CN105784695B - The measuring method of Soil protease - Google Patents
The measuring method of Soil protease Download PDFInfo
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- CN105784695B CN105784695B CN201610121495.2A CN201610121495A CN105784695B CN 105784695 B CN105784695 B CN 105784695B CN 201610121495 A CN201610121495 A CN 201610121495A CN 105784695 B CN105784695 B CN 105784695B
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- G—PHYSICS
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The measuring method of Soil protease, the present invention relates to the measuring methods of protease, more particularly to a kind of measuring method of Soil protease.The purpose of the present invention is to solve the lower problems of the accuracy of measurement proteinase activity.The measuring method of Soil protease of the invention sequentially includes the following steps: one, preparation of reagents；Two, phosphate buffer solution (PBS) is prepared；Three, gelatin solution is prepared；Four, copper salt solution is prepared；Five, the culture of soil sample；Six, it measures；Seven, the drafting of the preparation of glycine titer and standard curve；Eight: result calculates.The measuring method of Soil protease of the present invention improves the accuracy of Soil protease determination of activity.The measuring method of Soil protease of the invention is used for the measurement of protease.
Description
Technical field
The present invention relates to the measuring methods of protease, more particularly to a kind of measuring method of Soil protease.
Background technique
Protein, peptides can be decomposed into amino acid by protease, be the important ectoenzyme of one of soil, enzyme tool
There is isolated activity, the Nitrogen Cycling of soil can be participated in.In the organic fertilizer of plant residue and application for entering soil, contain
A certain number of protein substances, therefore Soil protease, which can carry out enzymatic decomposition to this substance, can characterize soil with protease
The Nitrogen Status and its conversion process of earth.
In the measurement active method of Soil protease --- Philip Seymour Hoffman (Hoffmann) method (being also mantoquita colorimetric method) is
The active method of measurement Soil protease more commonly used at present.This method uses 1% gelatin culture of water dissolution, then uses and adds
Enter the copper-phosphate solution mixed in proportion, the amino acid discharged after enzymatic hydrolysis reacts to form blue complex with mantoquita, with point
Photometer carries out colorimetric, calculates amino acid content, and then analyze proteinase activity.But there is many deficiencies in this method, it is first
First, the effect of 1% gelatin substrate culture is not good enough, and the result measured is not sufficiently stable, secondly, copper phosphate mixes according to a certain percentage
Get togather carry out reaction generate blue compound effect it is not obvious enough, cause measurement result it is not accurate enough.Soil protease
Be put to the test being affected of reagent during determination of activity, cause current Philip Seymour Hoffman method measurement proteinase activity accuracy compared with
It is low.
Summary of the invention
The purpose of the present invention is to solve the lower problems of the accuracy of measurement proteinase activity, provide a kind of soil
The measuring method of protease.
