Soil Amylase (S-AL) Activity Assay Kit (DNS, Micro Method)

Cat. No.: S1508358

GRADE & PURITY BioReagent, Sterile

Overview

Soil amylase (S‑AL) refers to the collective term for enzymes in soil that catalyze the hydrolysis of starch. It is primarily accumulated by microorganisms and can randomly cleave glycosidic bonds within starch, oligosaccharide, or polysaccharide molecules, producing maltose, oligosaccharides, glucose, etc., playing a significant role in the transformation of carbohydrate substances in soil.

Detection Principle

Amylase hydrolyzes starch to produce reducing sugars, which react with 3,5‑dinitrosalicylic acid to form a reddish‑brown product with a characteristic absorption peak at 540 nm. Within a certain range, the color intensity is proportional to the amount of reducing sugars.

Applicable Sample: Soil

Note: Before formal testing, it is recommended to perform a preliminary experiment with 2‑3 samples expected to show significant differences.

Reagents, consumables and Equipments not provided

• Microplate reader or visible spectrophotometer (capable of measuring absorbance at 540 nm)

• 96‑well plate or micro‑volume glass cuvette, Adjustable pipettes and tips

• Constant temperature water bath, Constant temperature shaker, Tabletop centrifuge, 30‑50 mesh sieve

• Deionized water, Toluene

Procedure

1. Reagent Preparation

Reagent Name	Preparation	Notes
Reagent Ⅰ	Ready‑to‑use. Equilibrate to room temperature before use.	Store at 4°C.
Working Reagent Ⅱ	Prepare immediately before use. For 48T, add 7.2 mL deionized water; for 96T, add 14.4 mL deionized water. Place in a 70°C water bath and stir or shake continuously until dissolved.	Unused reagent can be stored at 4°C for one week.
Reagent Ⅲ	Ready‑to‑use. Equilibrate to room temperature before use.	Store at 4°C.

Note: Reagent Ⅲ is somewhat irritating. Personal protection is recommended during use.

2. Standard Preparation

Standard: Prepare immediately before use. Add 1 mL deionized water to prepare a 10 mg/mL glucose standard solution. Store at 4°C protected from light for up to two weeks. Further dilute the standard as shown in the table below:

Tube	10mg/mL Standard（μL）	Deionized Water Volume (µL)	Concentration (mg/mL)
Std.1	40	160	2
Std.2	20	180	1
Std.3	10	190	0.5
Std.4	5	195	0.25
Std.5	2.5	197.5	0.125
Std.6	1.25	198.75	0.063
Blank	0	200	0

Note: A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.

3. Sample Preparation

Note: Fresh samples are recommended. If not used immediately, samples can be stored at ‑80°C for one month. Control the thawing temperature and time during measurement. When thawing at room temperature, complete the process within 4 hours.

Air‑dry fresh soil samples naturally or in a 37°C oven, then sieve through a 30‑50 mesh screen.

4. Experimental Steps

4.1 Preheat the microplate reader or visible spectrophotometer for 30 minutes and set the wavelength to 540 nm. Zero the visible spectrophotometer with deionized water.

4.2 Operation Table (perform in 1.5 mL EP tubes):

Reagent	Control Tube (µL)	Test Tube (µL)	Standard Tube (µL)	Blank Tube (µL)
Air‑dried soil (g)	0.04	0.04	0	0
Toluene	20	20	0	0

Let stand at 25°C for 15 min.

Reagent Ⅰ	100	100	0	0
Working Reagent Ⅱ	0	120	0	0
Deionized Water	120	0	0	0

Mix well, shake in a 37°C constant temperature shaker for 24 hours. Centrifuge at 25°C, 8,000 rpm for 10 minutes. Collect the supernatant.

Sample Supernatant	120	120	0	0
Standard	0	0	120	0
Deionized Water	0	0	0	120
Reagent Ⅲ	120	120	120	120

4.3 Mix thoroughly, incubate in a 90°C water bath for 5 minutes. After cooling, transfer 200 µL to a micro‑volume glass cuvette / 96‑well plate. Measure the absorbance at 540 nm, recorded as A_Control, A_Test, A_Standard, A_Blank. Calculate ΔA_Test = A_Test ‑ A_Control and ΔA_Standard = A_Standard ‑ A_Blank.

Note:

• The blank and standard tubes only need to be measured once. A control group must be set up for each experiment. It is recommended to perform a preliminary experiment with 2‑3 samples showing expected large differences before formal testing.

• Keep the cooling time after the 90°C water bath consistent for each experiment. Complete the measurement within 30 minutes.

5. Calculation of Results

Note: We provide two formula sets: the derived calculation formulas and the simplified formulas. They are completely equivalent. The simplified formulas are recommended as the final calculation formulas.

5.1 Plotting the Standard Curve

Plot the standard curve with the standard solution concentration as the x‑axis and ΔA_Standard as the y‑axis. Obtain the standard equation. Substitute ΔA_Test into the equation to obtain x (mg/mL).

5.2 Calculation of Enzyme Activity

Unit Definition: One soil amylase activity unit (U) is defined as the amount of enzyme that produces 1 mg of reducing sugar per day per gram of soil.

S‑AL (U/g soil) = x × V_{Total Reaction} ÷ W ÷ T = 6 × x

Parameter Description:

V_{Total Reaction}: Total volume of the enzymatic reaction, 0.24 mL;

W: Soil sample mass, 0.04 g;

T: Reaction time, 1 day.

6. Example Results         

Figure 1. Soil amylase activity determined by this kit

Notes

This product is for scientific research use only and is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.

Contents & Storage

S1508358	Component	48T	96T	Storage
S1508358A	Reagent Ⅰ	12 mL	24 mL	2-8℃
S1508358B	Reagent Ⅱ	1EA	1EA	2-8℃
S1508358C	Reagent Ⅲ	15.6 mL	31.2 mL	2-8℃
S1508358D	Standard	1EA	1EA	2-8℃. Store in the dark.