CN106501247A

CN106501247A - A kind of method of activity of acid phosphatase in measure soil

Info

Publication number: CN106501247A
Application number: CN201610963050.9A
Authority: CN
Inventors: 贺若阳; 徐振锋; 庄丽燕; 郑海峰; 陈亚梅
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2016-10-28
Filing date: 2016-10-28
Publication date: 2017-03-15

Abstract

The invention discloses a kind of method for determining activity of acid phosphatase in soil, this method is provided with multigroup sample and control, and make which after 30 DEG C of 45 60min of constant temperature culture, after adding 4-NPP solution, add NaOH solution terminating reaction, hydrolyzed under the catalysis of soil acidity phosphatase using which and generate paranitrophenol, add the characteristic developed the color after NaOH solution terminating reaction, the amount of the paranitrophenol generated after 405nm colorimetric determinations enzymatic reacts, characterizes the activity of acid phosphatase.The method includes preparing raw material, measurement soil dry weight, the Abs for preparing crude enzyme liquid, determination sample and matched group and calculates these steps of activity of acid phosphatase in soil, have that reagent type is few, consumption is little, simple to operate, time-consuming short, repeatability is good and determines quick and accurate advantage.

Description

A kind of method of activity of acid phosphatase in measure soil

Technical field

The present invention relates to the assay method of phosphatase activity, and in particular to activity of acid phosphatase in a kind of measure soil Method.

Background technology

Used as nutrient necessary to plant growing, major part is existed with the phosphorus of organic to soil phophorus（Account for full phosphorus 15%~80%）, and organophosphors are generally needed under the enzymatic catalysis of soil phosphorus loss, ability decomposition and inversion is plant available profit Form.Phosphatase as important hydrolase in soil, can with the hydrolysis of catalytic phosphatase ester and phosphoric anhydride, and wherein acid Property phosphomonoesterase is the closest with Mineralization of organic phosphorus and phosphorus nourishing relation.The strong and weak impact of activity of acid phosphatase in soil The decomposition of Organic phosphate, conversion and its biological effectiveness in soil phosphorus circulation.And it is catalyzed a series of bioids in soil Learn reaction, remote-effects to soil fertility and vegetable metabolic process etc..So, the activity of acid phosphatase in soil is studied, is had Beneficial to the conversion process, direction and the intensity that deeply understand Soil Phosphorus.

In soil, the activity of acid phosphatase is subject to soil physico-chemical property, such as moisture, temperature, the strong impact of PH, while Also the impact of forest management measure, different vegetation types etc. is subject to, and up to the present, the country is about activity of acid phosphatase Determine and be substantially with reference to Guan Songyin's《Soil enzyme and its research method》Write with nineteen ninety-five Kandeler et al. 《Methods in soil biology》Method in Deng book.Such method is comparatively laborious, and operating procedure is complicated, incubation time Long, and reagent dosage is more, colorimetric development is unstable etc., and defect causes the analysis that should not make large sample amount.

Content of the invention

Present invention aim to address the problems referred to above, there is provided a kind of reagent type for using is few, consumption is little, simple to operate, Time-consuming short, with good repeatability and the method that can quickly and accurately determine soil acidity phosphatase activity.

For achieving the above object, the technical solution used in the present invention is as follows：

In a kind of measure soil, the method for activity of acid phosphatase, comprises the following steps：

1）Prepare raw material：A fresh soil, acetate buffers of the pH between 4.8 ~ 5.2, concentration are 5.0 ~ 5.1umol/ Ml, the 4-NPP solution as substrate, NaOH solution of the concentration for 38 ~ 41mg/ml；

2）A pedotheque A is taken in fresh soil, fresh weight is weighed and record, and after being then baked to constant weight, is weighed simultaneously Record dry weight, is thus calculated the moisture content of the fresh soil；The soil-like of portion 1 ~ 5g weight is taken in the fresh soil again Product B, weighs and records its fresh weight；

3）The acetate buffer of 40ml is added in pedotheque B, is stirred, is obtained crude enzyme liquid, measures and record its body Product, continues stirring, keeps crude enzyme liquid to be in the state of mix homogeneously；

4）One piece of 96 hole elisa Plates is taken, enzyme mark version A is designated as, 3 ~ 6 groups of samples and control are set in enzyme mark version A；Every group of sample The crude enzyme liquid and 150ul substrate of 100ul mix homogeneously have all been pipetted in product as culture fluid；Per group of control includes one Substrate controls and a crude enzyme liquid control, the Substrate controls have all pipetted 150ul substrates and 100ul acetate buffer conducts Culture fluid, the crude enzyme liquid control have all pipetted the crude enzyme liquid and 150ul acetate buffers of 100ul mix homogeneously as culture Liquid；