The measuring method of Soil protease of the invention sequentially includes the following steps:
One, preparation of reagents: 0.10mol/L~0.15mol/L NaOH solution, 0.10mol/L~0.15mol/ are prepared respectively
L KH2PO4Solution；
Two, phosphate buffer solution (PBS) is prepared: the NaOH solution 790mL~795mL and KH for taking step 1 to prepare2PO4
Solution 1000mL~1010mL mixing, adjusts pH to 7.40~7.42；
Three, it prepares gelatin solution: gelatin being ground, and weighs 10.0g~10.4g, measure 700mL~710mL step
Rapid two phosphate buffer solutions (PBS) prepared are heated to 80 DEG C~85 DEG C, by phosphorus of the ground Gelatin after heating
Constant volume is cooled down in hydrochlorate buffer solution (PBS), after dissolution to 1L；
Four, copper salt solution is prepared: preparation 0.15mol/L~0.17mol/L CuCl first2Solution, then prepare 0.18mol/
L~0.19mol/L Na2HPO4Solution, to Na2HPO4The pH value that NaOH is used to adjust solution is added in solution, makes its pH 8.0
~8.5；Then 0.28mol/L~0.29mol/LNa is prepared2B4O7(anhydrous sodium tetraborate) solution, addition 0.08mol/L~
0.12mol/L hydrochloric acid makes its pH 6.0~6.5, set aside for use for adjusting the pH value of solution；
Five, the culture of soil sample: accurately weighing the 10.01~10.04g of Farmland Black Soil for picking up from field, and soil sample is made in drying, uses
Aperture is the sieve of 2mm, and then the soil sample after sieving is placed in container, ± 2 μ of toluene 4mL is accurately added with pipette
L places 13min~17min, and gelatin solution 39mL~41mL that step 3 is prepared then is added；Again with preservative film by vessel port
It seals, places in warmed-up 37 DEG C~38 DEG C insulating boxs, cultivate 23h~25h；
Six, it measures: the soil sample of step 5 culture being filtered, takes 10mL~12mL filtrate in 50mL~100mL centrifuge tube,
Add water 10mL~15mL, the CuCl of step 4 preparation is successively accurately added with pipette2± 2 μ L of solution 4mL, Na2HPO4Solution
8mL ± 2 μ L and Na2B4O7± 2 μ L of solution 8mL, centrifuge tube is covered, is mixed well, solution by blue become after bluish violet from
The heart, 4000r/min~4500r/min are centrifuged 4min~5min, after centrifugation, take supernatant at spectrophotometer 650nm
Measure absorbance value；
Seven, the drafting of the preparation of glycine titer and standard curve: (purpose: the size of proteinase activity is with sweet ammonia
The amount of acid indicates, so needing to be produced standard curve, draw the titer of glycine, marked with glycine configuration standard liquid
The value of prepare liquid is found on directrix curve it is known that proteinase activity size) accurately weigh 0.2679g glycine and be dissolved in steaming
Distilled water, and accurate dilutions, to 100mL, this is glycine standard solution, and 1mL contains 500 μ gNH3-N；It is accurately drawn with pipette sweet
Propylhomoserin titer 10mL is diluted with water to scale in 50mL volumetric flask to get 100 μ gNH3The titer of-N/mL, this solution
It should not deposit long；It draws respectively and dilutes 5 times of titers 0,2,4,6,8,10mL in 50mL~100mL centrifuge tube, be diluted with water to
20mL~25mL；CuCl is accurately successively added with pipette2± 2 μ L of solution 4mL, Na2HPO4± 2 μ L of solution 8mL, Na2B4O7It is molten
± 2 μ L of liquid 8mL；Centrifuge tube is covered, is mixed well, is centrifuged after becoming bluish violet by blue, 4000r/min~4500r/min
It is centrifuged 4~5min；After centrifugation, supernatant is taken to measure absorbance value at spectrophotometer 650nm；It is with glycine concentration
Abscissa, the absorbance value measured are that ordinate draws standard curve；
Eight: result calculates: drying soil sample in the NH of interior enzymolysis protein matter release for 24 hours with every gram3The quality (μ g) of-N carrys out table
Show proteinase activity (Pro): in Pro=aVn/m formula: a is the NH asked by standard curve3- N concentration (μ g/mL)；V is aobvious
Color liquid product (40mL)；N is to divide to take multiple；M is drying soil weight (g).
The calculation method of proteinase activity:
Soil sample is dried in the NH of interior enzymolysis protein matter release for 24 hours with every gram3The quality (μ g) of-N indicates proteinase activity
(Pro): in Pro=aVn/m formula: a is the NH asked by standard curve3- N concentration (μ g/mL)；V is developing solution volume
(40mL)；N is to divide to take multiple；M is drying soil weight (g).
