A method for detecting cellulase activity in soil

Abstract

The present invention provides a method for detecting the cellulase activity in soil, belonging to the technical field of soil detection. The present invention pre-treats the soil with a impurity remover containing potassium sulfate and sodium sulfate, which can effectively remove interfering factors such as metal ions, thereby improving the detection accuracy. Then, after the incubation of the sample to be tested with carboxymethyl cellulose solution, phosphate buffer and toluene is completed, potassium alum is added to avoid affecting filtration due to the excessive viscosity of the culture solution. The detection method of the present invention can accurately detect the cellulase activity in soil, and compared with the existing method for determination at 540 nm, the present invention measures the cellulase activity at 508 nm, showing a good linear relationship, and the change in color is more stable and reliable.

Description

Technical Field

[0001] The present invention relates to the technical field of soil detection, and particularly relates to a method for detecting the activity of cellulase in soil. Background Art

[0002] Soil cellulase (S-CL) mainly comes from soil microorganisms and is a complex enzyme that can catalyze the decomposition of cellulose into cellobiose and further into glucose, thereby providing available carbon source nutrients for microorganisms. Therefore, soil cellulase is an important enzyme in soil carbon metabolism. In agricultural and forestry production, soil enzyme activity is an important indicator for evaluating soil fertility and soil quality. As one of the key enzymes involved in the mineralization of organic matter in soil, the determination of its activity is crucial for understanding soil fertility and plant growth environment.

[0003] Currently, the detection methods for soil cellulase mainly include spectrophotometry, fluorescence method, enzyme-linked immunosorbent assay, etc. Among them, spectrophotometry mainly uses soil cellulase to catalyze the decomposition of cellulose to generate soluble sugars, and calculates the activity of soil cellulase by measuring the amount of generated sugars. However, during the determination process, it is easily affected by interference factors. For example, metal ions may react chemically with the detection reagents to form colored substances or precipitates, interfering with the normal color reaction, or hindering the normal binding of the substrate and the enzyme, reducing the catalytic activity of the enzyme, thereby affecting the accurate determination of the enzymatic hydrolysis products and resulting in deviations in the detection results of enzyme activity and interfering with the authenticity of the test results. Moreover, in a complex soil environment, cellulase is prone to non-specific adsorption, adsorbing to soil particles or other impurities, thus affecting the accuracy of the detection results. In addition, the fluorescence method and enzyme-linked immunosorbent assay are relatively complex in operation, require high requirements for operators, and have high equipment costs, and are not suitable for continuous use.

[0004] Therefore, if a detection method that reduces the influence of interference factors and has reliable results can be designed based on the existing methods, it will be beneficial to the development of cellulase detection technology. Summary of the Invention

[0005] In view of the defects of the prior art, the present invention designs a method for detecting the activity of cellulase in soil.

[0006] To achieve the above object, the present invention adopts the following technical solutions to solve the technical problems:

[0007] On the one hand, the present invention provides a method for detecting the activity of cellulase in soil, and the detection method includes the following steps:

[0008] S1. Prepare a glucose standard solution, subject the glucose standard solution and 3,5-dinitrosalicylic acid solution to a boiling water bath, cool, detect at a wavelength of 508 nm, and plot a standard curve;

[0009] S2. Pretreat the soil powder to remove impurities;

[0010] S3. Mix the pretreated soil with carboxymethyl cellulose solution, buffer solution, and toluene, and incubate at a constant temperature;

[0011] S4. After the incubation, add potassium alum, shake for 30 min, then filter. Take the filtrate and perform colorimetric determination at a wavelength of 508 nm according to the method in step S1.

[0012] In step S3, toluene is added to prevent the reaction from proceeding excessively, thereby ensuring the accuracy of the measurement results, reducing the oxidation or decomposition of the sample due to high temperature, and avoiding affecting the measurement results.

[0013] In some embodiments, in step S1, the pretreatment includes:

[0014] First, dry and grind the soil into powder, sieve it through a 50-mesh sieve, then add the soil powder to the impurity remover and stir at 150 rpm for 15 - 25 min. Then add 1 mmol / L hydrogen peroxide solution and continue to stir for 35 - 55 min. After stirring, filter, take the filtrate as the sample to be measured;

[0015] The impurity remover includes potassium sulfate and sodium sulfate.

