CN106248604B - Method for determining laccase enzyme activity in soil - Google Patents
Method for determining laccase enzyme activity in soil Download PDFInfo
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 63
- 239000002689 soil Substances 0.000 title claims abstract description 58
- 230000000694 effects Effects 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 12
- 238000002835 absorbance Methods 0.000 claims abstract description 42
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 claims abstract description 36
- 239000000693 micelle Substances 0.000 claims abstract description 33
- 238000000605 extraction Methods 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- -1 ABTS free radical Chemical class 0.000 claims abstract description 8
- 238000004737 colorimetric analysis Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 81
- 239000011259 mixed solution Substances 0.000 claims description 36
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 claims description 16
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 claims description 16
- 239000007974 sodium acetate buffer Substances 0.000 claims description 13
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 13
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 150000003254 radicals Chemical class 0.000 claims description 11
- 239000004094 surface-active agent Substances 0.000 claims description 10
- 238000007605 air drying Methods 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000007873 sieving Methods 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000003517 fume Substances 0.000 claims description 3
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 229920002521 macromolecule Polymers 0.000 abstract description 2
- 239000010410 layer Substances 0.000 description 12
- 239000000126 substance Substances 0.000 description 6
- 239000003960 organic solvent Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 108010075016 Ceruloplasmin Proteins 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 235000019738 Limestone Nutrition 0.000 description 1
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 239000006028 limestone Substances 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7769—Measurement method of reaction-produced change in sensor
Abstract
The invention belongs to the field of soil science and environmental science, and particularly relates to a method for determining laccase enzyme activity in soil, which is characterized by comprising the following steps of: (1) pretreating soil; (2) preparing a reverse micelle system; (3) solving a linear equation of the relation between the ABTS free radical concentration and the absorbance; (4) and (3) measuring the laccase enzyme activity by an ABTS colorimetric method. The invention adopts reverse micelles to extract laccase in soil, the reverse micelles are suitable for purifying biological macromolecules, and the laccase can enter a 'water pool' of the reverse micelles when contacting with the reverse micelles, so that the activity of the laccase in the extraction process can be effectively ensured.
Description
Technical Field
The invention belongs to the field of soil science and environmental science, and particularly relates to a method for determining laccase enzyme activity in soil.
Background
Laccase is a protein combined with a plurality of copper ions, belongs to ceruloplasmin oxidase, is water as a byproduct of molecular oxygen catalytic oxidation reaction, belongs to environment-friendly enzymes, is a multifunctional enzyme with excellent characteristics in various aspects due to wide catalytic organic substrates of the laccase, is applied to a plurality of disciplines such as biology, chemistry and environment, at present, more and more scholars begin to utilize the laccase to treat soil polluted by organic matters, and the enzyme activity of the laccase is a key factor influencing the organic matter treatment capacity, so that the determination of the change rule of the laccase activity in the soil is particularly important.
The reverse micelle is an orientation micelle formed by a surfactant in an organic solvent, the surfactant can form a polar inner core with a hydrophilic group facing inwards and a hydrophobic group facing outwards in the organic solvent, the polar inner core of the reverse micelle can form a water pool phenomenon after dissolving a proper amount of water, the reverse micelle can selectively dissolve some biological substances such as protein, nucleic acid, amino acid and the like due to the existence of the water pool phenomenon, the biological substances can not be in direct contact with the organic solvent due to the separation of the reverse micelle between the biological substances and the organic solvent, and the microenvironment of the water pool can protect the activity of the biological substances. The reverse micelle is used for extracting the biological substances, so that the extraction rate is high, the extraction has selectivity, and the activity of the biological substances in the extraction process can be kept.
Disclosure of Invention
The invention aims to provide a method for effectively determining the laccase activity in soil.
