CN106248604A - A kind of measure the method for laccase activity in soil - Google Patents
A kind of measure the method for laccase activity in soil Download PDFInfo
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- CN106248604A CN106248604A CN201610528833.4A CN201610528833A CN106248604A CN 106248604 A CN106248604 A CN 106248604A CN 201610528833 A CN201610528833 A CN 201610528833A CN 106248604 A CN106248604 A CN 106248604A
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 69
- 239000002689 soil Substances 0.000 title claims abstract description 41
- 230000000694 effects Effects 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 63
- 238000002835 absorbance Methods 0.000 claims abstract description 43
- 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 32
- 238000000605 extraction Methods 0.000 claims abstract description 24
- 239000007974 sodium acetate buffer Substances 0.000 claims abstract description 14
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 11
- 102000004190 Enzymes Human genes 0.000 claims abstract description 11
- 150000003254 radicals Chemical class 0.000 claims abstract description 7
- 238000004737 colorimetric analysis Methods 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 239000011259 mixed solution Substances 0.000 claims description 20
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 claims description 16
- -1 BTS free radical Chemical class 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 239000004094 surface-active agent Substances 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 6
- 230000009514 concussion Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 239000003517 fume Substances 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 4
- 239000007853 buffer solution Substances 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 description 7
- 239000003960 organic solvent Substances 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 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
- 102000006833 Multifunctional Enzymes Human genes 0.000 description 1
- 108010047290 Multifunctional Enzymes Proteins 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 229910052955 covellite Inorganic materials 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 125000001165 hydrophobic group Chemical group 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
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 239000000758 substrate 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
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- 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
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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
- 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
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- General Health & Medical Sciences (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to agrology and environmental science, be specifically related to a kind of measure the method for laccase activity in soil, it is characterised in that it comprises the following steps: the pretreatment of (1) soil；(2) preparation of reverse micelle system；(3) ABTS number of free radical and the linear equation of absorbance relation are solved；(4) ABTS colorimetric method for determining laccase activity.The present invention uses reverse micelle to extract the laccase in soil, reverse micelle is suitable for the purification of biomacromolecule, and in when laccase contacts with reverse micelle, laccase can enter reverse micelle " pond ", can effectively ensure that the activity of laccase during extraction, present invention laccase in acetic acid sodium-acetate buffer back extraction reverse micelle, buffer solution can make laccase be maintained in a metastable environment and make the enzyme of laccase live to reach optimum degree, in addition the laccase activity detection technique inverse micellar solution that is easy to be quick and that use that the present invention uses can be reused, it is suitable for batch detection.
Description
Technical field
The invention belongs to agrology and environmental science, be specifically related to a kind of measure the method for laccase activity in soil.
Background technology
Laccase is a kind of protein being combined with multiple copper ion, belongs to covellite oxidase, and laccase utilizes molecular oxygen catalysis
The by-product of oxidation reaction is water, belongs to environment-friendly type enzyme, and owing to the catalysis organic substrates of laccase is relatively broad so that paint
Enzyme has become a kind of multifunctional enzyme with many-sided good characteristic, and its application relates to multiple subjects such as biology, chemistry and environment
Field, has had increasing scholar to start with laccase treatment by the soil of Organic Pollution, and the enzyme of laccase has been lived
It is the key factor affecting Organic substance disposal ability, therefore measures the Changing Pattern of laccase activity in soil and seem and be even more important.
Reverse micelle is a kind of direction micelle that surfactant is formed in organic solvent, and surfactant is at organic solvent
Middle can form the hydrophilic group hydrophobic group polarity kernel with multimolecules reaction model outwardly inwardly, in the polarity of reverse micelle
" pond " phenomenon can be formed, due to the existence of " pond " phenomenon so that reverse micelle can be the most molten after karyolysis suitable quantity of water
Solve some biological substances, such as protein, nucleic acid and aminoacid etc., due between these biological substance and organic solvents across anti-glue
Group so that biological substance directly will not contact with organic solvent, simultaneously the activity of the biological substance that the microenvironment in pond is protected again.
