenyl-alpha-L-arabinofuranosidase with a to-be-detected sample, incubating, adjusting the pH value to 6.5-8 after the reaction is terminated, and determining. According to the method, p-nitrophenyl-alpha-L-arabinofuranosidase is adopted to catalyze p-nitrophenyl-alpha-L-arabinofuranoside to generate p-nitrophenol, p-nitrophenol has a characteristic absorption peak at 410nm, and the activity of the arabinofuranosidase under different dilution multiples can be calculated by measuring a 410nm light absorption value. The optimal enzyme activity of the alpha-L-arabinofuranosidase can be finally calculated by performing parabola fitting on different dilution multiples and enzyme activities. Wherein in a pH range of 6.5-8, p-nitrophenol can have a relatively strong color development effect, and a substrate blank is free of mixed color interference, so that the accuracy of detection by adopting a light absorption value can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme activity detection, and in particular to an activity detection method of arabinofuranosidase and an application thereof. Background Art

[0002] α-L-arabinofuranosidase α-L-Af is a glycosidase that can hydrolyze non-reducing furanose arabinose residues, causing the cell wall arabinogalactan, arabinomannan and other neutral sugars to continuously dissociate, thereby promoting the solubilization and degradation of pectin. α-L-arabinofuranosidase (α-L-AFase) belongs to the glycoside hydrolases (GH) family. It mainly catalyzes the hydrolysis of arabinofuranosyl groups linked by α-(1,2), α-(1,3) or α-(1,5) in arabinose polymers, removes side chain residues from the xylan backbone, and cooperates with other hemicellulase systems such as xylanase, β-xylosidase, ferulic acid esterase and acetylxylan esterase to complete the hydrolysis of xylan. It can be used for the production of biofuels, pulp bleaching, improving wine flavor, increasing feed digestibility, improving bread quality, clarifying juice, producing arabinose and increasing the filtration rate of wort in beer brewing.

[0003] However, there is currently no corresponding national standard for the determination of arabinofuranosidase activity. The existing detection methods are vague and confusing in terms of substrate, reaction temperature, pH, time, detection wavelength, and enzyme activity calculation, and have poor operability.

[0004] Therefore, how to realize a standard detection method for arabinofuranosidase activity that is simple and clear to operate and can measure rapidly and accurately has become a problem that needs to be solved urgently. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an activity detection method of arabinofuranosidase and its application. The method is fast and efficient in detection, highly accurate, easy to operate and applicable to many application scenarios.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting the activity of arabinofuranosidase, the method comprising the steps of: mixing p-nitrophenyl-α-L-arabinofuranoside with a sample to be tested, incubating the mixture, adjusting the pH value to 6.5-8 after terminating the reaction, and performing the assay. The 6.5-8 may be, for example, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, etc.

[0008] In the present invention, α-L-arabinofuranosidase (α-L-Af) is used to catalyze p-nitrophenyl-α-L-arabinofuranoside (pNPAf) to produce p-nitrophenol (pNP). p-nitrophenol has a characteristic absorption peak at 410nm. The arabinofuranosidase activity can be calculated by measuring the increase rate of light absorption at 410nm.

[0009] In the pH range of 6.5-8, p-nitrophenol can have a strong color development effect, which can significantly improve the accuracy of absorbance detection.

[0010] Preferably, the activity detection method further comprises data analysis.

[0011] Preferably, the data analysis process includes: according to OD 410 The average absorbance was calculated, and the corresponding pNP content was found on the standard curve to calculate the arabinofuranosidase activity.

[0012] Preferably, the calculation formula of the arabinofuranosidase activity is as follows:

[0013]

[0014] Wherein, G: arabinofuranosidase activity, unit is enzyme activity unit per gram (U / g); C: pNP concentration corresponding to the absorbance calculated according to the standard curve, unit is μmol / mL; V 0 : Volume of diluted enzyme solution added to the reaction, unit: mL; V 1 : Volume of reaction system, in mL; t: Enzyme hydrolysis reaction time, in min; n: Total dilution multiple of the sample.

[0015] Preferably, the calculation formula for the arabinofuranosidase activity in the sample to be tested is as follows:

[0016] X L =G×ρ.

[0017] Among them, X L : activity of arabinofuranosidase in the sample, U / mL; ρ: initial density of liquid enzyme, g / mL.

