CN101936909B - A kind of enzymatic activity testing method of microorganism degrading benzene ring - Google Patents

Abstract

The invention discloses an enzyme activity test method for degrading benzene rings by microorganisms, which comprises the following steps: 1. making a standard curve of the relation between the substrate concentration and the absorbance, and finding out a linear fitting equation between the substrate concentration and the absorbance; 2. preparing a substrate solution according to the catalysis conditions of the enzyme or the bacterial suspension with the activity to be detected; 3. respectively heating a substrate solution and an enzyme solution to respective optimal reaction temperatures in advance, then mixing and carrying out catalytic reaction under the optimal reaction conditions, and detecting the concentration change of the substrate after the reaction; 4. measuring the content of benzene rings in the reaction liquid: diluting the reaction solution before and after the catalytic reaction to enable the absorbance of the reaction solution to be within the range of a linear equation, testing the absorbance of the reaction solution under the maximum absorption wavelength, and then calculating the substrate concentration before and after the reaction according to the linear equation; 5. the calculation is according to formula : enzyme activity U =DELTAC/M/DELTAt X10³And calculating to obtain the enzyme activity of the microorganism for degrading the benzene ring.

Description

A kind of enzymatic activity testing method of microorganism degrading benzene ring

technical field

The invention relates to a technology for testing enzyme activity in the catalysis process of whole cells of microorganisms, in particular to a method for testing enzyme activity of microorganisms degrading benzene rings.

Background technique

Substances containing benzene rings are widely used in the chemical industry, so the amount of industrial wastewater formed is large, and benzene ring substances have certain toxic effects on the environment, and even have carcinogenic and teratogenic effects on organisms. Therefore, benzene ring substances degradation is a hot topic of research. Biodegradation is a commonly used method for the degradation of pollutants. Benzene rings have certain biodegradability. Therefore, there are many studies on microbial degradation of substances containing benzene rings.

During the degradation process of substances containing benzene rings, the opening of benzene rings is an important indicator for evaluating the degradation situation. Therefore, to evaluate a microorganism's ability to degrade pollutants, the key is to investigate the degradation rate of its biological enzymes on the benzene ring, that is, the enzyme activity that degrades the benzene ring. In the existing enzyme activity research methods, the enzyme activity is generally judged by examining the change of a certain side group chromophore, but due to the different chromophores of each substance, this kind of test method does not have good universality. Within the scope of the applicant's search, there is no report on the literature about directly adopting the benzene ring opening method to measure the enzyme activity.

Contents of the invention

Aiming at the limitations of the existing enzyme activity test methods, the technical problem to be solved by the present invention is to provide an enzyme activity test method for microbial degradation of benzene rings. This test method has good universality and directness, and is simple and easy. OK.

The technical solution of the present invention to solve the technical problem is to design a method for testing the enzyme activity of microorganisms degrading benzene rings, the test method comprising the following steps:

1. Make a standard curve of the relationship between substrate concentration and absorbance: prepare a series of substrate solutions with different concentrations of benzene rings, and perform full-wavelength scanning on a UV spectrophotometer to determine the substrates with different concentrations of benzene rings The maximum absorption wavelength of the solution, and then measure the absorbance of substrate solutions with different concentrations of benzene rings at the maximum absorption wavelength, draw the standard curve of the relationship between substrate concentration and absorbance, and find out the linearity between substrate concentration and absorbance The fitting degree reaches the substrate concentration range above 0.999, and the linear equation used to calculate the change of the benzene ring content in the degradation process is obtained by fitting; the substrate is a substance containing a benzene ring;

2. Prepare the substrate solution: prepare the substrate solution according to the catalytic conditions of the enzyme or bacterial suspension to be tested;

3. Catalytic reaction: Heat the substrate solution and enzyme solution to their respective optimum reaction temperatures in advance, then mix the substrate solution and enzyme solution into a reaction solution, and make it undergo a catalytic reaction under the optimum reaction conditions, and start Timing, after the time set by the catalytic reaction, take out the reaction solution, quickly inactivate it at high temperature, and detect the change of the substrate concentration;

4. Determination of the benzene ring content in the reaction solution: Dilute the reaction solution before and after the above-mentioned catalytic reaction by a corresponding multiple, so that the absorbance is within the range of the linear equation; the diluted reaction solution is tested at the maximum absorption wavelength before and after the reaction. And calculate the substrate concentration before and after the reaction according to the linear equation;

5. Calculate the enzyme activity of microorganisms to degrade benzene rings: Calculate the enzyme activities of microorganisms to degrade benzene rings according to the following formula (1):

Enzyme activity U=△C/M/△t×10 ³ (1)

(1) In the formula, △C represents the concentration change of the substrate in the reaction solution before and after the catalytic reaction, in g/L; M represents the relative molecular weight of the substrate, in g/mol; △t represents the catalytic reaction time, in units of min; 10 ³ is the correction amount between each amount unit and the specified enzyme activity unit.