[0016] In some embodiments, the mass ratio of potassium sulfate to sodium sulfate is (1 - 5):(3 - 7).

[0017] In some embodiments, the mass ratio of potassium sulfate to sodium sulfate is 3:5.

[0018] In some embodiments, the concentration of the glucose standard solution is 0 - 1.2 mg / mL.

[0019] In some embodiments, in step S3, the incubation time is 48 - 96 h.

[0020] In some embodiments, in step S3, the incubation temperature is 35 - 40 °C.

[0021] In some embodiments, the buffer solution is a 0.2 mol / L phosphate buffer solution with a pH value of 5.5.

[0022] In some embodiments, the mass percentage of the carboxymethyl cellulose solution is 1%.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention pre - treats the soil with a impurity - removing agent to remove interfering factors such as metal ions, thereby improving the detection accuracy. The impurity - removing agent includes potassium sulfate and sodium sulfate. During the pre - treatment process, sodium sulfate and potassium sulfate adjust the ionic strength of the solution, improve the stability of cellulase, and reduce the non - specific adsorption of cellulase, avoiding the limitation of the contact between cellulase and the substrate due to adsorption on soil particles or impurities. At the same time, sulfate ions can also precipitate with metal ions that interfere with the activity of cellulase, reducing the interference of metal ions on the detection results. Then, the present invention adds potassium alum after culturing the pre - treated sample to be tested with 1% carboxymethyl cellulose solution, phosphate buffer solution and toluene, which is beneficial to the filtration of the culture solution.

[0025] Using the detection method of the present invention, the cellulase activity in the soil can be accurately detected. And compared with the existing method measured at 540nm, the present invention measures the cellulase activity at 508nm, showing a good linear relationship, and the color change is more stable and reliable. Brief Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions of the present invention, the drawings required for the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0027] Figure 1 It is the standard curve graph of Example 1. Detailed Embodiments

[0028] The following combines specific embodiments to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0029] In the present invention, regarding numerical ranges, unless otherwise specified, the above - mentioned numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub - ranges subsumed therein.

[0030] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0031] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained commercially.

[0032] 1% carboxymethyl cellulose solution: Prepared by dissolving carboxymethyl cellulose in distilled water.

[0033] Phosphate buffer (pH = 5.5): Prepared by mixing 0.06M KH2PO4 and Na2HPO4 solutions.

[0034] Detection principle: Cellulase catalyzes the degradation of cellulose to produce reducing sugars, which further react with 3,5-dinitrosalicylic acid to form a brownish-red amino compound. The product has a characteristic absorption peak at 508 nm, and the activity of soil cellulase can be characterized by the change in absorbance value.

[0035] Example 1

[0036] A method for detecting the activity of cellulase in soil, the method comprising the following steps:

[0037] I. Plotting the standard curve

[0038] 1. Respectively take 1 mL of glucose standard solutions with different concentrations and put them into 25 mL volumetric flasks. Add 3 mL of 3,5-dinitrosalicylic acid solution and heat in a boiling water bath for 5 min, then quickly cool to room temperature, make up the volume to 25 mL with distilled water. After 15 min, perform colorimetric determination at a wavelength of 508 nm on a spectrophotometer. Use the blank tube to zero and perform colorimetry at a wavelength of 508 nm. Take the absorbance value as the ordinate and the glucose concentration (mg / mL) as the abscissa to plot the standard curve, as Figure 1 shown.

[0039] Prepare glucose standard solutions with distilled water, with concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1, 1.2 mg / mL respectively.

[0040] The linear equation measured under standard conditions is: y = 6.7848x - 0.0019 (1), R 2 = 0.9963, where x is the concentration of the standard product (mg / mL) and y is the absorbance value.

[0041] II. Soil pretreatment

[0042] 1. Dry the fresh soil to constant weight, then grind it into powder and pass through a 50-mesh sieve.