The present invention compared with the existing technology the advantage is that:
It prepares gelatin substrate by using phosphate buffer to replace preparing gelatin with water, copper-salting liquid is also by successively distinguishing
It is added after the original first mixing in proportion of addition substitution, colour developing mode is also by mixing centrifugation colorimetric instead of originally three in centrifuge tube
Mixing colour developing filtering, improves the accuracy of Soil protease determination of activity in the bottle of angle.Simultaneously using this method with it is original general
Circulation method measures same Soil protease activity, the value that the method for the present invention obtains is respectively 793.47,787.70,789.16,
781.48, standard value is 788.85 ± 4.75, original commonsense method measure to obtain value respectively 638.45,622.96,666.78,
671.63,558.72, standard value is that 631.71 ± 45.48 present invention measurement Soil protease activity collimations are good, the coefficient of variation
Small, method feasibility is high.
Detailed description of the invention:
Fig. 1 is the standard curve of Glycine Levels.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: the measuring method of the Soil protease of present embodiment sequentially includes the following steps:
One, preparation of reagents: 0.10mol/L~0.15mol/L NaOH solution, 0.10mol/L~0.15mol/ are prepared respectively
L KH2PO4Solution；
Two, phosphate buffer solution is prepared: the NaOH solution 790mL~795mL and KH for taking step 1 to prepare2PO4Solution
1000mL~1010mL mixing, adjusts pH to 7.40~7.42；
Three, it prepares gelatin solution: gelatin is ground, measure the phosphate-buffered that 700mL~710mL step 2 is prepared
Solution is heated to 80 DEG C~85 DEG C, weighs the phosphate buffer solution that ground gelatin 10.0g~10.4g is dissolved in after heating
In, constant volume is cooled down after dissolution to 1L；
Four, copper salt solution is prepared: preparation 0.15mol/L~0.17mol/L CuCl first2Solution, then prepare 0.18mol/
L~0.19mol/L Na2HPO4Solution, to Na2HPO4NaOH is added in solution, makes its pH 8.0~8.5；Then it prepares
0.28mol/L~0.29mol/L Na2B4O7Solution, to Na2B4O70.08mol/L~0.12mol/L hydrochloric acid is added in solution, makes
Its pH is 6.0~6.5, set aside for use；
Five, the culture of soil sample: accurately weighing the 10.01~10.04g of Farmland Black Soil for picking up from field, and soil sample is made in drying, uses
Aperture is the sieve of 2mm, and then the soil sample after sieving is placed in container, ± 2 μ of toluene 4mL is accurately added with pipette
L places 13min~17min, and gelatin solution 39mL~41mL that step 3 is prepared then is added；Again with preservative film by vessel port
It seals, places in warmed-up 37 DEG C~38 DEG C insulating boxs, cultivate 23h~25h；
Six, it measures: the soil sample of step 5 culture being filtered, takes 10mL~12mL filtrate in 50mL~100mL centrifuge tube,
Add water 10mL~15mL, the CuCl of step 4 preparation is successively accurately added with pipette2± 2 μ L of solution 4mL, Na2HPO4Solution
8mL ± 2 μ L and Na2B4O7± 2 μ L of solution 8mL, centrifuge tube is covered, is mixed well, solution by blue become after bluish violet from
The heart, 4000r/min~4500r/min are centrifuged 4min~5min, after centrifugation, take supernatant at spectrophotometer 650nm
Measure absorbance value；
Seven, the drafting of the preparation of glycine titer and standard curve: glycine titer is prepared, is with glycine concentration
Abscissa, the absorbance value measured are that ordinate draws standard curve；
Eight: result calculates: drying soil sample in the NH of interior enzymolysis protein matter release for 24 hours with every gram3The quality (μ g) of-N carrys out table
Show proteinase activity (Pro): in Pro=aVn/m formula: a is the NH asked by standard curve3- N concentration (μ g/mL)；V is aobvious
Color liquid product (40mL)；N is to divide to take multiple；M is drying soil weight (g).
Specific embodiment 2: the present embodiment is different from the first embodiment in that, toluene 4mL is added in step 5.
Other steps and parameter are same as the specific embodiment one.
Specific embodiment 3: the present embodiment is different from the first embodiment in that, step 5 is added step 3 and matches
The gelatin solution 40mL of system.Other steps and parameter are same as the specific embodiment one.