[0018] In the present invention, the activity of arabinofuranosidase can be calculated by the above formula, and the activity of arabinofuranosidase in the sample solution to be tested can be further obtained. The formula is simple and easy to calculate.

[0019] Preferably, the data analysis further comprises enzyme concentration-enzyme activity parabola fitting and calculation of optimal enzyme activity.

[0020] Preferably, the calculation method of the data analysis includes using a quadratic formula to fit the test results to a parabola of enzyme concentration-enzyme activity and obtain constants a, b and c, and substituting them into the calculation formula of optimal enzyme activity to obtain the optimal enzyme activity.

[0021] Preferably, the calculation formula for the enzyme concentration-enzyme activity parabola fitting is:

[0022] Y = ax + bx + c;

[0023] Preferably, the calculation formula for the dilution multiple corresponding to the optimal enzyme activity is:

[0024]

[0025] Preferably, the calculation formula for the optimal enzyme activity is:

[0026]

[0027] Preferably, the preparation method of p-nitrophenyl-α-L-arabinofuranoside comprises: dissolving p-nitrophenyl-α-L-arabinofuranoside in sodium citrate buffer.

[0028] Preferably, the concentration of the sodium citrate buffer is 0.01-0.5 mol / L, and the pH value is 4-6. The 0.01-0.5 mol / L may be, for example, 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L or 0.5 mol / L, etc. The pH value is 4-6, for example, 4, 4.5, 5, 5.5 or 6, etc.

[0029] In the present invention, sodium citrate buffer is used to dissolve p-nitrophenyl-α-L-arabinofuranoside, which can ensure the effectiveness of the substrate and provide suitable and stable pH conditions for subsequent enzymatic hydrolysis reactions. Sodium citrate has excellent solubility, is colorless and does not react with p-nitrophenyl-α-L-arabinofuranoside. In addition, sodium citrate is a weak acid and strong base salt with good pH adjustment and buffering properties, is non-toxic and stable, is friendly to experimenters, and has good biodegradability and is friendly to the ecological environment.

[0030] Preferably, the p-nitrophenyl-α-L-arabinofuranoside and the sample to be tested are pre-incubated separately before detection.

[0031] Preferably, the mass ratio of p-nitrophenyl-α-L-arabinofuranoside to the sample to be tested is (5.4-216):1, and the (5.4-216) can be, for example, 5.4, 6, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 or 216, etc.

[0032] Preferably, the pre-incubation temperature is 45-55°C and the time is 2-8 min. The 45-55°C may be, for example, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C, etc. The 2-8 min may be, for example, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min or 8 min, etc.

[0033] Preferably, the incubation temperature is 45-55°C and the incubation time is 5-15 min. The 45-55°C may be, for example, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C, etc. The 5-15 min may be, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.

[0034] In the present invention, the substrate and the sample to be tested are first pre-incubated to make the substrate and the sample in a stable state, and then the reaction system is constructed, which can significantly reduce the system error. Pre-incubating the sample to be tested can better enable the arabinofuranosidase in the sample to be tested to reach its best activity state, which can further improve the accuracy of the detection.

[0035] Preferably, the method for terminating the reaction comprises: adding a terminating reagent and / or an ice bath.

[0036] In the present invention, there are two methods for terminating the reaction. Using the above methods to terminate the reaction can reduce the impact of the reaction on the detection process, thereby improving the accuracy of the detection.

[0037] Preferably, the stop reagent comprises any one of 10-30% sodium carbonate, PBS or Tris-HCl buffer or a combination of at least two thereof. The 10-30% may be, for example, 10%, 15%, 20%, 25% or 30%.

[0038] Preferably, the temperature of the ice bath is 0-4°C and the time is 2-8 min. The 0-4°C may be, for example, 0°C, 1°C, 2°C, 3°C or 4°C. The 2-8 min may be, for example, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min or 8 min.

[0039] Preferably, the reagent for adjusting the pH value comprises PBS and / or Tris-HCl buffer.

[0040] Preferably, the oscillation time is 10-60s and the frequency is 5-20Hz. The 10-60s may be, for example, 10s, 20s, 30s, 40s, 50s or 60s, etc. The 5-20Hz may be, for example, 5Hz, 11Hz or 20Hz, etc.

