Method for detecting activity of cellulase in soil

Abstract

The invention provides a method for detecting the activity of cellulase in soil, and belongs to the technical field of soil detection. According to the method, soil is pretreated through the impurity removal agent containing potassium sulfate and sodium sulfate, interference factors such as metal ions can be effectively removed, so that the detection accuracy is improved, then aluminum potassium sulfate is added after culture of the sample to be detected, the carboxymethyl cellulose solution, the phosphate buffer solution and methylbenzene is finished, and the situation that filtration is affected due to the fact that the culture solution is too thick is avoided. The detection method provided by the invention can accurately detect the activity of cellulase in soil, and compared with the existing method for determination at 540nm, the method provided by the invention has the advantages that the cellulase activity is tested at 508nm, a good linear relationship is shown, and the color change is more stable and reliable.

Description

Technical Field

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

[0002] Soil cellulase (S-CL) is mainly derived from soil microorganisms. It is a complex enzyme that can catalyze the decomposition of cellulose into cellobiose and further decompose it into glucose, thereby providing microorganisms with available carbon source nutrients. 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 the soil, the determination of cellulase activity is crucial to understanding soil fertility and plant growth environment.

[0003] At present, the main methods for detecting soil cellulase include spectrophotometry, fluorescence, and enzyme-linked immunosorbent assay. Among them, spectrophotometry mainly uses soil cellulase to catalyze cellulose decomposition to generate soluble sugars, and calculates the activity of soil cellulase by measuring the amount of sugar generated. However, it is easily affected by interference factors during the determination process. For example, metal ions may react chemically with the detection reagent to generate colored substances or precipitation, interfering with the normal color development reaction, or hindering the normal combination of substrate and enzyme, reducing the catalytic activity of the enzyme, thereby affecting the accurate determination of the enzymatic hydrolysis product, resulting in deviations in the enzyme activity detection results, and interfering with the authenticity of the test results; and in complex soil environments, cellulase is prone to non-specific adsorption, adsorbed to soil particles or other impurities, thereby affecting the accuracy of the test results. In addition, the fluorescence method and enzyme-linked immunosorbent assay are relatively complicated to operate, have 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 provides reliable results can be designed based on 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 cellulase activity in soil.

[0006] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions:

[0007] In one aspect, the present invention provides a method for detecting cellulase activity in soil, the detection method comprising the following steps:

[0008] 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;

[0009] S2. Pre-treating the soil powder to remove impurities;

[0010] S3. The pretreated soil was mixed with carboxymethyl cellulose solution, buffer solution and toluene and cultured at a constant temperature;

[0011] S4. After the culture is completed, add potassium aluminum sulfate and shake for 30 minutes, then filter, and take the filtrate and perform colorimetric determination at a wavelength of 508nm according to the method in step S1.

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

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

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

[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 of the embodiments, in step S3, the culturing time is 48-96 hours.

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

[0021] In some embodiments, the buffer 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 uses an impurity remover to pre-treat the soil to remove interference factors such as metal ions, thereby improving the detection accuracy. The impurity remover includes potassium sulfate and sodium sulfate. During the pre-treatment process, sodium sulfate and potassium sulfate improve the stability of cellulase by adjusting the ionic strength of the solution, and reduce the non-specific adsorption of cellulase, thereby avoiding the cellulase from being adsorbed on soil particles or impurities and limiting its contact with the substrate; at the same time, sulfate ions can also precipitate with metal ions that interfere with the activity of cellulase, thereby reducing the interference of metal ions on the detection results. Then, the present invention adds potassium aluminum sulfate after the pre-treated sample to be tested is cultured with 1% carboxymethyl cellulose solution, phosphate buffer and toluene, which is conducive to the filtration of the culture solution.

[0025] The detection method of the present invention can accurately detect the cellulase activity in the soil, and compared with the existing method of measuring at 540nm, the present invention tests 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 solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 It is the standard curve diagram of Example 1. DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

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

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

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

[0033] Phosphate buffer (pH=5.5): a mixture of 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 generate brown-red amino compounds. The product has a characteristic absorption peak at 508nm. The change in absorbance can be used to characterize the activity of soil cellulase.

[0035] Example 1

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

[0037] 1. Draw a standard curve

[0038] 1. Take 1mL of glucose standard solution of different concentrations and put it into a 25mL volumetric flask, add 3mL of 3,5-dinitrosalicylic acid solution and boil it in a water bath for 5min, then quickly cool it to room temperature, dilute it to 25mL with distilled water, and perform colorimetric determination at a wavelength of 508nm on a spectrophotometer after 15min. Use a blank tube to adjust the color at a wavelength of 508nm, and draw a standard curve with absorbance as the ordinate and glucose concentration (mg / mL) as the abscissa. Figure 1 shown.