5）By step 4）In install ELISA Plate A after culture fluid and be placed in 45 ~ 60min of constant temperature culture in 30 DEG C of incubator；

6）First take clean and water white 96 hole elisa Plates, be designated as ELISA Plate B, with microplate reader at the 405nm wavelength ratio Color, detects ELISA Plate B per the corresponding Abs in hole, is designated as blank Abs；

Then take out culture terminate after ELISA Plate A, pipette respectively 100ul sample and control supernatant to ELISA Plate B Corresponding hole position, and be separately added into the NaOH solution terminating reaction of 10ul, finally inserts microplate reader colorimetric at the 405nm wavelength, Obtain sample Abs, Substrate controls Abs and crude enzyme liquid control Abs；

7）Calculate OD values：

The OD=（Sample Abs- blank Abs）-[（Substrate controls Abs- blank Abs）+（Control Abs- is empty for crude enzyme liquid White control Abs）],

Wherein, the blank Abs deducted by every group of sample Abs and control Abs, is that the hole with which in ELISA Plate A is relative The blank Abs in the hole in ELISA Plate B that answers；The OD deducts the absorbance after control absorbance for sample absorbance；

According to the OD values that this step can calculate remaining sample；

8）It is calculated activity of acid phosphatase：

Enzymatic activity (umol h^-1·gDOM^-1)=（OD×0.25×V）/（EC×h×m×0.1）

m=m_Fresh×(m_{A does}/m_{A is fresh})

Wherein, dry weights of the m for pedotheque B, unit is g；m_FreshFor the fresh weight of pedotheque B, unit is g；m_{A does}Refer to sample A's Dry weight, unit are g, m_{A is fresh}Refer to the fresh weight of sample A, unit is g；0.25 refers to that ELISA Plate single hole nutrient solution volume is 0.25ml；V For crude enzyme liquid volume, unit is ml；EC is extinction coefficient；H is the time that culture fluid is cultivated in incubator, and unit is hour； 0.1 is volume 0.1ml of the taken supernatant of colorimetric.

Specifically, the acetate buffer is mixed by sodium acetate trihydrate, glacial acetic acid and deionized water.

Specifically, the pH of the acetate buffer is 5.0.

Specifically, the step 2）In, the baking step is that soil is placed on the baking oven that temperature is 60 ~ 105 DEG C In carry out fooling dry, a length of 36 ~ 48h when roars of laughter are dry.

Specifically, the step 3）In, the configuration process of crude enzyme liquid is：Pedotheque B is first placed in the wide-mouth of 50ml In triangular flask, then the acetate buffer of 40ml is added in wide-mouth triangular flask, be then put into a stirrer thereto, and will The wide-mouth triangular flask is placed on above magnetic stirring apparatuss and is stirred so as to mix homogeneously, obtains crude enzyme liquid, and continues to stir, and makes Crude enzyme liquid is in the state of mix homogeneously.

Further, the step 5）In, before ELISA Plate A is put into incubator, one layer of preservative film of lid above it.

Alternatively, the step 4）In per group of control both increase a buffer control, and each buffering Liquid control has all pipetted 250ul acetate buffers and has been cultivated as culture fluid；Meanwhile, step 6）Middle pipette 100ul respectively The supernatant of buffer control is to corresponding hole position in ELISA Plate B, then is separately added into the NaOH solution terminating reaction of 10ul, finally With other group of sample and insert microplate reader colorimetric at the 405nm wavelength together with compareing, obtain 3 ~ 6 buffer control Abs.1st） ~3）I.e. the 5th）Step is identical with abovementioned steps.

Further, the step 7）In,

The OD=（Sample Abs- blank Abs）-[（Buffer control Abs- blank Abs）+（Substrate controls Abs- is empty White control Abs）+（Crude enzyme liquid compares Abs- blank Abs）],

According to the OD values that this step can calculate remaining sample and matched group.Then, according to abovementioned steps 8）Mode can meter Calculation obtains soil acidity phosphatase activity.