The detection method of the present invention adopts the bacterial suspension as the catalytic medium, and the substance containing the benzene ring as the substrate, draws the corresponding standard curve through the change of the absorbance of the benzene ring during the catalytic degradation process, and calculates through a specific formula to obtain Corresponding enzyme activity data. Compared with the prior art, the test method of the present invention has good universality for the detection of enzyme activity in the degradation process of substances containing benzene rings, and is applicable to the direct determination of enzyme activities in the degradation process of most compounds containing benzene rings, and The test process is simple, does not require expensive instruments and equipment, and is very suitable for actual test use. the

Description of drawings

  Fig. 1 is the graph that a kind of embodiment (relationship between concentration of terephthalic acid and absorbance) draws of the enzymatic activity testing method of microbial degradation benzene ring of the present invention;

Fig. 2 is a graph drawn by another embodiment (relationship between protocatechuic acid concentration and absorbance) of the enzyme activity test method for microbial degradation of benzene rings of the present invention.

Detailed ways

The test method of the present invention will be further described below in conjunction with embodiment:

The enzyme activity test method (abbreviation test method) of the microbial degradation benzene ring designed by the present invention, this test method comprises the following steps:

1. Make a standard curve of the relationship between the concentration and absorbance of a substrate containing a benzene ring (hereinafter referred to as the substrate): prepare a series of solutions of substances containing a benzene ring at different concentrations, and perform full-wavelength scanning on a UV spectrophotometer to determine The maximum absorption wavelength of different concentrations of benzene ring-containing substance solutions, and then measure the absorbance of different concentrations of benzene ring-containing substance solutions at its maximum absorption wavelength, draw a standard curve of the relationship between substrate concentration and absorbance, and find out the substrate The linear fitting degree between the concentration and the absorbance reaches the substrate concentration range above 0.999, and the linear equation used to calculate the change of the benzene ring content during the degradation process is obtained by fitting;

2. Prepare the substrate solution: prepare the substrate solution according to the catalytic conditions of the enzyme or bacterial suspension to be tested; the catalytic conditions refer to 3-4g/L potassium dihydrogen phosphate and 6-7g/L phosphoric acid Sodium hydrogen disodium is used as a buffer solution to maintain a stable pH value of the system = 6.8-7.0, and finally a substrate containing a benzene ring, including terephthalic acid or protocatechuic acid, is added to form a 1-2g/L solution.

Studies have shown that adding 1-3 g/L of yeast extract powder as an organic nitrogen source to the catalytic reaction solution can effectively promote the consumption rate of microorganisms on carbon sources, thereby increasing enzyme activity.

3. Catalytic reaction: Heat the substrate solution and enzyme solution to their respective optimum reaction temperatures (for example, 37°C) in advance, and then mix the substrate solution and enzyme solution to form a reaction solution. The optimum reaction conditions refer to temperature, rotation speed The rotation speed of the shaking incubator, etc., for example: 37°C, 200r/min, to cause a catalytic reaction to occur, and start timing. After the time set by the catalytic reaction, such as 30min, take out the reaction solution, quickly inactivate it at high temperature, and detect the bottom. Changes in the concentration of substances;

4. Determination of the benzene ring content in the reaction solution: Dilute the reaction solution before and after the above catalytic reaction by a corresponding multiple, such as 50-100 times, so that the absorbance is within the range of the linear equation; the diluted reaction solution is at the maximum absorption wavelength The absorbance before and after the reaction was measured, and the substrate concentration before and after the reaction was calculated according to the linear equation.

5. Calculate the enzymatic activity of microorganisms degrading benzene rings: In the test method of the present invention, the definition of enzyme activity is: 1ml of enzyme solution or bacterial suspension can degrade benzene rings in 1 μmol of substances containing benzene rings within 1 minute, defined as One enzyme activity unit U/ml. According to the following formula (1), the enzyme activity of microbial degradation of benzene ring was calculated:

Enzyme activity U=△C/M/△t×10 ³ (1)

(1) In the formula, △C represents the concentration change of the substrate in the reaction solution before and after the catalytic reaction, in g/L; M represents the relative molecular weight of the substrate, in g/mol; △t represents the catalytic reaction time, in units of min; 10 ³ is the correction amount between each amount unit and the specified enzyme activity unit.