Specific embodiment 4: the present embodiment is different from the first embodiment in that, step 6 pipette is accurate
The CuCl that step 4 is prepared is added2Solution 4mL, Na2HPO4Solution 8mL and Na2B4O7Solution 8mL.Other steps and parameter and tool
Body embodiment one is identical.
Specific embodiment 5: the present embodiment is different from the first embodiment in that, step 7 prepares glycine mark
Quasi- liquid sequentially includes the following steps: the accurate 0.2679g glycine that weighs and is dissolved in distilled water, and accurate dilutions, to 100mL, this is sweet ammonia
Sour standard solution, 1mL contain 500 μ gNH3-N；Glycine titer 10mL is accurately drawn with pipette in 50mL volumetric flask, is used
Water is diluted to scale to get 100 μ gNH3The titer of-N/mL；Then 5 times of 0,2,4,6,8 and of titer of dilution are drawn respectively
10mL is diluted with water to 20mL~25mL in 50mL~100mL centrifuge tube；Step 4 preparation is successively accurately added with pipette
CuCl2± 2 μ L of solution 4mL, Na2HPO4Solution 8mL ± 2 μ L and Na2B4O7± 2 μ L of solution 8mL；Centrifuge tube is covered, sufficiently
It mixes, is centrifuged after becoming bluish violet by blue, 4000r/min~4500r/min is centrifuged 4~5min；After centrifugation, supernatant is taken
Liquid measures absorbance value at spectrophotometer 650nm.Other steps and parameter are same as the specific embodiment one.
Embodiment 1
The measuring method of the Soil protease of the present embodiment sequentially includes the following steps:
One, preparation of reagents: 0.10mol/LNaOH solution, 0.10KH are prepared respectively2PO4Solution；
Two, phosphate buffer solution (PBS) is prepared: what the NaOH solution 790mL and step 1 for taking step 1 to prepare were prepared
KH2PO4Solution 1000mL mixing, adjusting pH is 7.40；
Three, it prepares gelatin solution: gelatin being ground, and weighs 10.0g, measure the phosphoric acid that 700mL step 2 is prepared
Salt buffer solution (PBS) is heated to 80 DEG C, by ground Gelatin in the phosphate buffer solution (PBS) after heating,
Constant volume is cooled down after dissolution to 1L；
Four, copper salt solution is prepared: preparation 0.15mol/L CuCl first2Solution, then prepare 0.18mol/LNa2HPO4, add
Enter the pH value that 7.2gNaOH is used to adjust solution.Then 0.28mol/LNa is prepared2B4O7(anhydrous sodium tetraborate) solution is added
0.1mol/L hydrochloric acid 100mL, for adjusting the pH value of solution, set aside for use；
Five, the culture of soil sample: two parts of fresh soil (crossing 2mm sieve) for being equivalent to 10.0g drying soil weight is weighed, is respectively placed in
In 200mL triangular flask.A addition toluene 4mL therein, places 15min, the gelatin solution prepared with PBS is then added
40mL.Toluene 4mL is added into another weighed soil sample simultaneously and places 15min, 40mL phosphate buffer solution PBS generation is added
For matrix as control.By load weighted soil sample, container closure is sealed with preservative film, is placed in warmed-up 37 DEG C of insulating boxs, training
It supports for 24 hours；
Six, it measures: after culture, suspension being filtered, takes 10mL filtrate in 50mL centrifuge tube, adds water 10mL, successively
CuCl is added2Solution 4mL, Na2HPO4Solution 8mL, Na2B4O7Solution 8mL, centrifuge tube is covered, and is mixed well, and solution becomes completely
It is centrifuged after color, 4500r/min is centrifuged 5min, after centrifugation, supernatant is taken to measure absorbance at spectrophotometer 650nm
Value；
Seven, the preparation of glycine titer: accurately weighing 0.2679g glycine is dissolved in distilled water, and is diluted to 100mL,
This is glycine standard solution, and 1mL contains 500 μ gNH3-N.Glycine titer 10mL is drawn in 50mL volumetric flask, it is dilute with water
The titer to scale to get 100 μ gNH3-N/mL is released, this solution should not be deposited long；Draw respectively dilute 5 times of titers 0,2,4,
6,8,10mL is diluted with water to 20mL in 50mL centrifuge tube；CuCl is accurately successively added with pipette2Solution 4mL, Na2HPO4
Solution 8mL, Na2B4O7Solution 8mL；Centrifuge tube is covered, is mixed well, centrifugation after developing the color completely, 4500r/min is centrifuged 5min.