In order to achieve the aim, the technical scheme adopted by the invention is that the method for measuring the laccase enzyme activity in the soil is characterized by comprising the following steps:
(1) pretreating soil:
taking back a soil sample to be detected, putting the soil sample into a fume hood for air drying, and grinding the air-dried soil and sieving the ground soil with a 20-mesh sieve for later use;
(2) preparation of reverse micelle system:
dissolving a surfactant AOT in isooctane to prepare an AOT/isooctane mixed solution with the concentration of 0.05-0.08 g/mL, adding KCl and an acetic acid-sodium acetate buffer solution into the AOT/isooctane mixed solution, and then carrying out ultrasonic treatment on the mixed solution until the surfactant AOT is completely dissolved, and clarifying and transparent the mixed solution to prepare a reverse micelle system;
(3) solving a linear equation of the relation between the ABTS free radical concentration and the absorbance:
respectively adding 0mL, 0.5mL, 1mL, 1.5mL, 2mL and 2.5mL of ABTS solution into 6 cuvettes with plugs, wherein the concentration of the ABTS solution is 4mmol/L, then respectively adding 1mL of laccase solution into 6 cuvettes with plugs, the enzyme activity of the laccase solution is 1U/mL, then adding distilled water into the 6 cuvettes with plugs to 25mL scale, then placing the 6 cuvettes with plugs uniformly for one hour, after placing for one hour, measuring the absorbance of the solution in the 6 cuvettes with plugs by using an ultraviolet spectrophotometer at the wavelength of 420nm and drawing a standard curve to obtain a linear equation of the relation between the concentration of ABTS free radicals of the mixed solution in the cuvettes with plugs and the absorbance:
Y＝aX+b，
in the formula: y is the concentration of ABTS free radicals (μmol/L); x is the absorbance (A); a. b is a constant to be solved;
(4) determining laccase enzyme activity by ABTS colorimetric method:
weighing 1.0-3.0 g of ground soil, placing the ground soil into a centrifuge tube, adding 10mL of a reverse micelle system into the centrifuge tube, placing the centrifuge tube into a shaking table to shake for 1-2 hours, after the shaking is finished, placing the centrifuge tube on a centrifuge, centrifuging for 15-30 minutes at the speed of 4000r/min, taking out the solution on the upper layer after the centrifuging is finished, placing the solution into another clean centrifuge tube for later use, continuously adding the soil on the lower layer into the 10mL of the reverse micelle system, repeating the shaking and centrifuging operations for two times, and making three parallel samples for each sample;
adding an acetic acid-sodium acetate buffer solution with the same volume into the taken solution at the upper layer, oscillating for 15-20 minutes, then placing the mixed solution into a centrifuge, centrifuging for 30-40 minutes at the speed of 4000r/min, taking out the laccase solution at the lower layer after centrifugal layering to realize reverse extraction of the laccase, adding 1.5mL of the laccase solution after reverse extraction and 1.5mL of ABTS solution into a cuvette, wherein the concentration of the ABTS solution is 0.5mmol/L, then measuring the absorbance of the mixed solution in the cuvette every 15s within 2 minutes by using an ultraviolet spectrophotometer at the wavelength of 420nm, and solving a linear equation of the relationship between the absorbance of the mixed solution in the cuvette and the time:
S＝cT+d，
in the formula: s is the absorbance (A); t is time (min); c. d is a constant to be solved;
the calculation formula of the enzyme activity of the soil laccase to be detected is as follows:
in the formula: e is the slope of a linear equation of the relation between the ABTS free radicals and the absorbance, namely e is equal to the constant a obtained in the step (3); f is the slope of a linear equation of the relationship between the absorbance and the time, namely f is equal to the constant c obtained in the step (4); v1Volume of laccase solution after back extraction (mL); v2Is the volume (mL) of laccase solution after addition to the cuvette for back extraction; m is the soil mass (g); v3Volume of ABTS solution (mL) added to the cuvette.
Furthermore, the concentration of the added KCl solution is 0.1mol/L, and 0.5-0.8 mL of KCl solution is added into every 10mL of AOT/isooctane mixed solution.