What is not mentioned in the present invention is applicable to the prior art.

The specific examples of the testing method of the present invention are given below: the specific examples are only to further describe the present invention in detail, and do not constitute limitations to the claims of the present invention.

Example 1

The enzymatic activity of Bacillus F4 degrading terephthalic acid with terephthalic acid as the sole carbon source.

Use phosphate buffer (3g/L potassium dihydrogen phosphate and 7g/L disodium hydrogen phosphate) to prepare terephthalic acid solutions with concentrations of 1, 2, 5, 10, 20, 30, 40mg/L (standard solution), carry out full-wavelength scanning on the prepared terephthalic acid solution on an ultraviolet spectrophotometer, determine that its maximum absorption wavelength is at 240nm, and then measure the absorbance of the terephthalic acid solution with different concentrations at a wavelength of 240nm , corresponding to the concentration-absorbance curve (see Figure 1), find out that the concentration range of its linear fitting degree (fitting degree reaches 0.99986) is 1-40mg/L, and the linear equation (2) is obtained by fitting: Y = 0.08342 +0.07584 ×X, where X represents the concentration of terephthalic acid, Y represents the UV absorbance, and the linear equation (2) is used to calculate the change of benzene ring content during the degradation process of this example.

For the catalytic conditions of the bacterial suspension to be tested for enzyme activity, it is necessary to add 3g/L potassium dihydrogen phosphate and 7g/L disodium hydrogen phosphate as a buffer solution to maintain a stable pH value of 6.8, and use this buffer solution to prepare 1g/L L of terephthalic acid solution.

After sterilizing the substrate solution, heat it to 37°C, suspend the centrifuged bacteria in the sterilized solution, mix well, shake the reaction at 37°C, 200r/min, and start timing, after 30min , take out the reaction solution, quickly inactivate it in a water bath at 95°C for 5 minutes, and detect the change of the substrate:

The reaction solution before and after the above reaction was diluted 50 times to make it within the concentration range of the linear equation. The absorbance of the diluted solution before and after the test reaction at a wavelength of 240nm was 1.607 and 1.472, respectively. According to the linear equation, the concentration of terephthalic acid before and after the reaction is calculated as 1.004g/L and 0.915g/L respectively; the concentration value of terephthalic acid obtained is substituted into the enzyme activity formula (1), and the enzyme activity U=△C/M /Δt×10 ³ , wherein, M is 166.13, and Δt is 30, that is, the enzyme activity of the obtained bacterial suspension is: U=0.018.

Example 2

Yeast powder was added as an organic nitrogen source, and the enzyme activity of Bacillus F4 degraded terephthalic acid.

The determination of the terephthalic acid standard solution and the corresponding concentration-absorbance curve plot are the same as in Example 1 (see Figure 1), and the linear equation (2) is obtained by fitting: Y = 0.08342 + 0.07584 × X, where X represents p-phenylene Diformic acid concentration, Y represents the UV absorbance, and the linear equation (2) can be used to calculate the change of benzene ring content during the degradation process of this example.

Adding yeast powder as an organic nitrogen source in the catalytic reaction solution can effectively promote the consumption rate of the carbon source by microorganisms, thereby increasing the enzyme activity. In order to determine the degree of improvement of enzyme activity, 1g/L yeast extract powder was added to the reaction solution, and 3g/L potassium dihydrogen phosphate and 7g/L disodium hydrogen phosphate were added as a buffer solution to maintain a stable pH value of the system = 6.8, finally add terephthalic acid to make a 1g/L solution.

After sterilizing the substrate solution, heat it to 37°C, suspend the centrifuged cells in the sterilized solution, mix well, shake the reaction at 37°C, 200r/min, and start timing. After 30 minutes, the reaction solution was taken out and quickly inactivated in a water bath at 95°C for 5 minutes to detect changes in the substrate.

The reaction solution before and after the above reaction was diluted 50 times to make it within the concentration range of the linear equation. The absorbance of the diluted solution was 1.607 and 1.341 before and after the test at a wavelength of 240 nm. According to the linear equation, the concentrations of terephthalic acid before and after the reaction were 1.004g/L and 0.829g/L, respectively.

Substitute into formula (1), enzyme activity U=△C/M/△t·10 ³ , (wherein M is 166.13, △t is 30), and the enzyme activity of bacterial suspension is: U=0.035. Compared with the results in Example 1, it was found that adding yeast powder can nearly double the enzyme activity of the bacterial suspension.