Using reverse micelle extraction biological substance, not only extraction yield is high, and extraction has selectivity, additionally it is possible to keep biological substance in extraction process
Activity.
Summary of the invention
It is an object of the invention to provide and a kind of can effectively measure the method for laccase activity in soil.
For achieving the above object, the present invention adopts the technical scheme that, a kind of measures the method for laccase activity in soil, its
It is characterised by that it comprises the following steps:
(1) pretreatment of soil:
After pedotheque to be measured is fetched, put in fume hood and air-dry, then by after air-dried ground 20 mesh sieves of soil
Standby；
(2) preparation of reverse micelle system:
Surfactant AOT is dissolved in isobutyltrimethylmethane., is made into the AOT/ isobutyltrimethylmethane. that concentration is 0.05～0.08g/mL and mixes
Close liquid, in AOT/ isobutyltrimethylmethane. mixed liquor, then add KCl and Acetic acid-sodium acetate buffer, then mixed liquor is carried out ultrasonic,
Until surfactant AOT is completely dissolved, and mixed solution clear, prepare reverse micelle system；
(3) ABTS number of free radical and the linear equation of absorbance relation are solved:
The ABTS being separately added into 0mL, 0.5mL, 1mL, 1.5mL, 2mL, 2.5mL in the color-comparison tube of 6 25mL is molten
Liquid, the concentration of ABTS solution is 4mmol/L, then 6 equipped with the color-comparison tube of ABTS solution in each to add 1mL laccase molten
Liquid, the enzyme of laccase solution is lived as 1U/mL, then all adds distilled water in 6 color-comparison tubes to 25mL scale, then by 6
Color-comparison tube is placed one hour after all shaking up, and after placing one hour, use ultraviolet spectrophotometer is under the wavelength of 420nm
Measure the absorbance of solution in 6 color-comparison tubes and draw standard curve, obtain in color-comparison tube mixed solution A BTS from
By base concentration and the linear equation of absorbance relation:
Y=aX+b,
In formula: Y is the concentration (μm ol/L) of ABTS free radical；X is absorbance (A)；A, b are constant to be asked；
(4) ABTS colorimetric method for determining laccase activity:
Soil after grinding weighs 1.0～3.0g and puts in centrifuge tube, and adds 10mL reverse micelle body in centrifuge tube
System, is then put into centrifuge tube shaking table concussion 1～2 hours, after having shaken, is placed on by centrifuge tube on centrifuge with 4000r/
The centrifugation 15～30min of min, puts in the cleanest centrifuge tube standby by the solution taking-up being in upper strata after being centrifuged
With, the reverse micelle system that the soil being in lower floor continuously adds 10mL repeats above-mentioned concussion and centrifugally operated, repeats two
Secondary, and each sample does three parallel samples；
Add isopyknic Acetic acid-sodium acetate buffer to being in the solution on upper strata of taking-up, and shake 15～20 points
Clock, is then placed in a centrifuge mixed solution, with the centrifugation 30 of 4000r/min～40 minutes, takes out after centrifugal layering
Be in the laccase solution of lower floor, it is achieved the back extraction of laccase, add in cuvette the laccase solution after 1.5mL back extraction and
The ABTS solution of 1.5mL, wherein the concentration of ABTS solution is 0.5mmol/L, then utilizes ultraviolet spectrophotometer 420nm's
Under wavelength, in 2 minutes, measure the absorbance of mixed solution in a cuvette every 15s, obtain mixed solution in cuvette
Absorbance and the linear equation of time relationship:
S=cT+d,
In formula: S is absorbance (A)；T is time (min)；C, d are constant to be asked；
The computing formula of soil laccase activity to be measured is as follows:
In formula: e is ABTS free radical and the slope of the linear equation of absorbance relation, i.e. e is equal to trying to achieve in step (3)
Constant a；F is the slope of absorbance and the linear equation of time relationship, i.e. f is equal to the constant c tried to achieve in step (4)；V1For instead
The volume (mL) of laccase solution after extraction；V2For joining the volume (mL) of laccase solution after back extraction in cuvette；M is soil
Quality (g)；V3For adding the volume (mL) of ABTS solution in cuvette.