Compared with prior art, the invention has the advantages that：

（1）The inventive method adopts 4-NPP for substrate, with traditional method using disodium phenyl phosphate as substrate Compare, its incubation time is shorter, and coloration method is simpler, short the time required to colour developing, and sensitivity is higher, develops the color more steady Fixed, the accuracy of measurement result while test period is substantially reduced, can be effectively improved.Simultaneously as ELISA Plate itself Contrast color have certain impact, therefore this method can be before prepare liquid colorimetric first by empty ELISA Plate（Blank control group）Colorimetric is carried out, is made For blank, then culture plate supernatant is moved to color board colorimetric, afterwards with imposite data deduct hollow plate data obtain more accurate OD values, effectively increase the accuracy of measurement result.

（2）The culture experiment of the inventive method is carried out in 96 hole elisa Plates, and during colorimetric be using imposite colorimetric, Colorimetric faster, is not required to the centrifuge tube referred to previous methods, teat glass etc., and the experimental apparatus for being adopted are few, decrease examination The triviality of step is tested, experiment progress is accelerated, is improve efficiency.

（3）The inventive method reagent dosage is few, can determine multiple sample sets of multiple soil simultaneously at identical conditions, And soil is not related to due to Substrate controls, therefore the Substrate controls of 3-6 groups that can be with multiple soil only in corresponding same ELISA Plate, Relative to the mode of replication, human error is reduced, make measurement result more accurate, continuous mode is easier, expended Time is also shorter.

（4）The inventive method determines soil acidity phosphatase activity fast and accurately, and the reagent type that the method is used Few, consumption is little, simple to operate, time-consuming short, with good repeatability.

Specific embodiment

With reference to embodiment, the invention will be further described, and the mode of the present invention includes but are not limited to following enforcement Example.

Embodiment 1

The pedotheque used by this example 1-4 is dark brown earth, picks up from and finishes canopy ditch " height in Sichuan Agricultural University's Aba Prefecture Li County Mountain forest ecosystem Position Research station " Man-made spruce forest.The soil sample for being used 5-8 is yellow earth, picks up from the old Jun Mountain state in Yibin Family's nature reserve area Metasequoia Glyptostroboides Plantation.Two place soil collecting times are in June, 2016, gather 0-15 cm pedotheques.Survey In the fixed soil, the method for activity of acid phosphatase, comprises the following steps：

（1）Prepare raw material

1）Configuration acetate buffer：The sodium acetate trihydrate of 4.374g is weighed, 1.1ml glacial acetic acid and 800ml is added thereto to Deionized water, after solid dissolving, is settled to 1L, and acetate buffers of the pH between 4.8 ~ 5.2 is obtained；

2）Configuration NaOH solution：4gNaOH is weighed, 100ml deionized waters are dissolved in, and concentration is obtained for 38 ~ 41mg/ml's NaOH solution；

3）Configuration substrate：Weigh the 4-NPP of 185.6mg（No. CAS：333338-18-4, Sigma company purchases Put）, then sodium-acetate buffer 100ml in A is measured, in the 100ml sodium-acetate buffers, i.e., 4-NPP is dissolved in Obtain substrate 4-NPP solution.

（2）Soil dry weight

1）By taking soil sample 1 as an example, 1 moisture content of soil sample is calculated：Weigh 9.1645g and the fresh soil of 2mm sieves is crossed in aluminum box, be designated as soil Earth sample A, weighs and records aluminum box weight 11.5911g, and aluminum box is 20.7556g with fresh sample gross weight, and being then placed in temperature is Dry in 60 ~ 105 DEG C of baking oven to constant weight, drying time period is 36 ~ 48 hours, then takes out, weighs and record aluminum box and drying Sample gross weight is 16.9961g, according to formula M_{A does}/M_{A is fresh}=(16.9961-11.5911)/9.1645=0.5898, is calculated Water Content Tests in Soil Samples is 69.56%.

2）By taking soil sample 1 as an example, soil sample sample dry weight is calculated：The fresh soil that 3.2478g crosses 2mm sieves is weighed, soil is designated as Sample B, according to 1）Middle moisture content can be calculated dry weight m=m of pedotheque B_Fresh×(M_{A does}/M_{A is fresh})=3.2478×0.5898= 1.9156g.