[0043] 2. Add 10 g of soil powder to 100 mL of distilled water, then add 1.5 g of impurity remover and stir at 150 rpm for 20 min. Then add 3 mL of hydrogen peroxide solution (1 mmol / L) and continue stirring for 45 min. After stirring is completed, filter to remove impurities, and take the filtrate as the sample to be tested.

[0044] The impurity remover is prepared from potassium sulfate and sodium sulfate with a mass ratio of 3:5.

[0045] III. Test (Nitrosalicylic acid colorimetric method)

[0046] 1. Place the sample to be tested in a 50 mL Erlenmeyer flask, add 20 mL of 1% carboxymethyl cellulose solution, 5 mL of phosphate buffer (pH = 5.5), and 1.5 mL of toluene. Place the Erlenmeyer flask in an incubator at 37 °C for 72 h.

[0047] 2. After the incubation is completed, add 1 g of potassium alum, shake at 150 rpm for 30 min, then filter. Take the filtrate and make up the volume to 25 mL with distilled water.

[0048] 3. Take 1 mL of the filtrate and measure the absorbance value A by colorimetry according to the standard curve color development method. Set a matrix-free control for each soil sample.

[0049] Cellulase activity calculation formula:

[0050]

[0051] In the formula, X is the cellulase activity in the dilution (mg / d / g). Generating 1 mg of glucose per g of soil sample per day is defined as one enzyme activity unit; ΔA = absorbance of the measurement tube A - absorbance of the control A. Substitute the measured ΔA into the standard curve formula to obtain x (mg / mL); V is the total volume of the enzymatic hydrolysis reaction (mL), 26.5 mL; M is the mass of the soil sample (g), 10 g; T is the enzymatic hydrolysis reaction time (d), 72 h = 3 d.

[0052] Example 2

[0053] The difference between Example 2 and Example 1 is that the soil pretreatment is different, and the others are the same.

[0054] Soil pretreatment

[0055] 1. Dry the fresh soil to a constant weight, then grind it into powder and pass through a 50-mesh sieve.

[0056] 2. Add 10 g of soil powder to 200 mL of distilled water, then add 1 g of impurity remover and stir at 150 rpm for 15 min. Then add 3 mL of hydrogen peroxide solution (1 mmol / L) and continue stirring for 55 min. After stirring is completed, filter to remove impurities, and take the filtrate as the sample to be tested.

[0057] The impurity remover is prepared from potassium sulfate and sodium sulfate with a mass ratio of 5:3.

[0058] Example 3

[0059] The difference between Example 3 and Example 1 is that the soil pretreatment is different, and the others are the same.

[0060] Soil pretreatment

[0061] 1. Dry the fresh soil to a constant weight, and then grind it into powder and pass through a 50-mesh sieve.

[0062] 2. Take 10 g of soil powder, add 200 mL of distilled water, then add 2 g of the impurity remover and stir at 150 rpm for 25 min. Then add 3 mL of hydrogen peroxide solution (1 mmol / L) and continue to stir for 35 min. After stirring, filter and take the filtrate as the test sample.

[0063] The impurity remover is prepared from potassium sulfate and sodium sulfate with a mass ratio of 1:7.

[0064] Comparative Example 1

[0065] The difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, the pretreatment is to grind the soil into powder, and the others are the same.

[0066] Take the fresh soil, dry it to a constant weight, and then grind it into powder and pass through a 50-mesh sieve.

[0067] Comparative Example 2

[0068] The difference between Comparative Example 2 and Example 1 is that in Comparative Example 2, an equal amount of distilled water is used to replace potassium sulfate, and the others are the same.

[0069] Comparative Example 3

[0070] The difference between Comparative Example 3 and Example 1 is that in the test of step three, after the cultivation in Comparative Example 3, it is directly filtered without adding potassium alum, and the others are the same.

[0071] Comparative Example 4

[0072] The difference between Comparative Example 4 and Example 1 is that in Comparative Example 4, an acetic acid-sodium acetate buffer solution with a pH of 5.0 is used to replace the phosphate buffer solution, and the others are the same.

[0073] Comparative Example 5

[0074] The difference between Comparative Example 5 and Example 1 is that in Comparative Example 5, citric acid-sodium citrate with a pH of 6.6 is used to replace the phosphate buffer solution, and the others are the same.