[0041] In the present invention, the sample is mixed before detection, thereby reducing mechanical errors and improving detection accuracy.

[0042] Preferably, the measuring step comprises: measuring the absorbance value at a wavelength of 410 nm.

[0043] In the present invention, the absorbance of each well is measured at 410 nm with the standard blank as the control. The absorbance of the test sample needs to be subtracted from the absorbance of the enzyme blank sample and the substrate blank to obtain the final test result.

[0044] Preferably, the activity detection method further comprises: detecting a series of standard solutions and preparing a standard curve.

[0045] Preferably, the standard solution series is a p-nitrophenol standard solution.

[0046] Preferably, the concentration range of the standard series solutions is 0-0.2 μmol / mL, for example, 0, 0.025 μmol / mL, 0.05 μmol / mL, 0.075 μmol / mL, 0.1 μmol / mL, 0.125 μmol / mL, 0.15 μmol / mL, 0.175 μmol / mL or 0.2 μmol / mL, etc.

[0047] In a second aspect, the present invention provides a use of the method for detecting the activity of arabinofuranosidase according to the first aspect in detecting the activity of arabinofuranosidase.

[0048] Compared with the prior art, the present invention has at least the following beneficial effects:

[0049] 1. In the present invention, α-L-arabinofuranosidase (α-L-Af) is used to catalyze p-nitrophenyl-α-L-arabinofuranoside (pNPAf) to produce p-nitrophenol (pNP). p-Nitrophenol has a characteristic absorption peak at 410 nm. The arabinofuranosidase activity can be calculated by measuring the increase rate of light absorption at 410 nm.

[0050] In the pH range of 6.5-8, p-nitrophenol can have a strong color development effect, which can significantly improve the accuracy of absorbance detection.

[0051] 2. In the present invention, a program for enzyme concentration-enzyme activity parabola fitting and optimal enzyme activity calculation is constructed according to the activity detection method. It is tested that the enzyme concentration-enzyme activity parabola and the optimal enzyme activity can be obtained by simply inputting the detection data into the program, which greatly reduces the labor cost and improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the standard curve of p-nitrophenol.

[0053] Figure 2 It is the color development picture after the reaction is terminated, among which Picture A is the color development picture of Example 1, and Picture B is the color development picture of Comparative Example 1. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0055] Sources of reagents used in the following examples:

[0056] p-Nitrophenyl-α-L-arabinofuranoside: MacLean N861461;

[0057] Arabinofuranosidase: Novozymes China cellic ctec3 HS;

[0058] p-Nitrophenol: Aladdin N130840;

[0059] Cellulase: Novozymes China cellic ctec3 HS.

[0060] Example 1

[0061] In this example, the activity of arabinofuranosidase was detected.

[0062] (1) Pre-incubation

[0063] Weigh 0.0108 g of p-nitrophenyl-α-L-arabinofuranoside standard product and dilute to 10 mL with 0.1 mol / L, pH 5 sodium citrate buffer to obtain a 4 mM p-nitrophenyl-α-L-arabinofuranoside solution.

[0064] 10 mL of p-nitrophenyl-α-L-arabinofuranoside solution was incubated at 50° C. for 5 min. 2000 μg / mL of arabinofuranosidase was diluted to 5000, 10000, 20000, 50000, 80000, 100000, 150000, and 200000, and then incubated at 50° C. for 5 min.

[0065] (2) Reaction

[0066] 0.2 mL of p-nitrophenyl-α-L-arabinofuranoside solution and 0.2 mL of arabinofuranosidase solution were mixed and incubated at 50° C. for 10 min.

[0067] (3) Termination of reaction and adjustment of pH

[0068] 0.8 mL of sodium carbonate with a mass concentration of 20% in the reaction system was added, the reaction volume was 1.2 mL and the pH was adjusted to 7 with Tris-HCl buffer.

[0069] (4) Determination

[0070] The above solution was shaken at a frequency of 11 Hz for 30 seconds and then mixed, and the absorbance at a wavelength of 410 nm was measured.

[0071] Example 2

[0072] In this example, the activity of arabinofuranosidase was detected.

[0073] (1) Pre-incubation

[0074] Weigh 0.0108 g of p-nitrophenyl-α-L-arabinofuranoside standard product and dilute to 10 mL with 0.01 mol / L, pH 6 sodium citrate buffer to obtain a 4 mM nitrophenyl-α-L-arabinofuranoside solution.