Example 3

Enzyme activity of bacteria TJ degrading protocatechuic acid with protocatechuic acid as the sole carbon source.

Use phosphate buffer (4g/L potassium dihydrogen phosphate and 6g/L disodium hydrogen phosphate) to prepare protocatechuic acid solutions with concentrations of 1, 2, 5, 10, 20, 30, 40mg/L, respectively. Wavelength scanning, to determine the maximum absorption wavelength at 250nm, measure the absorbance of solutions with different concentrations at the wavelength of 250nm, draw a graph corresponding to the concentration-absorbance (see Figure 2), and find out that the linear fit is up to standard (the fit reaches 0.99971 ) concentration range is 1-40mg/L, and the linear equation (3) is obtained by fitting: Y = 0.07613 +0.07579 × X, where X represents the concentration of protocatechuic acid, Y represents the UV absorbance, and the linear equation (3) can be used to calculate Changes in benzene ring content during degradation.

The catalytic conditions of the bacterial suspension to be tested for enzyme activity need to add 4g/L potassium dihydrogen phosphate and 6g/L disodium hydrogen phosphate as a buffer solution to maintain a stable pH value of about 7. Therefore, a buffer solution is used to prepare 2g /L of protocatechuic acid solution.

After sterilizing the substrate solution, heat it to 37°C, suspend the centrifuged cells in the sterilized solution, mix well, shake the reaction at 37°C, 200r/min, and start timing. After 30 minutes, the reaction solution was taken out and quickly inactivated in a water bath at 95°C for 5 minutes to detect changes in the substrate.

The reaction solution before and after the above reaction was diluted 100 times to make it within the concentration range of the linear equation. The absorbance of the diluted solution was 1.592 and 1.491 before and after the test at a wavelength of 250 nm. According to the linear equation, the concentrations of terephthalic acid before and after the reaction were 2.000g/L and 1.867g/L, respectively.

Substituting into the formula (1), the enzyme activity U=△C/M/△t×10 ³ , (wherein M is 154.12, △t is 30), and the enzyme activity of the bacterial suspension is: U=0.029.

Example 4

Yeast powder was added as organic nitrogen source, and the enzyme activity of bacteria TJ degraded protocatechuic acid.

The determination of the standard solution of protocatechuic acid and the corresponding concentration-absorbance curve are the same as in Example 3, and the linear equation (3) is obtained by fitting: Y = 0.07613 + 0.07579×X, where X represents the concentration of protocatechuic acid, and Y represents UV absorbance. The linear equation (3) can be used to calculate the change of benzene ring content during the degradation process of this example.

Adding yeast powder as an organic nitrogen source in the catalytic reaction solution can effectively promote the consumption rate of the carbon source by microorganisms, thereby increasing the enzyme activity. In order to determine the degree of improvement of enzyme activity, 3g/L yeast extract powder was added to the reaction solution, and 4g/L potassium dihydrogen phosphate and 6g/L disodium hydrogen phosphate were added as a buffer solution to maintain a stable pH of the system. 7 or so, and finally add protocatechuic acid to make a 2g/L solution.

After sterilizing the substrate solution, heat it to 37°C, suspend the centrifuged cells in the sterilized solution, mix well, shake the reaction at 37°C, 200r/min, and start timing. After 30 minutes, the reaction solution was taken out and quickly inactivated in a water bath at 95°C for 5 minutes to detect changes in the substrate.

The reaction solution before and after the above reaction was diluted 100 times to make it within the concentration range of the linear equation. The absorbance of the diluted solution was 1.592 and 1.388 before and after the test at a wavelength of 250 nm. According to the linear equation, the concentrations of terephthalic acid before and after the reaction were 2.000g/L and 1.731g/L, respectively.

Substituting the formula (1), the enzyme activity U=△C/M/△t·10 ³ , (where M is 154.12, and △t is 30), the enzyme activity of the bacterial suspension is: U=0.058. Compared with the results in Example 3, it was found that adding yeast powder can more than double the enzyme activity of the bacterial suspension.

Example 5

Yeast powder was added as organic nitrogen source, and the enzyme activity of bacteria TJ degraded protocatechuic acid.

The determination of the standard solution of protocatechuic acid and the corresponding concentration-absorbance curve are the same as in Example 3, and the linear equation (3) is obtained by fitting: Y = 0.07613 + 0.07579×X, where X represents the concentration of protocatechuic acid, and Y represents UV absorbance. The linear equation (3) can be used to calculate the change of benzene ring content during the degradation process of this example.