[0039] Use distilled water to prepare glucose standard solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1, and 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 standard concentration (mg / mL) and y is the absorbance value.

[0041] 2. Soil Pretreatment

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

[0043] 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 hydrogen peroxide solution (1mmol / L) and continue stirring for 45min. After stirring, filter and remove impurities, and take the filtrate as the sample to be tested.

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

[0045] 3. 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, and place the Erlenmeyer flask in a 37°C incubator for 72 h.

[0047] 2. After the culture is completed, add 1g of potassium aluminum sulfate, shake at 150rpm for 30min, filter, take the filtrate and make up to 25mL with distilled water.

[0048] 3. Take 1 mL of the filtrate and determine the absorbance value A by colorimetric method according to the standard curve. Set up a no-matrix control for each soil sample.

[0049] Calculation formula of cellulase activity:

[0050]

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

[0052] Example 2

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

[0054] Soil pretreatment

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

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

[0057] The impurity remover is prepared from potassium sulfate and sodium sulfate in 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 other aspects are the same.

[0060] Soil pretreatment

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

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

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

[0064] Comparative Example 1

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

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

[0067] Comparative Example 2

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

[0069] Comparative Example 3

[0070] The difference between Comparative Example 3 and Example 1 is that, in the step 3 test, Comparative Example 3 is directly filtered after the cultivation is completed without adding potassium aluminum sulfate, and the other conditions are the same.

[0071] Comparative Example 4

[0072] The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 uses an acetic acid-sodium acetate buffer solution with a pH of 5.0 instead of a phosphate buffer, and the other contents are the same.

[0073] Comparative Example 5

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

[0075] Comparative Example 6

[0076] The difference between Example 6 and Example 1 is that Example 6 uses a soil cellulase (Solid-cellulase, S-CL) activity assay kit (anthrone colorimetric test), and the rest are the same.

[0077] Comparative Example 7

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

[0079] Test Example 1

[0080] The cellulase activity in the soil was detected according to the methods of Example 1 and Comparative Examples 1-6, and the effects of different treatment methods on the enzyme activity were analyzed.

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

[0082]

[0083]

[0084] As can be seen from Table 1, different detection methods detect the activity of cellulase in the same soil. Compared with Comparative Examples 1-6, the method of Example 1 detects the activity of cellulase in the soil with the highest activity. Specifically, it can be seen from Comparative Example 1 and Example 1 that after pretreatment with impurity remover, the interference of factors such as metal ions can be removed, which is convenient for subsequent detection of enzyme activity. It can be further seen from Comparative Example 2 that the appropriate ionic strength contributes to the stability of cellulase, which is conducive to subsequent activity detection; and in Comparative Example 3, the sample to be tested is directly filtered after the reaction is completed, which is easy to filter out the solution due to the viscous culture solution, and the enzyme activity cannot be detected. This is because the 1% carboxymethyl cellulose solution needs to be heated to dissolve, and becomes very viscous at room temperature, and adding potassium aluminum sulfate can reduce its viscosity, so that it can be filtered out for normal detection, which can be used for the detection of different types of soils, with high flexibility and versatility. It can be seen from Comparative Examples 3 and 4 that the cellulase has the highest enzyme activity when phosphate is used as a buffer solution, 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 activity of enzymes in soil. Moreover, the present invention is measured at a colorimetric wavelength of 508nm, which has better linearity and more stable colorimetry than measuring absorbance at 540nm.

[0086] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes 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 soil was mixed with carboxymethyl cellulose solution, buffer solution and toluene and cultured at a constant temperature; S4. After the culture is completed, add potassium aluminum sulfate and shake for 30 minutes, then filter, and take the filtrate and perform colorimetric determination at a wavelength of 508nm according to the method in step S1.

2. The method for detecting cellulase activity in soil according to claim 1, characterized in that: In step S1, the preprocessing includes: First, dry the soil and grind it into powder and pass it through a 50-mesh sieve. Then, add the soil powder to the impurity remover and stir at 150 rpm for 15-25 minutes. Then, add 1 mmol / L hydrogen peroxide solution and continue stirring for 35-55 minutes. After stirring, filter and take the filtrate as the sample to be tested. The impurity remover includes potassium sulfate and sodium sulfate.

3. The method for detecting cellulase activity in soil according to claim 2, characterized in that: The mass ratio of potassium sulfate to sodium sulfate is (1-5):(3-7).

4. The method for detecting cellulase activity in soil according to claim 3, characterized in that: The mass ratio of potassium sulfate to sodium sulfate is 3:

5.

5. 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.

6. 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.

7. 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.

8. The method for detecting cellulase activity in soil according to claim 1, characterized in that: The buffer solution is a 0.2 mol / L phosphate buffer solution with a pH value of 5.

5.

9. 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%.

Images