[0075] Comparative Example 6

[0076] Example 6 is different from Example 1 in that Comparative Example 6 uses a soil cellulase (S-CL) activity assay kit (tested by the anthrone colorimetric method), and the others are the same.

[0077] Comparative Example 7

[0078] Commercially available soil cellulase (S-CL) activity detection kit (visible spectrophotometry).

[0079] Test Example 1

[0080] Detect the cellulase activity in the soil according to the methods of Example 1 and Comparative Examples 1-6 respectively, and analyze the effects of different treatment methods on the enzyme activity.

[0081] Table 1 Results of soil cellulase activity detected by different detection methods

[0082]

[0083]

[0084] As can be seen from Table 1, when detecting the cellulase activity in the same soil by different detection methods, compared with Comparative Examples 1-6, the method of Example 1 has the highest detected cellulase activity in the soil. Specifically, from Comparative Example 1 and Example 1, it can be seen that after pretreatment with the impurity remover, the interference of factors such as metal ions can be removed, which is convenient for subsequent enzyme activity detection. Further, from Comparative Example 2, it can be seen that an appropriate ionic strength contributes to the stability of cellulase and is beneficial to subsequent activity detection; and in Comparative Example 3, after the reaction of the sample to be tested is completed, it is directly filtered, and it is easy to fail to filter out the solution due to the too viscous culture solution, resulting in the inability to detect the enzyme activity. This is because the 1% carboxymethyl cellulose solution needs to be heated to dissolve and becomes very viscous at room temperature, while adding potassium alum can reduce its viscosity, so that it can be filtered out for normal detection and can be used for the detection of different types of soil, with high flexibility and versatility. Again, from Comparative Examples 3 and 4, it can be seen that when phosphate is used as the buffer solution, the enzyme activity is the highest, indicating that the phosphate buffer solution provides the most suitable reaction environment for cellulase and can maintain a relatively stable pH value during the cellulase detection process.

[0085] Therefore, compared with other methods for detecting cellulase, the method of the present invention has higher sensitivity and accuracy, and can more effectively capture and measure the enzyme activity in the soil. Moreover, the present invention measures at a colorimetric wavelength of 508 nm. Compared with measuring the absorbance at 540 nm, its linearity is better and the colorimetry is more stable.

[0086] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment contains only one independent technical solution. This narrative style of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for detecting cellulase activity in soil, characterized in that: The detection method comprises the following steps: S1. Prepare a glucose standard solution, place the glucose standard solution and 3,5-dinitrosalicylic acid solution in a boiling water bath, detect at a wavelength of 508nm after cooling, and draw a standard curve; S2. Pre-treating the soil powder to remove impurities; S3. The pretreated sample to be tested was mixed with carboxymethyl cellulose solution, 0.2 mol / L phosphate buffer solution with a pH value of 5.5, and toluene and cultured at a constant temperature; S4. After the incubation, potassium aluminum sulfate was added and shaken for 30 min, and then filtered. The filtrate was colorimetrically determined at a wavelength of 508 nm according to the method in step S1; The pre-processing comprises:

1. Dry the fresh soil to constant weight, grind it into powder and pass it through a 50-mesh sieve; 2. Take 10g soil powder and add 100mL distilled water, then add 1.5g impurity remover and stir at 150rpm for 20min, then add 3mL 1mmol / L hydrogen peroxide solution and continue stirring for 45min. After stirring, filter and remove impurities, and take the filtrate as the sample to be tested; The impurity remover is prepared from potassium sulfate and sodium sulfate in a mass ratio of 3:

5.

2. The method for detecting cellulase activity in soil according to claim 1, characterized in that: The concentration of the glucose standard solution is 0-1.2 mg / mL.

3. The method for detecting cellulase activity in soil according to claim 1, characterized in that: In step S3, the culture time is 48-96 hours.

4. The method for detecting cellulase activity in soil according to claim 1, characterized in that: In step S3, the culture temperature is 35-40°C.

5. The method for detecting cellulase activity in soil according to claim 1, characterized in that: The mass percentage of the carboxymethyl cellulose solution is 1%.

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