Further, the pH value of the added acetic acid-sodium acetate buffer solution is 2.0-6.0, and the concentration is 0.1 mol/L.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention adopts the reverse micelle to extract the laccase in the soil, the reverse micelle is suitable for purifying biological macromolecules, and the laccase can enter a 'water pool' of the reverse micelle when contacting with the reverse micelle, so that the activity of the laccase in the extraction process can be effectively ensured.
2. The invention uses acetic acid-sodium acetate buffer solution to back extract the laccase in the reverse micelles, and the buffer solution can keep the laccase in a relatively stable environment and ensure that the enzyme activity of the laccase reaches the optimum degree.
3. The laccase enzyme activity detection technology used in the invention is simple, convenient and rapid, and the used reverse micelle solution can be repeatedly used, thus being suitable for batch detection.
Drawings
FIG. 1 is a graph of the linear relationship between ABTS radicals and absorbance in example 1;
FIG. 2 is a graph showing the linear relationship between absorbance and time in example 1;
FIG. 3 is a graph showing the linear relationship between ABTS radicals and absorbance in example 2;
FIG. 4 is a graph of the linear relationship of absorbance versus time in example 2;
wherein R is2Is a linear correlation index.
Detailed Description
The process of the present invention is further described below with reference to specific embodiments, without limiting the scope of protection.
Examples 1
A method for measuring laccase enzyme activity in soil is characterized by comprising the following steps:
(1) pretreating soil:
collecting brown soil on the surface of the limestone soil, putting the brown soil into a fume hood for air drying, and grinding the air-dried soil and sieving the ground soil with a 20-mesh sieve for later use;
(2) preparation of reverse micelle system:
dissolving a surfactant AOT in isooctane to prepare an AOT/isooctane mixed solution with the concentration of 0.05g/mL, adding KCl and an acetic acid-sodium acetate buffer solution into the AOT/isooctane mixed solution, and then carrying out ultrasonic treatment on the mixed solution until the surfactant AOT is completely dissolved, and clarifying and transparent the mixed solution to prepare a reverse micelle system;
(3) solving a linear equation of the relation between the ABTS free radical concentration and the absorbance:
respectively adding 0mL, 0.5mL, 1mL, 1.5mL, 2mL and 2.5mL of ABTS solution into 6 cuvettes with plugs, wherein the concentration of the ABTS solution is 4mmol/L, then respectively adding 1mL of laccase solution into 6 cuvettes with plugs, the enzyme activity of the laccase solution is 1U/mL, then adding distilled water into the 6 cuvettes with plugs to 25mL scale, then placing the 6 cuvettes with plugs uniformly for one hour after shaking uniformly, measuring the absorbance of the solution in the 6 cuvettes with plugs by using an ultraviolet spectrophotometer at the wavelength of 420nm after placing for one hour, drawing a standard curve (see figure 1), and solving a linear equation of the relation between the concentration of ABTS free radicals and the absorbance of the mixed solution in the cuvettes with plugs:
Y＝aX+b，
in the formula: y is the concentration of ABTS free radicals (μmol/L); x is the absorbance (A); a. b is a constant obtained, a is 192.1, b is 2.950, and R is a linear correlation index2＝0.998；
(4) Determining laccase enzyme activity by ABTS colorimetric method:
weighing 1.0g of ground soil, placing the ground soil into a centrifuge tube, adding 10mL of reverse micelle system into the centrifuge tube, placing the centrifuge tube into a shaking table to shake for 1 hour, placing the centrifuge tube on a centrifuge to centrifuge for 15 minutes at the speed of 4000r/min after shaking is finished, taking out the solution on the upper layer after centrifugation is finished, placing the solution into another clean centrifuge tube for later use, continuously adding the soil on the lower layer into the 10mL of reverse micelle system, repeating the shaking and centrifuging operations for two times, and making three parallel samples for each sample;