[0075] 10 mL of p-nitrophenyl-α-L-arabinofuranoside solution was incubated at 55° C. for 8 min. 2000 μg / mL of arabinofuranosidase was diluted to 5000, 10000, 20000, 50000, 80000, 100000, 150000, and 200000, and then incubated at 55° C. for 5 min.

[0076] (2) Reaction

[0077] 0.2 mL of p-nitrophenyl-α-L-arabinofuranoside solution and 0.2 mL of arabinofuranosidase solution were mixed and incubated at 55° C. for 5 min.

[0078] (3) Termination of reaction and adjustment of pH

[0079] 0.8 mL of sodium carbonate with a mass concentration of 10% in the reaction system was added, the reaction volume was 1.2 mL and the pH was adjusted to 6 with Tris-HCl buffer.

[0080] (4) Determination

[0081] The above solution was shaken at a frequency of 5 Hz for 10 seconds and then mixed, and the absorbance at a wavelength of 410 nm was measured.

[0082] Example 3

[0083] In this example, the activity of arabinofuranosidase was detected.

[0084] (1) Pre-incubation

[0085] Weigh 0.0108 g of p-nitrophenyl-α-L-arabinofuranoside standard product and dilute to 10 mL with 0.5 mol / L, pH 4 sodium citrate buffer to obtain a 4 mM p-nitrophenyl-α-L-arabinofuranoside solution.

[0086] 10 mL of p-nitrophenyl-α-L-arabinofuranoside solution was incubated at 45° C. for 2 min. 2000 μg / mL of arabinofuranosidase was diluted to 5000, 10000, 20000, 50000, 80000, 100000, 150000, and 200000, and then incubated at 45° C. for 5 min.

[0087] (2) Reaction

[0088] 0.2 mL of p-nitrophenyl-α-L-arabinofuranoside solution and 0.2 mL of arabinofuranosidase solution were mixed and incubated at 45° C. for 15 min.

[0089] (3) Termination of reaction and adjustment of pH

[0090] 0.8 mL of sodium carbonate with a mass concentration of 30% in the reaction system was added, the reaction volume was 1.2 mL and the pH was adjusted to 8 with Tris-HCl buffer.

[0091] (5) Determination

[0092] The above solution was shaken at a frequency of 20 Hz for 60 seconds and then mixed, and the absorbance at a wavelength of 410 nm was measured.

[0093] Example 4

[0094] This example detects the activity of arabinofuranosidase, which differs from Example 1 only in the steps of terminating the reaction and adjusting the pH. The operation of terminating the reaction is to place the reaction system in an ice bath at 4°C for 5 minutes and then add PBS to adjust the pH. The rest is the same as Example 1.

[0095] Example 5

[0096] This example detects the activity of arabinofuranosidase, which is different from Example 1 only in that the incubation temperature is 40° C., and the rest is the same as Example 1.

[0097] Example 6

[0098] This example detects the activity of arabinofuranosidase, which is different from Example 1 only in that the incubation temperature is 60° C., and the rest is the same as Example 1.

[0099] Example 7

[0100] This example detects the activity of arabinofuranosidase, which is different from Example 1 only in that no pre-incubation is performed, and the rest is the same as Example 1.

[0101] Example 8

[0102] This example is used to detect the activity of arabinofuranosidase. The only difference from Example 1 is that acetic acid-sodium acetate is used as the termination reagent. The rest is the same as Example 1.

[0103] Example 9

[0104] This example is used to detect the activity of arabinofuranosidase. The only difference from Example 1 is that deionized water is used to dissolve p-nitrophenyl-α-L-arabinofuranoside. The rest is the same as Example 1.

[0105] Comparative Example 1

[0106] The comparative example was used to detect the activity of arabinofuranosidase, which was different from Example 1 only in that the pH was adjusted to 5, and the rest was the same as Example 1.

[0107] Comparative Example 2

[0108] The comparative example was used to detect the activity of arabinofuranosidase, which was different from Example 1 only in that the pH was adjusted to 9, and the rest was the same as Example 1.