Further, the concentration of the KCl solution of described addition is 0.1mol/L, and addition is that the AOT/ isobutyltrimethylmethane. of every 10mL mixes
Close the KCl solution adding 0.5～0.8ml in liquid.
Further, the pH of the Acetic acid-sodium acetate buffer of described addition is 2.0～6.0, and concentration is 0.1mol/L.
The present invention compared with prior art, has the following advantages and effect:
1, the present invention uses reverse micelle to extract the laccase in soil, and reverse micelle is suitable for the purification of biomacromolecule, works as laccase
In when contacting with reverse micelle, laccase can enter reverse micelle " pond ", it is possible to the activity of laccase during effective guarantee extraction.
2, present invention laccase in Acetic acid-sodium acetate buffer back extraction reverse micelle, buffer solution can make laccase protect
Hold in a metastable environment and make the enzyme of laccase live and reach optimum degree.
3, the laccase activity detection technique that the present invention uses inverse micellar solution that is easy to be quick and that use can be reused,
It is suitable for batch detection.
Accompanying drawing explanation
Fig. 1 is ABTS free radical and the linear relationship chart of absorbance relation in embodiment 1；
Fig. 2 is the linear relationship chart in embodiment 1 for absorbance with time relationship；
Fig. 3 is ABTS free radical and the linear relationship chart of absorbance relation in embodiment 2；
Fig. 4 is absorbance and the linear relationship chart of time relationship in embodiment 2；
Wherein, R2For linear correlation index.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the inventive method is further described, not the limit to its protection domain
System.
Embodiment 1
A kind of measure the method for laccase activity in soil, it is characterised in that it comprises the following steps:
(1) pretreatment of soil:
Gather brown calcareous soil topsoil and put in fume hood air-dried, then by after air-dried ground 20 mesh sieves of soil
Standby；
(2) preparation of reverse micelle system:
Surfactant AOT is dissolved in isobutyltrimethylmethane., is made into the AOT/ isobutyltrimethylmethane. mixed liquor that concentration is 0.05g/mL, so
After in AOT/ isobutyltrimethylmethane. mixed liquor, add KCl and Acetic acid-sodium acetate buffer, then mixed liquor is carried out ultrasonic, until table
Face activating agent AOT is completely dissolved, and mixed solution clear, prepares reverse micelle system；
(3) ABTS number of free radical and the linear equation of absorbance relation are solved:
The ABTS being separately added into 0mL, 0.5mL, 1mL, 1.5mL, 2mL, 2.5mL in the color-comparison tube of 6 25mL is molten
Liquid, the concentration of ABTS solution is 4mmol/L, then 6 equipped with the color-comparison tube of ABTS solution in each to add 1mL laccase molten
Liquid, the enzyme of laccase solution is lived as 1U/mL, then all adds distilled water in 6 color-comparison tubes to 25mL scale, then by 6
Color-comparison tube is placed one hour after all shaking up, and after placing one hour, use ultraviolet spectrophotometer is under the wavelength of 420nm
Measure the absorbance of solution in 6 color-comparison tubes and draw standard curve (see Fig. 1), obtaining mixed solution in color-comparison tube
ABTS number of free radical and the linear equation of absorbance relation:
Y=aX+b,
In formula: Y is the concentration (μm ol/L) of ABTS free radical；X is absorbance (A)；A, b are the constant tried to achieve, and a=
192.1, b=2.950, linear correlation index R2=0.998；