3）Measure and be calculated the data of other seven soil samples as stated above, as shown in table 1：

1 soil sample tables of data of table

Soil sample	Sample A fresh weights（g）	Sample A dry weights（g）	Moisture content（%）	Sample B fresh weights（g）	Sample B dry weights（g）
						Soil sample 1	9.1645	5.4050	69.56	3.2478	1.9155
Soil sample 2	10.4313	7.3888	41.18	3.4142	2.4184
						Soil sample 3	9.4015	6.4405	45.97	3.3771	2.3135
Soil sample 4	9.0535	5.8264	55.39	3.3473	2.1542
						Soil sample 5	10.3350	5.8050	78.04	3.8350	2.1541
Soil sample 6	10.3500	6.3550	62.86	3.7976	2.3318
						Soil sample 7	10.3150	6.5800	56.76	3.8477	2.4545
Soil sample 8	10.4150	6.6500	56.62	3.8180	2.4378

（3）Crude enzyme liquid processed

It is placed in the wide-mouth triangular flask of 50ml in pedotheque B, adds the acetate buffer of 40ml, measure and record its volume Afterwards, a stirrer is put into, then wide-mouth triangular flask is placed on magnetic stirring apparatuss, magnetic stirring apparatuss are adjusted to high speed shape State, stirs 2min so as to mix homogeneously, obtains crude enzyme liquid；

In order to keep crude enzyme liquid to have been at the state of mix homogeneously, magnetic stirring apparatuss speed is turned down, continue stirring, make thick enzyme Liquid is less than the state of wide-mouth triangular flask bottleneck radius in mix homogeneously, the vortex diameter being internally formed.

（4）Determine Abs

2 96 hole elisa Plates schematic diagram of table

1

2

3

4

5

6

7

8

9

10

11

12

a

Bottom

Enzyme 8

Sample 8

b

Enzyme 1

Sample 1

Enzyme 9

Sample 9

c

Enzyme 2

Sample 2

Enzyme 10

Sample 10

d

Enzyme 3

Sample 3

Enzyme 11

Sample 11

e

Enzyme 4

Sample 4

Enzyme 12

Sample 12

f

Enzyme 5

Sample 5

Enzyme 13

Sample 13

g

Enzyme 6

Sample 6

Enzyme 14

Sample 14

h

Enzyme 7

Sample 7

Enzyme 15

Sample 15

Note：Sample represents sample well；Bottom represents Substrate controls hole；Enzyme represents crude enzyme liquid control wells；Numeral represents sample number, an enzyme Target can determine 15 samples, 3 groups of repetitions.

Table 3 operates table

	Blank control wells	Substrate controls hole	Crude enzyme liquid control wells	Sample well
					Acetate buffer	Vacant	100ul	150ul	-
Crude enzyme liquid	-	-	100ul	100ul
					Substrate	-	150ul	-	150ul

1）Two pieces of 96 intact transparent hole elisa Plates as shown in table 2 are taken, is described for convenience, is designated as ELISA Plate A1 and enzyme Target A2, arranges sample and the control of 8 parts of soil wherein（3 repetitions）, Li County and Yibin are respectively in ELISA Plate A1 and enzyme mark Plate A2 is cultivated.

In ELISA Plate A1, Li County plate does 1-4 holes, and No. 5-15 vacant, and soil sample numbering is 1-4；

In ELISA Plate A2, Yibin plate does 1-4 holes, and No. 5-15 vacant, and soil sample numbering is 5-8；

Wherein, every part of soil sample all corresponds to 3 sample wells, and 3 crude enzyme liquid control wells, and all samples share 6 Substrate controls holes, And the Substrate controls hole is set to the a1-a6 holes in ELISA Plate；As shown in table 3, the acetate that pipettes respective volume with pipettor Buffer, crude enzyme liquid and substrate are in corresponding hole；In order to prevent crude enzyme liquid blocking pipettor, the suction nozzle tip of the pipettor One section is cut so as to a diameter of 1 ~ 2mm.

2）By step 1）In add and respectively cover one layer of preservative film above ELISA Plate A1 and ELISA Plate A2 of solution, then by which It is placed in constant temperature culture 1h in 30 DEG C of incubator；If culture failure, then should suitably increase crude enzyme liquid concentration or Extending culture Time；

3）ELISA Plate A1 after culture terminates and ELISA Plate A2 is taken out, in the sample well and control wells with crude enzyme liquid, soil is micro- Grain is deposited in ELISA Plate bottom, and top is clarified；96 intact, clean, transparent and colourless hole elisa Plates are first selected, enzyme mark is designated as Plate B, and by ELISA Plate B microplate reader at the 405 nm wavelength colorimetric, obtain each hole corresponding A bs value in ELISA Plate B, obtain sky White control Abs；No matter determining blank Abs is because that ELISA Plate is how clean, more or less can all affect our samples and The measure of the absorbance of control, therefore, in this step, by the absorbance measurement of blank ELISA Plate out now, then to be measured After setting the absorbance of ELISA Plate with culture fluid, the absorbance of the ELISA Plate of blank is deducted, to eliminate empty ELISA Plate itself Impact of the absorbance to them.