[0109] Test Example 1

[0110] This test example prepares the standard curve

[0111] Dry the p-nitrophenol standard (pNP) at 50°C in a constant temperature drying oven to constant weight, accurately weigh 0.0140g in a 100mL beaker, dissolve it with a small amount of distilled water, transfer it to a 100mL volumetric flask and dilute it to 100mL with distilled water, and mix thoroughly. Use an ELISA plate, select numbers 1-8, add standard pNP solution and distilled water according to Table 1, and prepare a series of pNP solutions of different concentrations. After shaking thoroughly, add 100μL of 20% (w / w) sodium carbonate solution to each well plate. At a wavelength of 410nm, use the solution in well 1 as the blank control, adjust the zero point, measure the optical density values ​​of the solutions in other wells and record the results. Use the pNP content as the horizontal axis and the OD as the horizontal coordinate. 410 The absorbance value is plotted as the ordinate, which is the standard curve. The fitting equation and R 2 The specific standard curve is as follows: Figure 1 shown.

[0112] Table 1

[0113]

[0114]

[0115] Test Example 2

[0116] This test example is used to test the detection effect of the above examples 1-9 and comparative examples 1-2

[0117] The above examples and comparative examples were tested, and three groups of blank controls were set up respectively. The standard blank control was a mixed solution of 25 μL of water, 25 μL of sodium citrate (the specific concentration and pH were consistent with the sodium citrate standard for dissolving p-nitrophenyl-α-L-arabinofuranoside in each example) and 100 μL of sodium carbonate (the specific concentration was consistent with the concentration of sodium carbonate in each example).

[0118] The substrate blank group was a mixed solution of 25 μL pNPAf solution, 25 μL sodium citrate (the specific concentration and pH were consistent with the sodium citrate used to dissolve the p-nitrophenyl-α-L-arabinofuranoside standard in each example) and 100 μL sodium carbonate (the specific concentration was consistent with the concentration of sodium carbonate in each example).

[0119] The enzyme solution blank group was a mixed solution of 25 μL diluted enzyme solution, 25 μL sodium citrate (the specific concentration and pH were consistent with the sodium citrate standard for dissolving p-nitrophenyl-α-L-arabinofuranoside in each example) and 100 μL sodium carbonate (the specific concentration was consistent with the concentration of sodium carbonate in each example).

[0120] According to OD 410The absorbance average value is measured at 410 nm with the standard blank as the control. The absorbance of the test sample needs to be subtracted from the absorbance of the enzyme blank sample and the substrate blank to obtain the final test result. The corresponding pNP content is checked on the standard curve to calculate the arabinofuranosidase activity. The calculation formula of the arabinofuranosidase activity is as follows:

[0121]

[0122] Wherein, G: arabinofuranosidase activity; C: pNP concentration corresponding to the absorbance calculated according to the standard curve; V 0 : Volume of diluted enzyme solution added to the reaction; V 1 : volume of reaction system; t: enzymatic reaction time; n: total dilution multiple of the sample.

[0123] The specific results are shown in Tables 2 and 3. The standard product used is cellulose complex enzyme (because pure arabinofuranosidase standard product cannot be purchased, and Novozymes cellulase is a mature commercial complex enzyme, which contains a quantitative amount of arabinofuranosidase, so it is used as a standard enzyme instead), the arabinofuranosidase activity is 2000U / g, and it is diluted 5000, 10000, 20000, 50000, 80000, 100000, 150000 and 200000 respectively. The corresponding enzyme addition amount is 40μg, 20μg, 10μg, 4μg, 2.5μg, 2, 1.33μg and 1μg respectively. And the enzyme activity is the highest point calculated by parabola fitting as the final enzyme activity.