(4) ABTS colorimetric method for determining laccase activity:
Soil after grinding weighs 1.0g and puts in centrifuge tube, and adds 10mL reverse micelle system in centrifuge tube, so
After centrifuge tube be put into shaking table shake 1 hour, after having shaken, centrifuge tube is placed on centrifuge the speed with 4000r/min
The centrifugal 15min of degree, puts in the cleanest centrifuge tube standby by the solution taking-up being in upper strata after being centrifuged, will be in down
The soil of layer continuously adds the reverse micelle system of 10mL and repeats above-mentioned concussion and centrifugally operated, is repeated twice, and each
Sample does three parallel samples；
It is in the solution on upper strata adds isopyknic Acetic acid-sodium acetate buffer to take out, and shake 15 minutes, so
After mixed solution is placed in a centrifuge, with the centrifugation 30 minutes of 4000r/min, take out after centrifugal layering and be in lower floor
Laccase solution, it is achieved the back extraction of laccase, adds the laccase solution after 1.5mL back extraction and the ABTS of 1.5mL in cuvette
Solution, wherein the concentration of ABTS solution is 0.5mmol/L, then utilize ultraviolet spectrophotometer under the wavelength of 420nm, 2
Measuring the absorbance of mixed solution in a cuvette every 15s in minute, with absorbance as vertical coordinate, the time is that abscissa is painted
Absorption curve processed (Fig. 2), obtains the linear equation of mixed solution absorbance and time relationship in cuvette:
S=cT+d,
In formula: S is absorbance (A)；T is time (min)；C, d are the constant tried to achieve, and c=0.358, d=0.058, line
Property index of correlation R2=0.999；
In soil, the enzyme of laccase is lived and is calculated as follows:
In formula: e is ABTS free radical and the slope of the linear equation of absorbance relation, i.e. e is equal to trying to achieve in step (3)
Constant a, e=192.1；F is the slope of absorbance and the linear equation of time relationship, i.e. f is equal to the constant tried to achieve in step (4)
C, f=0.358；V1For the volume (mL) of laccase solution, V after back extraction1=30mL；V2For joining in cuvette after back extraction
The volume (mL) of laccase solution, V2=1.5mL；M is soil quality (g), m=1g；V3For adding as ABTS solution in cuvette
Volume (mL), V3=1.5mL.
Further, the concentration of the KCl solution of described addition is 0.1mol/L, and addition is that the AOT/ isobutyltrimethylmethane. of every 10mL mixes
Close the KCl solution adding 0.5～0.8ml in liquid.
Further, the pH of the Acetic acid-sodium acetate buffer of described addition is 2.0～6.0, and concentration is 0.1mol/L.
Embodiment 2
A kind of measure the method for laccase activity in soil, it is characterised in that it comprises the following steps:
(1) pretreatment of soil:
Air-dry the Subtropical Red Soil topsoil gathered, and soil is ground air-dry after cross after 20 mesh sieves standby；
(2) preparation of reverse micelle system:
Surfactant AOT is dissolved in isobutyltrimethylmethane., is made into the AOT/ isobutyltrimethylmethane. mixed liquor that concentration is 0.08g/mL, so
After in AOT/ isobutyltrimethylmethane. mixed liquor, add KCl and Acetic acid-sodium acetate buffer, then mixed liquor is carried out ultrasonic, until table
Face activating agent AOT is completely dissolved, and mixed solution clear, prepares reverse micelle system；
(3) ABTS number of free radical and the linear equation of absorbance relation are solved:
The ABTS being separately added into 0mL, 0.5mL, 1mL, 1.5mL, 2mL, 2.5mL in the color-comparison tube of 6 25mL is molten