adding an equal volume of acetic acid-sodium acetate buffer solution into the extracted solution at the upper layer, shaking for 15 minutes, then placing the mixed solution into a centrifuge, centrifuging for 30 minutes at the speed of 4000r/min, extracting the laccase solution at the lower layer after centrifugal layering to realize the back extraction of laccase, adding 1.5mL of the laccase solution after back extraction and 1.5mL of ABTS solution into a cuvette, wherein the concentration of the ABTS solution is 0.5mmol/L, then measuring the absorbance of the mixed solution in the cuvette every 15s within 2 minutes by using an ultraviolet spectrophotometer at the wavelength of 420nm, drawing an absorption curve (figure 2) by using the absorbance as a vertical coordinate and the time as a horizontal coordinate, and solving a linear equation of the relationship between the absorbance of the mixed solution in the cuvette and the time:
S＝cT+d，
in the formula: s is the absorbance (A); t is time (min); c. d is a constant obtained, c is 0.358, d is 0.058, and R is a linear correlation index2＝0.999；
The enzyme activity of laccase in soil is calculated according to the following formula:
in the formula: e is the slope of the linear equation of the relation between ABTS free radical and absorbanceA rate, i.e., e is equal to the constant a obtained in step (3), e is 192.1; f is the slope of the linear equation of the absorbance versus time, i.e., f is equal to the constant c obtained in step (4), and f is 0.358; v1Volume (mL) of laccase solution after back extraction, V1＝30mL；V2Volume (mL) of laccase solution after back extraction for addition to the cuvette, V21.5 mL; m is soil mass (g), and m is 1 g; v3Is added as the volume (mL) of ABTS solution in the cuvette, V3＝1.5mL。
Furthermore, the concentration of the added KCl solution is 0.1mol/L, and 0.5-0.8 mL of KCl solution is added into every 10mL of AOT/isooctane mixed solution.
Further, the pH value of the added acetic acid-sodium acetate buffer solution is 2.0-6.0, and the concentration is 0.1 mol/L.
EXAMPLES example 2
A method for measuring laccase enzyme activity in soil is characterized by comprising the following steps:
(1) pretreating soil:
air-drying the collected subtropical red soil surface layer soil, grinding the soil, air-drying, and sieving with a 20-mesh sieve for later use;
(2) preparation of reverse micelle system:
dissolving a surfactant AOT in isooctane to prepare an AOT/isooctane mixed solution with the concentration of 0.08g/mL, adding KCl and an acetic acid-sodium acetate buffer solution into the AOT/isooctane mixed solution, and then carrying out ultrasonic treatment on the mixed solution until the surfactant AOT is completely dissolved, and clarifying and transparent the mixed solution to prepare a reverse micelle system;
(3) solving a linear equation of the relation between the ABTS free radical concentration and the absorbance:
respectively adding 0mL, 0.5mL, 1mL, 1.5mL, 2mL and 2.5mL of ABTS solution into 6 cuvettes with plugs, wherein the concentration of the ABTS solution is 4mmol/L, then respectively adding 1mL of laccase solution into 6 cuvettes with plugs, the enzyme activity of the laccase solution is 1U/mL, then adding distilled water into the 6 cuvettes with plugs to 25mL scale, then placing the 6 cuvettes with plugs uniformly for one hour after shaking uniformly, measuring the absorbance of the solution in the 6 cuvettes with plugs by using an ultraviolet spectrophotometer at the wavelength of 420nm after placing for one hour, drawing a standard curve (see FIG. 3), and solving a linear equation of the relation between the concentration of ABTS free radicals of the mixed solution in the cuvettes with plugs and the absorbance:
Y＝aX+b，
in the formula: y is the concentration of ABTS free radicals (μmol/L); x is the absorbance (A); a. b is a constant obtained by a 193.7 and b is 0.105, and a linear correlation index R2＝0.999；
(4) Determining laccase enzyme activity by ABTS colorimetric method:
weighing 2.0g of ground soil, placing the ground soil into a centrifuge tube, adding 10mL of reverse micelle system into the centrifuge tube, placing the centrifuge tube into a shaking table to vibrate for 2 hours, placing the centrifuge tube on a centrifuge to centrifuge for 30 minutes at the speed of 4000r/min after vibration, taking out the solution on the upper layer after centrifugation, placing the solution into another clean centrifuge tube for later use, continuously adding the soil on the lower layer into the 10mL of reverse micelle system, repeating the vibration and centrifugation operation twice, and making three parallel samples for each sample;
adding an equal volume of acetic acid-sodium acetate buffer solution into the extracted solution at the upper layer, shaking for 20 minutes, then placing the mixed solution into a centrifuge, centrifuging for 40 minutes at the speed of 4000r/min, extracting the laccase solution at the lower layer after centrifugal layering to realize the back extraction of laccase, adding 1.5mL of the laccase solution after back extraction and 1.5mL of ABTS solution into a cuvette, wherein the concentration of the ABTS solution is 0.5mmol/L, then measuring the absorbance of the mixed solution in the cuvette every 15s within 2 minutes by using an ultraviolet spectrophotometer at the wavelength of 420nm, drawing an absorption curve by using the absorbance as a vertical coordinate and using the time as a horizontal coordinate (figure 4), and solving a linear equation of the relationship between the absorbance of the mixed solution in the cuvette and the time:
S＝cT+d，
in the formula: s is the absorbance (A); t is time (min); c. d is a constant obtained, c is 0.666, d is 0.107, and R is a linear correlation index2＝0.999；
The enzyme activity of laccase in soil is calculated according to the following formula:
in the formula: e is the slope of the linear equation of the relationship between ABTS free radicals and absorbance, namely, e is equal to the constant a obtained in the step (3), and e is 193.7; f is the slope of the linear equation of the absorbance versus time, i.e., f is equal to the constant c obtained in step (4), and f is 0.666; v1Volume (mL) of laccase solution after back extraction, V1＝30mL；V2Volume (mL) of laccase solution after back extraction for addition to the cuvette, V21.5 mL; m is soil mass (g), and m is 2 g; v3Is added as the volume (mL) of ABTS solution in the cuvette, V3＝1.5mL。
Furthermore, the concentration of the added KCl solution is 0.1mol/L, and 0.5-0.8 mL of KCl solution is added into every 10mL of AOT/isooctane mixed solution.
Further, the pH value of the added acetic acid-sodium acetate buffer solution is 2.0-6.0, and the concentration is 0.1 mol/L.
Claims (3)
1. A method for measuring laccase enzyme activity in soil is characterized by comprising the following steps:
(1) pretreating soil:
taking back a soil sample to be detected, putting the soil sample into a fume hood for air drying, and grinding the air-dried soil and sieving the ground soil with a 20-mesh sieve for later use;
(2) preparation of reverse micelle system:
dissolving a surfactant AOT in isooctane to prepare an AOT/isooctane mixed solution with the concentration of 0.05-0.08 g/mL, adding KCl and an acetic acid-sodium acetate buffer solution into the AOT/isooctane mixed solution, and then carrying out ultrasonic treatment on the mixed solution until the surfactant AOT is completely dissolved, and clarifying and transparent the mixed solution to prepare a reverse micelle system;
(3) solving a linear equation of the relation between the ABTS free radical concentration and the absorbance:
respectively adding 0mL, 0.5mL, 1mL, 1.5mL, 2mL and 2.5mL of ABTS solution into 6 cuvettes with plugs, wherein the concentration of the ABTS solution is 4mmol/L, then respectively adding 1mL of laccase solution into 6 cuvettes with plugs, the enzyme activity of the laccase solution is 1U/mL, then adding distilled water into the 6 cuvettes with plugs to 25mL scale, then placing the 6 cuvettes with plugs uniformly for one hour, after placing for one hour, measuring the absorbance of the solution in the 6 cuvettes with plugs by using an ultraviolet spectrophotometer at the wavelength of 420nm and drawing a standard curve to obtain a linear equation of the relation between the concentration of ABTS free radicals of the mixed solution in the cuvettes with plugs and the absorbance:
Y＝aX+b，