Adding yeast powder as an organic nitrogen source in the catalytic reaction solution can effectively promote the consumption rate of the carbon source by microorganisms, thereby increasing the enzyme activity. In order to determine the degree of improvement of enzyme activity, 2g/L of yeast extract powder was added to the reaction solution, and 4g/L of potassium dihydrogen phosphate and 6g/L of disodium hydrogen phosphate were added as a buffer solution to maintain a stable pH of the system. 7 or so, and finally add protocatechuic acid to make a 1.5g/L solution.

After sterilizing the substrate solution, heat it to 37°C, suspend the centrifuged cells in the sterilized solution, mix well, shake the reaction at 37°C, 200r/min, and start timing. After 30 minutes, the reaction solution was taken out and quickly inactivated in a water bath at 95°C for 5 minutes to detect changes in the substrate.

The reaction solution before and after the above reaction was diluted 75 times to make it within the concentration range of the linear equation. The absorbance of the diluted solution was 1.592 and 1.324 before and after the test at a wavelength of 250 nm. According to the linear equation, the concentrations of terephthalic acid before and after the reaction were calculated to be 1.500g/L and 1.235g/L, respectively.

Substituting into the formula (1), the enzyme activity U=△C/M/△t·10 ³ , (wherein M is 154.12, △t is 30), and the enzyme activity of the bacterial suspension is: U=0.057. Compared with the results in Example 3, it was found that adding yeast powder can nearly double the enzyme activity of the bacterial suspension.

Claims (3)

1. a kind of enzymatic activity test method of microbial degradation benzene ring, this test method comprises the following steps: (1). Make a standard curve of the relationship between substrate concentration and absorbance: Prepare a series of substrate solutions with different concentrations of benzene rings, and perform full-wavelength scanning on a UV spectrophotometer to determine the concentration of different benzene rings. The maximum absorption wavelength of the substrate solution, and then measure the absorbance of the substrate solution with different concentrations of benzene rings at the maximum absorption wavelength, draw the standard curve of the relationship between the substrate concentration and the absorbance, and find out the relationship between the substrate concentration and the absorbance. The linear fitting degree reaches the substrate concentration range above 0.999, and the linear equation used to calculate the change of the benzene ring content in the degradation process is obtained by fitting; the substrate is a substance containing a benzene ring; (2). Preparation of substrate solution: according to the catalytic conditions of the enzyme or bacterial suspension to be tested, prepare the substrate solution; (3). Catalytic reaction: heat the substrate solution and enzyme solution to their respective optimum reaction temperatures in advance, then mix the substrate solution and enzyme solution into a reaction solution, and make it undergo a catalytic reaction under the optimum reaction conditions. And start timing, after the time set by the catalytic reaction, take out the reaction solution, quickly inactivate it at high temperature, and detect the change of the substrate concentration; (4). Determination of the benzene ring content in the reaction solution: Dilute the reaction solution before and after the above-mentioned catalytic reaction by a corresponding multiple, so that the absorbance is within the range of the linear equation; the diluted reaction solution is tested at the maximum absorption wavelength before and after the reaction. Absorbance, and calculate the substrate concentration before and after the reaction according to the linear equation; (5). Calculating the enzyme activity of microbial degradation of benzene ring: according to the following formula (1), calculate the enzyme activity of microbial degradation of benzene ring: Enzyme activity U=ΔC/M/Δt×10 ³ (1) (1) In the formula, ΔC represents the concentration change of the substrate in the reaction solution before and after the catalytic reaction, and the unit is g/L; M represents the relative molecular weight of the substrate, and the unit is g/mol, and Δt represents the catalytic reaction time, and the unit is min; 10 ³ is the correction amount between each amount unit and the specified enzyme activity unit. 2. the enzyme activity testing method of microbial degradation benzene ring according to claim 1 is characterized in that described substrate is terephthalic acid or protocatechuic acid; Described catalytic condition refers to 3-4g/L Potassium dihydrogen phosphate and disodium hydrogen phosphate of 6-7g/L are used as a buffer solution to maintain a stable pH value of the system=6.8-7.0, and finally the substrate is added to form a solution of 1-2g/L; the optimal reaction The temperature is 37°C; the optimum reaction conditions refer to a temperature of 37°C and a rotating speed of the shaking incubator at 200r/min; the time set for the catalytic reaction is 30min; and the corresponding dilution ratio is 50-100 times. 3. The enzyme activity test method for microbial degradation of benzene ring according to claim 1 or 2, characterized in that 1-3g/L of yeast extract powder is added to the catalytic reaction liquid.

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