[0124] Table 2

[0125] Dilution multiple / group Example 1 Example 2 Example 3 Example 4 Example 5 5000 713.21 627.62 641.89 791.66 427.92 Difference from standard product 64.34％ 68.62％ 67.91％ 60.42％ 78.60％ 10000 901.02 774.87 792.89 946.07 522.59 Difference from standard product 54.95％ 61.26％ 60.36％ 52.70％ 73.87％ 20000 1231.88 1071.73 1071.73 1231.88 702.17 Difference from standard product 38.41％ 46.41％ 46.41％ 38.41％ 64.89％ 50000 1866.41 1661.10 1661.10 1903.74 1138.51 Difference from standard product 6.68％ 16.94％ 16.94％ 4.81％ 43.07％ 80000 1963.86 1728.20 1767.47 2062.05 1080.12 Difference from standard product 1.81％ 13.59％ 11.63％ 3.10％ 45.99％ 100000 1730.45 1488.19 1505.49 1782.37 986.36 Difference from standard product 13.48％ 25.59％ 24.73％ 10.88％ 50.68％ 150000 102.62 90.30 90.30 105.69 56.44 Difference from standard product 94.87％ 95.48％ 95.48％ 94.72％ 97.18％ 200000 -3017.10 -2564.54 -2685.22 -3137.79 -1749.92 Difference from standard product 50.86％ 228.23％ 234.26％ 256.89％ 187.50％ Curve fitting value 1991.11 1752.18 1792.00 2090.67 1194.67 Difference from standard product 0.44％ 12.39％ 10.40％ 4.53％ 40.27％

[0126] Table 3

[0127]

[0128]

[0129] The test results show that:

[0130] (1) It can be seen from Examples 1 to 2-3 that α-L-arabinofuranosidase (α-L-Af) is used to catalyze p-nitrophenyl-α-L-arabinofuranoside (pNPAf) to produce p-nitrophenol (pNP), and p-nitrophenol has a characteristic absorption peak at 410 nm. The 410 nm light absorption value can be measured to calculate the arabinofuranosidase activity. The accuracy of the detection can be significantly improved by adjusting the pH.

[0131] (2) By comparing Example 1 with Example 4, it can be seen that both the ice bath and the addition of a stop reagent have good effects.

[0132] (3) By comparing Example 1 with Examples 5-6, it can be seen that when the incubation temperature is outside the range specified in this application, the maximum enzyme activity cannot be obtained, thereby reducing the accuracy of the detection.

[0133] (4) By comparing Example 1 with Example 7, it can be seen that without pre-incubation, the reaction system will be unstable, the mechanical error will be increased, and the detection results will be affected.

[0134] (5) By comparing Example 1 with Example 8, it can be seen that the effect is poor when other termination reagents are used.

[0135] (6) By comparing Example 1 with Example 9, it can be seen that not using sodium citrate as the dissolving liquid will lead to significant differences in the experimental results.

[0136] (7) It can be seen from Example 1 and Comparative Example 1 that Figure 2 As shown, if the pH value is too low, the reaction is not terminated, and the color of p-nitrophenol is too light, and the color reaction cannot be accurately detected, resulting in detection distortion.

[0137] (8) By comparing Example 1 with Comparative Example 2, it can be seen that using too high a pH value will result in a darker color reaction, causing the detection device to be unable to accurately identify, thereby reducing the accuracy of the detection.

[0138] Test Example 3

[0139] This test example performs enzyme concentration-enzyme activity parabola fitting and optimal enzyme activity calculation on Example 1.

[0140] According to the method of the present invention, the enzyme concentration-enzyme activity parabola fitting and the optimal enzyme activity calculation of the α-L-arabinofuranosidase in Example 1 were performed. First, the standard curve was fitted according to the concentration and absorbance values ​​of the series of pNP standard solutions, and the standard equation Y=aX+b and the standard curve R were output. 2 And perform R 2 ≥0.99 verification. The pNP concentration corresponding to the absorbance at different dilution multiples was calculated using the standard curve, and the enzyme activity at different dilution multiples was calculated according to the enzyme activity formula.

[0141] Y=6.5364X-0.0061R 2 =0.9991

[0142] The parabola equation Y = aX was fitted using the dilution multiple and enzyme activity results. 2+bX+c, and then use the parabola vertex formula to solve the optimal dilution multiple and the corresponding optimal enzyme activity.

[0143] Y=-3E-07x 2 +0.042x+510.48

[0144]

[0145]

[0146] According to the above test results, it can be concluded that the enzyme activity of Example 1 is 1991.11 after curve fitting, as shown in Table 4 below, and the standard enzyme activity of α-L-arabinofuranosidase in Novozymes cellulase (cellic ctec3 HS) is 2000, with a difference of only 0.44%, which proves that the detection method of the present application has high accuracy.