After centrifugation, supernatant is taken to measure at spectrophotometer 650nm.
Eight: result calculates: drying soil sample in the NH of interior enzymolysis protein matter release for 24 hours with every gram3The quality (μ g) of-N carrys out table
Show proteinase activity (Pro): in Pro=aVn/m formula: a is the NH asked by standard curve3- N concentration (μ g/mL)；V is aobvious
Color liquid product (40mL)；N is to divide to take multiple；M is drying soil weight (g).
Embodiment 2
The present embodiment weighs the gelatin dissolved after soil sample plus with water in step 5 unlike the first embodiment.Other steps
It is same as Example 1.
Embodiment 3
The present embodiment presses 1:2:2 (volume fraction) unlike the first embodiment in step 6 plus when copper salt solution, using preceding
By CuCl2Solution, Na2HPO4Solution and Na2B4O7(anhydrous sodium tetraborate) solution carry out mixing be added sample in, other steps with
Embodiment 1 is identical.
Embodiment 4
The amount that the present embodiment weighs soil sample in step 6 unlike the first embodiment is to weigh the fresh native (mistake of 5.0g drying
2mm sieve), other steps are same as Example 1.
The effective splitting ratio color value of table 1
Table 1 is effective splitting ratio color value of four embodiments.Wherein sample number into spectrum A is 5 measured using 1 method of embodiment
Part active test result of standard sample Soil protease, sample number into spectrum B are the 5 parts of standards measured using the method for embodiment 2
The test result of samples-soil proteinase activity, sample C are the 5 portions of standard sample soil eggs measured using the method for embodiment 3
The test result of white enzymatic activity, D are the 5 parts of active tests of standard sample Soil protease measured using the method for embodiment 4
As a result.It uses embodiment 1 to measure obtained Soil protease active variation coefficient as 0.60%, shows preferable accuracy,
Fail to generate effective spectrophotometric colo numerical value using embodiment 3.
Claims (5)
1. the measuring method of Soil protease, it is characterised in that: the measuring method sequentially includes the following steps:
One, preparation of reagents: 0.10mol/L~0.15mol/L NaOH solution, 0.10mol/L~0.15mol/L are prepared respectively
KH2PO4Solution；
Two, phosphate buffer solution is prepared: the NaOH solution 790mL~795mL and KH for taking step 1 to prepare2PO4Solution 1000mL
~1010mL mixing, adjusts pH to 7.40~7.42；
Three, it prepares gelatin solution: gelatin is ground, measure the phosphate buffer solution that 700mL~710mL step 2 is prepared
80 DEG C~85 DEG C are heated to, is weighed in the phosphate buffer solution after ground gelatin 10.0g~10.4g is dissolved in heating,
Constant volume is cooled down after dissolution to 1L；
Four, copper salt solution is prepared: preparation 0.15mol/L~0.17mol/L CuCl first2Solution, then prepare 0.18mol/L~
0.19mol/L Na2HPO4Solution, to Na2HPO4NaOH is added in solution, makes its pH 8.0~8.5；Then it prepares
0.28mol/L~0.29mol/L Na2B4O7Solution, to Na2B4O70.08mol/L~0.12mol/L hydrochloric acid is added in solution, makes
Its pH is 6.0~6.5, set aside for use；
Five, the culture of soil sample: accurately weighing the 10.01~10.04g of Farmland Black Soil for picking up from field, and drying is made soil sample, uses aperture