Cultured ELISA Plate A1 and ELISA Plate A2 is then taken out, and pipettes the sample of 100 ul and the supernatant of control respectively The hole position with culture fluid on liquid to ELISA Plate A1 and ELISA Plate A2, and the NaOH solution terminating reaction of 10 ul is separately added into, Finally with microplate reader at the 405 nm wavelength colorimetric, obtain the corresponding Abs values in each hole, then deduct the blank of corresponding aperture Abs, you can obtain sample Abs, Substrate controls Abs and crude enzyme liquid control Abs；Its specific data is as shown in table 4：

4 each group sample of table and the Abs data of control

（5）Calculate

1）Calculate OD values

According to the OD that the data in table 3 can be calculated corresponding group respectively,（By taking in soil sample 1 first group as an example）

The OD=samples Abs-（Average Substrate controls Abs+ crude enzyme liquids control Abs）=0.137-（0.004167+0.093）= 0.0398,

Wherein, as Substrate controls do not add soil, which is determined without the need for being corresponded with sample, therefore is only provided with 6 groups altogether, because This, when calculating, it is only necessary to be averaged value and calculate, bring formula into.

The OD deducts the absorbance after control absorbance for sample absorbance；

According to the OD values that this formula can calculate each group sample in other each soil samples.

2）Calculate activity of acid phosphatase：

Enzymatic activity（umol·h^-1·gDOM^-1）=（OD×0.25×V）/（EC×h×m×0.1）=（0.0398×0.25× 43）/（1.0444×1×1.9156×0.1）=2.1386；

Wherein, dry weights of the m for pedotheque B, unit is g；m_FreshFor the fresh weight of pedotheque B, unit is g；0.25 refers to enzyme mark Plate single hole nutrient solution volume is 0.25ml；V is crude enzyme liquid volume, and unit is ml；EC is extinction coefficient；H is that culture fluid is being cultivated The time that cultivates in case, unit is hour；0.1 is volume 0.1ml of the taken supernatant of colorimetric；

According to the enzymatic activity value that this formula can calculate each group in other each soil samples, and record as shown in table 5：

5 activity of acid phosphatase of table

In the present embodiment, the calculating of extinction coefficient is to adopt paranitrophenol to be configured to the titer of 1.00umol/ml for base fluid, Totally, variable concentrations are diluted in bright ELISA Plate（Concrete operations are：Be separately added in A1-A6 holes 0ul, 20ul, 40ul, 60ul, 80ul, 100ul titer, is subsequently adding sodium-acetate buffer and is supplemented to 100ul, obtains concentration and is respectively 0 Umol/ml, 0.2 umol/ml, 0.4 umol/ml, 0.6 umol/ml, 0.8 umol/ml and 1umol/ml paranitrophenol molten Liquid）, then after being added thereto to the colour developing of 10ulNaOH solution terminating reaction respectively, colorimetric at ELISA Plate 405nm.Molten with nitrophenols The concentration of liquid is abscissa, and the absorbance for obtaining is that vertical coordinate draws standard curve, and its slope is just extinction coefficient.

From embodiment 1, the inventive method adopts 4-NPP for substrate, adopts phosphoric acid with traditional method Benzene disodium is compared as substrate, and which is simple to operate, and the reagent type for using is few, and consumption is little, and incubation time is shorter, coloration method letter Single, required time is short, with good repeatability；And, colorimetric is carried out using ELISA Plate, can be made simultaneously multigroup to having a competition Test, be not required to the centrifuge tube referred to previous methods, teat glass etc., greatly reduce the triviality of test procedure.