[0147] Table 4

[0148]

[0149] In summary, in the present invention, α-L-arabinofuranosidase (α-L-Af) is used to catalyze p-nitrophenyl-α-L-arabinofuranoside (pNPAf) to produce p-nitrophenol (pNP), p-nitrophenol has a characteristic absorption peak at 410 nm, and the arabinofuranosidase activity can be calculated by measuring the light absorption value at 410 nm.

[0150] In the pH range of 6-8, p-nitrophenol can have a strong color development effect, which can significantly improve the accuracy of absorbance detection.

[0151] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting the activity of arabinofuranosidase, characterized in that: The activity detection method comprises the following steps: mixing p-nitrophenyl-α-L-arabinofuranoside with a sample to be tested, incubating the mixture, adjusting the pH value to 6.5-8 after terminating the reaction, and conducting the test.

2. The activity detection method according to claim 1, characterized in that: The activity detection method also includes data analysis; Preferably, the data analysis process includes: according to OD 410 The average absorbance was used to check the corresponding p-nitrophenol content on the standard curve and calculate the arabinofuranosidase activity; Preferably, the calculation formula of the arabinofuranosidase activity is as follows: Wherein, G: arabinofuranosidase activity; C: the concentration of p-nitrophenol corresponding to the absorbance calculated according to the standard curve; V0: the volume of the reaction plus diluted enzyme solution; V1: the volume of the reaction system; t: the enzymatic reaction time; n: the total dilution multiple of the sample; Preferably, the calculation formula for the arabinofuranosidase activity in the sample to be tested is as follows: X L =G×ρ; Among them, X L : Activity of arabinofuranosidase in the sample; ρ: initial density of liquid enzyme.

3. The activity detection method according to claim 2, characterized in that: The data analysis also includes enzyme concentration-enzyme activity parabola fitting and calculation of optimal enzyme activity.

4. The activity detection method according to claim 3, characterized in that: The calculation method of the data analysis includes fitting the enzyme concentration-enzyme activity parabola to the test results using a quadratic formula and obtaining constants a, b and c, and inserting them into the calculation formula of the optimal enzyme activity to obtain the optimal enzyme activity; Preferably, the calculation formula for the enzyme concentration-enzyme activity parabola fitting is: Y=ax 2 +bx+c; Preferably, the calculation formula for the dilution multiple corresponding to the optimal enzyme activity is: Preferably, the calculation formula for the optimal enzyme activity is:

5. The activity detection method according to any one of claims 1 to 4, characterized in that: The preparation method of p-nitrophenyl-α-L-arabinofuranoside comprises: dissolving p-nitrophenyl-α-L-arabinofuranoside in sodium citrate buffer; Preferably, the concentration of the sodium citrate buffer is 0.01-0.5 mol / L, and the pH value is 4-6; Preferably, the p-nitrophenyl-α-L-arabinofuranoside and the sample to be tested are pre-incubated separately before detection; Preferably, the mass ratio of the p-nitrophenyl-α-L-arabinofuranoside to the sample to be tested is (5.4-216):1; Preferably, the pre-incubation temperature is 45-55° C. and the time is 2-8 min.

6. The activity detection method according to any one of claims 1 to 5, characterized in that: The incubation temperature is 45-55°C and the incubation time is 5-15 minutes.

7. The activity detection method according to any one of claims 1 to 6, characterized in that: The method for terminating the reaction comprises: adding a terminating reagent and / or an ice bath; Preferably, the stop reagent comprises any one or a combination of at least two of 10-30% sodium carbonate, PBS or Tris-HCl buffer; Preferably, the temperature of the ice bath is 0-4°C and the time is 2-8 min; Preferably, the reagent for adjusting the pH value comprises PBS and / or Tris-HCl buffer.

8. The activity detection method according to any one of claims 1 to 7, characterized in that: The reaction system is shaken and mixed before the determination; Preferably, the oscillation time is 10-60s and the frequency is 5-20Hz; Preferably, the measuring step comprises: measuring the absorbance value at a wavelength of 410 nm.

9. The activity detection method according to any one of claims 1 to 8, characterized in that: The activity detection method further comprises: detecting a series of standard solutions and preparing a standard curve; Preferably, the standard series solution is a p-nitrophenol standard solution; Preferably, the concentration range of the standard series solutions is 0-0.2 μmol / mL.

10. Use of the method for detecting the activity of arabinofuranosidase according to any one of claims 1 to 9 in detecting the activity of arabinofuranosidase.

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