Liquid, the concentration of ABTS solution is 4mmol/L, then 6 equipped with the color-comparison tube of ABTS solution in each to add 1mL laccase molten
Liquid, the enzyme of laccase solution is lived as 1U/mL, then all adds distilled water in 6 color-comparison tubes to 25mL scale, then by 6
Color-comparison tube is placed one hour after all shaking up, and after placing one hour, use ultraviolet spectrophotometer is under the wavelength of 420nm
Measure the absorbance of solution in 6 color-comparison tubes and draw standard curve (see Fig. 3), obtaining mixed solution in color-comparison tube
ABTS number of free radical and the linear equation of absorbance relation:
Y=aX+b,
In formula: Y is the concentration (μm ol/L) of ABTS free radical；X is absorbance (A)；A, b are the constant tried to achieve, and a=
193.7, b=0.105, linear correlation index R2=0.999；
(4) ABTS colorimetric method for determining laccase activity:
Soil after grinding weighs 2.0g and puts in centrifuge tube, and adds 10mL reverse micelle system in centrifuge tube, so
After centrifuge tube be put into shaking table shake 2 hours, after having shaken, centrifuge tube is placed on centrifuge the speed with 4000r/min
The centrifugal 30min of degree, puts in the cleanest centrifuge tube standby by the solution taking-up being in upper strata after being centrifuged, will be in down
The soil of layer continuously adds the reverse micelle system of 10mL and repeats above-mentioned concussion and centrifugally operated, is repeated twice, and each
Sample does three parallel samples；
It is in the solution on upper strata adds isopyknic Acetic acid-sodium acetate buffer to take out, and shake 20 minutes, so
After mixed solution is placed in a centrifuge, with the centrifugation 40 minutes of 4000r/min, take out after centrifugal layering and be in lower floor
Laccase solution, it is achieved the back extraction of laccase, adds the laccase solution after 1.5mL back extraction and the ABTS of 1.5mL in cuvette
Solution, wherein the concentration of ABTS solution is 0.5mmol/L, then utilize ultraviolet spectrophotometer under the wavelength of 420nm, 2
Measuring the absorbance of mixed solution in a cuvette every 15s in minute, with absorbance as vertical coordinate, the time is that abscissa is painted
Absorption curve processed (Fig. 4), obtains the linear equation of mixed solution absorbance and time relationship in cuvette:
S=cT+d,
In formula: S is absorbance (A)；T is time (min)；C, d are the constant tried to achieve, and c=0.666, d=0.107, line
Property index of correlation R2=0.999；
In soil, the enzyme of laccase is lived and is calculated as follows:
In formula: e is ABTS free radical and the slope of the linear equation of absorbance relation, i.e. e is equal to trying to achieve in step (3)
Constant a, e=193.7；F is the slope of absorbance and the linear equation of time relationship, i.e. f is equal to the constant tried to achieve in step (4)
C, f=0.666；V1For the volume (mL) of laccase solution, V after back extraction1=30mL；V2For joining in cuvette after back extraction
The volume (mL) of laccase solution, V2=1.5mL；M is soil quality (g), m=2g；V3For adding as ABTS solution in cuvette
Volume (mL), V3=1.5mL.
Further, the concentration of the KCl solution of described addition is 0.1mol/L, and addition is that the AOT/ isobutyltrimethylmethane. of every 10mL mixes
Close the KCl solution adding 0.5～0.8ml in liquid.
Further, the pH of the Acetic acid-sodium acetate buffer of described addition is 2.0～6.0, and concentration is 0.1mol/L.