For the sieve of 2mm, then the soil sample after sieving is placed in container, ± 2 μ L of toluene 4mL is accurately added with pipette, puts
13min~17min is set, gelatin solution 39mL~41mL that step 3 is prepared then is added；Vessel port is sealed with preservative film again,
It places in warmed-up 37 DEG C~38 DEG C insulating boxs, cultivates 23h~25h；
Six, it measures: the soil sample of step 5 culture being filtered, takes 10mL~12mL filtrate in 50mL~100mL centrifuge tube, adds water
The CuCl of step 4 preparation is successively accurately added in 10mL~15mL with pipette2± 2 μ L of solution 4mL, Na2HPO4Solution 8mL ± 2
μ L and Na2B4O7± 2 μ L of solution 8mL, centrifuge tube is covered, is mixed well, and solution is centrifuged after becoming bluish violet by blue,
4000r/min~4500r/min is centrifuged 4min~5min, after centrifugation, supernatant is taken to measure at spectrophotometer 650nm
Absorbance value；
Seven, the drafting of the preparation of glycine titer and standard curve: preparing glycine titer, with glycine concentration for horizontal seat
Mark, the absorbance value measured are that ordinate draws standard curve；
Eight: result calculates: drying soil sample in the NH of interior enzymolysis protein matter release for 24 hours with every gram3The quality of-N indicates protease activity
Property Pro:Pro=aVn/m, in formula: a is the NH asked by standard curve3- N concentration, unit are μ g/mL；V is colour developing liquid
Product, unit mL；N is to divide to take multiple；M is drying soil weight, unit g.
2. the measuring method of Soil protease according to claim 1, it is characterised in that: toluene 4mL is added in step 5.
3. the measuring method of Soil protease according to claim 1, it is characterised in that: step 5 is added step 3 and prepares
Gelatin solution 40mL.
4. the measuring method of Soil protease according to claim 1, it is characterised in that: step 6 pipette accurately adds
Enter the CuCl of step 4 preparation2Solution 4mL, Na2HPO4Solution 8mL and Na2B4O7Solution 8mL.
5. the measuring method of Soil protease according to claim 1, it is characterised in that: step 7 prepares glycine standard
Liquid, which sequentially includes the following steps:, accurately to be weighed 0.2679g glycine and is dissolved in distilled water, and accurate dilutions, to 100mL, this is glycine
Standard solution, 1mL contain 500 μ gNH3-N；Glycine titer 10mL is accurately drawn with pipette in 50mL volumetric flask, uses water
Scale is diluted to get 100 μ gNH3The titer of-N/mL；Then 5 times of 0,2,4,6,8 and 10mL of titer of dilution are drawn respectively
In 50mL~100mL centrifuge tube, it is diluted with water to 20mL~25mL；Step 4 preparation is successively accurately added with pipette
CuCl2± 2 μ L of solution 4mL, Na2HPO4Solution 8mL ± 2 μ L and Na2B4O7± 2 μ L of solution 8mL；Centrifuge tube is covered, it is sufficiently mixed
It is even, it is centrifuged after becoming bluish violet by blue, 4000r/min~4500r/min is centrifuged 4~5min；After centrifugation, supernatant is taken
Absorbance value is measured at spectrophotometer 650nm.
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| NH4+-N部分代替NO3－-N对番茄生育中后期氮代谢相关酶活性的影响;董园园等;《土壤学报》;20060331;第43卷(第2期);第1.5节 * |
| 自生固氮菌对玉米、小麦根际微生态调控机制的研究;陈胜男;《中国博士学位论文全文数据库》;20121015;正文第79-80页 * |
| 重组大肠杆菌产淀粉分支酶的发酵条件探索;鲍春辉等;《食品工业科技》;20141231;第35卷(第15期);第155-159页 * |
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