Embodiment 2

The present embodiment is similar to Example 1, only exists following difference：

Step（4）In, respectively newly increase in ELISA Plate A1 and A2 in 6 groups of buffer control groups, and the buffer control group Acetate buffer of the culture fluid for 250ul, remaining operation are identical with Substrate controls, obtain buffer control Abs, record number According to as shown in table 6：

The Abs data of 6 buffer control of table


							Li County buffer control	-0.001	0	-0.003	-0.001	0.002	0.002
Yibin buffer control	0	-0.002	-0.001	0.001	-0.001	0.001

Step（5）In

OD value computing formula are：

OD=sample Abs-（The average Substrate controls Abs+ crude enzyme liquids of buffer control Abs+ compare Abs）=0.137-（0.0005+ 0.004167+0.093）=0.0393

Enzymatic activity value is calculated to obtain by carrying it into following formula：

Enzymatic activity (umol h^-1·gDOM^-1)=（OD×0.25×V）/（EC×h×m×0.1）=（0.0400×0.25× 43）/（1.0444×1×1.92×0.1）=2.1493；

According to the enzymatic activity value that this formula can calculate each group in other each soil samples, and record as shown in table 7：

7 activity of acid phosphatase of table

Above-described embodiment is only the preferred embodiment of the present invention, should not be taken to limit protection scope of the present invention, as long as The body design thought of the present invention and the change or polishing of having no essential meaning mentally made, its technical problem for being solved Still consistent with the present invention, should be included within protection scope of the present invention.

Claims

1. a kind of determine soil in activity of acid phosphatase method, it is characterised in that comprise the following steps：

7）Calculate OD values：

According to the OD values that this step can calculate remaining sample；

8）It is calculated activity of acid phosphatase：

Enzymatic activity (umol h^-1·gDOM^-1)=（OD×0.25×V）/（EC×h×m×0.1）

m=m_Fresh×(m_{A does}/m_{A is fresh})

Wherein, dry weights of the m for pedotheque B, unit is g；m_FreshFor the fresh weight of pedotheque B, unit is g；m_{A does}Refer to that sample A's is dry Weight, unit is g, m_{A is fresh}Refer to the fresh weight of sample A, unit is g；0.25 refers to that ELISA Plate single hole nutrient solution volume is 0.25ml；V is Crude enzyme liquid volume, unit are ml；EC is extinction coefficient；H is the time that culture fluid is cultivated in incubator, and unit is hour；0.1 It is volume 0.1ml of the taken supernatant of colorimetric.

2. according to claim 1 a kind of determine soil in activity of acid phosphatase method, it is characterised in that the vinegar Phthalate buffer is mixed by sodium acetate trihydrate, glacial acetic acid and deionized water.

3. according to claim 2 a kind of determine soil in activity of acid phosphatase method, it is characterised in that the vinegar The pH of phthalate buffer is 5.0.

4. according to claim 1 a kind of determine soil in activity of acid phosphatase method, it is characterised in that the step Rapid 2）In, the baking step is to be placed on to carry out fooling in the baking oven that temperature is 60 ~ 105 DEG C by soil to do, and roars of laughter are a length of when dry 36~48h.

5. according to claim 1 a kind of determine soil in activity of acid phosphatase method, it is characterised in that the step Rapid 3）In, the configuration process of crude enzyme liquid is：First pedotheque B is placed in the wide-mouth triangular flask of 50ml, then to wide-mouth triangular flask The acetate buffer of middle addition 40ml, is then put into a stirrer thereto, and the wide-mouth triangular flask is placed on magnetic force and stir Mix so as to mix homogeneously, obtain crude enzyme liquid, and continue to stir, make crude enzyme liquid be in the shape of mix homogeneously State.

6. according to claim 1 a kind of determine soil in activity of acid phosphatase method, it is characterised in that the step Rapid 5）In, before ELISA Plate A is put into incubator, one layer of preservative film of lid above it.

7. according to claim 1 a kind of determine soil in activity of acid phosphatase method, it is characterised in that the step Rapid 4）In per group of control both increase a buffer control, and each buffer control has pipetted 250ul acetate salt buffers Liquid is cultivated as culture fluid；Meanwhile, step 6）The middle supernatant for pipetting 100ul buffer controls respectively is to ELISA Plate B Corresponding hole position, then be separately added into the NaOH solution terminating reaction of 10ul, finally inserts enzyme with other group of sample and together with compareing Mark instrument colorimetric at the 405nm wavelength, obtains 3 ~ 6 buffer control Abs.

8. according to claim 7 a kind of determine soil in activity of acid phosphatase method, it is characterised in that the step Rapid 7）In, the OD=（Sample Abs- blank Abs）-[（Buffer control Abs- blank Abs）+（Substrate controls Abs- blank Abs）+（Crude enzyme liquid compares Abs- blank Abs）],

According to the OD values that this step can calculate remaining sample.