in the formula: y is the concentration of ABTS free radicals (μmol/L); x is the absorbance (A); a. b is a constant to be solved;
(4) determining laccase enzyme activity by ABTS colorimetric method:
weighing 1.0-3.0 g of ground soil, placing the ground soil into a centrifuge tube, adding 10mL of a reverse micelle system into the centrifuge tube, placing the centrifuge tube into a shaking table to shake for 1-2 hours, after the shaking is finished, placing the centrifuge tube on a centrifuge, centrifuging for 15-30 minutes at the speed of 4000r/min, taking out the solution on the upper layer after the centrifuging is finished, placing the solution into another clean centrifuge tube for later use, continuously adding the soil on the lower layer into the 10mL of the reverse micelle system, repeating the shaking and centrifuging operations for two times, and making three parallel samples for each sample;
adding an acetic acid-sodium acetate buffer solution with the same volume into the taken solution at the upper layer, oscillating for 15-20 minutes, then placing the mixed solution into a centrifuge, centrifuging for 30-40 minutes at the speed of 4000r/min, taking out the laccase solution at the lower layer after centrifugal layering to realize reverse extraction of the laccase, adding 1.5mL of the laccase solution after reverse extraction and 1.5mL of ABTS solution into a cuvette, wherein the concentration of the ABTS solution is 0.5mmol/L, then measuring the absorbance of the mixed solution in the cuvette every 15s within 2 minutes by using an ultraviolet spectrophotometer at the wavelength of 420nm, and solving a linear equation of the relationship between the absorbance of the mixed solution in the cuvette and the time:
S＝cT+d，
in the formula: s is the absorbance (A); t is time (min); c. d is a constant to be solved;
the calculation formula of the enzyme activity of the soil laccase to be detected is as follows:
in the formula: e is the slope of a linear equation of the relation between the ABTS free radicals and the absorbance, namely e is equal to the constant a obtained in the step (3); f is the slope of a linear equation of the relationship between the absorbance and the time, namely f is equal to the constant c obtained in the step (4); v1Volume of laccase solution after back extraction (mL); v2Is the volume (mL) of laccase solution after addition to the cuvette for back extraction; m is the soil mass (g); v3Volume of ABTS solution (mL) added to the cuvette.
2. The method for determining laccase enzyme activity in soil according to claim 1, wherein the concentration of the added KCl solution is 0.1mol/L, and 0.5-0.8 mL of KCl solution is added into every 10mL of AOT/isooctane mixed solution.
3. The method for determining laccase enzyme activity in soil according to claim 1, wherein the pH of the acetate-sodium acetate buffer is 2.0-6.0, and the concentration is 0.1 mol/L.
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| WO1996038577A1 (en) * | 1995-06-01 | 1996-12-05 | Biotechnology Research And Development Corp. | Enzyme catalysis in organic solutions |
| CN101270385A (en) * | 2007-03-21 | 2008-09-24 | 中国科学院沈阳应用生态研究所 | Analysis method for testing soil denitrification enzyme liveness |
| CN102533688A (en) * | 2012-03-07 | 2012-07-04 | 湖南大学 | Method for extracting and purifying lignin peroxidase by using reverse micelles |
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| WO1996038577A1 (en) * | 1995-06-01 | 1996-12-05 | Biotechnology Research And Development Corp. | Enzyme catalysis in organic solutions |
| CN101270385A (en) * | 2007-03-21 | 2008-09-24 | 中国科学院沈阳应用生态研究所 | Analysis method for testing soil denitrification enzyme liveness |
| CN102533688A (en) * | 2012-03-07 | 2012-07-04 | 湖南大学 | Method for extracting and purifying lignin peroxidase by using reverse micelles |
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Application publication date: 20161221 Assignee: Wuhan University of science and Technology Business Incubator Co., Ltd Assignor: WUHAN University OF SCIENCE AND TECHNOLOGY Contract record no.: X2020420000008 Denomination of invention: A method for determination of laccase activity in soil Granted publication date: 20200310 License type: Common License Record date: 20201022 |