Claims (3)
1. one kind measures the method for laccase activity in soil, it is characterised in that it comprises the following steps:
(1) pretreatment of soil:
After pedotheque to be measured is fetched, put in fume hood and air-dry, then by standby after air-dried ground 20 mesh sieves of soil；
(2) preparation of reverse micelle system:
Surfactant AOT is dissolved in isobutyltrimethylmethane., is made into the AOT/ isobutyltrimethylmethane. mixed liquor that concentration is 0.05～0.08g/mL,
Then in AOT/ isobutyltrimethylmethane. mixed liquor, add KCl and Acetic acid-sodium acetate buffer, then mixed liquor is carried out ultrasonic, until
Surfactant AOT is completely dissolved, and mixed solution clear, prepares reverse micelle system；
(3) ABTS number of free radical and the linear equation of absorbance relation are solved:
The ABTS solution of 0mL, 0.5mL, 1mL, 1.5mL, 2mL, 2.5mL it is separately added in the color-comparison tube of 6 25mL,
The concentration of ABTS solution is 4mmol/L, then 6 equipped with the color-comparison tube of ABTS solution in each add 1mL laccase solution,
The enzyme of laccase solution is lived as 1U/mL, then all adds distilled water in 6 color-comparison tubes to 25mL scale, then by 6 tools
Plug color comparison tube is placed one hour after all shaking up, and measures 6 with ultraviolet spectrophotometer after placing one hour under the wavelength of 420nm
The absorbance of solution draw standard curve in color-comparison tube, obtains mixed solution A BTS free radical in color-comparison tube dense
The linear equation of degree and absorbance relation:
Y=aX+b,
In formula: Y is the concentration (μm ol/L) of ABTS free radical；X is absorbance (A)；A, b are constant to be asked；
(4) ABTS colorimetric method for determining laccase activity:
Soil after grinding weighs 1.0～3.0g and puts in centrifuge tube, and adds 10mL reverse micelle system in centrifuge tube, so
After centrifuge tube is put into shaking table concussion 1～2 hours, after having shaken, centrifuge tube is placed on centrifuge with 4000r/min's
Centrifugation 15～30min, puts in the cleanest centrifuge tube standby by the solution taking-up being in upper strata after being centrifuged, will
It is in the soil of lower floor to continuously add the reverse micelle system of 10mL and repeat above-mentioned concussion and centrifugally operated, is repeated twice, and
And each sample does three parallel samples；
It is in the solution on upper strata adds isopyknic Acetic acid-sodium acetate buffer to take out, and shake 15～20 minutes, so
After mixed solution is placed in a centrifuge, with the centrifugation 30 of 4000r/min～40 minutes, take out after centrifugal layering and be in down
The laccase solution of layer, it is achieved the back extraction of laccase, adds the laccase solution after 1.5mL back extraction and 1.5mL in cuvette
ABTS solution, wherein the concentration of ABTS solution is 0.5mmol/L, then utilize ultraviolet spectrophotometer under the wavelength of 420nm,
In 2 minutes, measure the absorbance of mixed solution in a cuvette every 15s, obtain in cuvette mixed solution absorbance with
The linear equation of time relationship:
S=cT+d,
In formula: S is absorbance (A)；T is time (min)；C, d are constant to be asked；
The computing formula of soil laccase activity to be measured is as follows:
In formula: e is the slope of ABTS free radical and the linear equation of absorbance relation, i.e. e is equal to the constant tried to achieve in step (3)
a；F is the slope of absorbance and the linear equation of time relationship, i.e. f is equal to the constant c tried to achieve in step (4)；V1For back extraction
The volume (mL) of rear laccase solution；V2For joining the volume (mL) of laccase solution after back extraction in cuvette；M is soil quality
(g)；V3For adding the volume (mL) of ABTS solution in cuvette.
The most according to claim 1 a kind of measure the method for laccase activity in soil, it is characterised in that the KCl of described addition
The concentration of solution is 0.1mol/L, addition be every 10mL AOT/ isobutyltrimethylmethane. mixed liquor in add 0.5～0.8ml KCl molten
Liquid.
The pH of the Acetic acid-sodium acetate buffer added the most according to claim 1 is 2.0～6.0, and concentration is 0.1mol/L.
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| CN112345413A (en) * | 2020-09-18 | 2021-02-09 | 武汉大学 | Method for testing global distribution of infiltration water and inorganic solute under micro-spray irrigation condition |
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| WO1996038577A1 (en) * | 1995-06-01 | 1996-12-05 | Biotechnology Research And Development Corp. | Enzyme catalysis in organic solutions |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112345413A (en) * | 2020-09-18 | 2021-02-09 | 武汉大学 | Method for testing global distribution of infiltration water and inorganic solute under micro-spray irrigation condition |
| CN112345413B (en) * | 2020-09-18 | 2021-12-17 | 武汉大学 | Method for testing global distribution of infiltration water and inorganic solute under micro-spray irrigation condition |
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