Amylase activity detection method

Amylase activity detection is of great significance in many fields such as food, fermentation, enzyme preparations, and medical diagnosis. The following are some common methods for detecting amylase activity:

1. DNS method (3,5-dinitro salicylic acid method)

Principle: Amylase catalyzes starch degradation to produce reducing sugars. DNS reagent reacts with these reducing sugars to produce reddish-brown compounds. By measuring the absorbance of the compound, the activity of amylase can be quantified.

Advantages: It is easy to operate and is one of the most commonly used methods for detecting amylase activity.

2. Iodine-starch colorimetry

Principle: Starch and iodine form a blue complex. Under the action of amylase, starch gradually degrades and the blue gradually weakens. The activity of amylase can be calculated by measuring the change in absorbance with a colorimeter or spectrophotometer.

Application: Commonly used for the determination of amylase activity in serum or urine.

3. Capillary electrophoresis method

Principle: Efficiently separate the products of amylase-catalyzed degradation, measure the peak area of ​​each product in the sample, and indirectly deduce the activity of amylase.

Advantages: High resolution, fast, suitable for the analysis of complex samples.

4. ELISA (Enzyme-Linked Immunosorbent Assay)

Principle: Specific amylase can be detected by quantitative analysis of the signal generated by the antigen-antibody reaction.
Advantages: High sensitivity, suitable for detecting low concentrations of amylase.
Disadvantages: Specific antibodies are required and the cost is high.

5. HPLC (High-Performance Liquid Chromatography)

Principle: Used to separate and quantify the degradation products after the action of amylase, thereby indirectly measuring the activity of amylase.
Advantages: High accuracy, suitable for analyzing complex samples.
Disadvantages: Expensive equipment and complicated operation.

6. Other methods

Titration: Use the glucose produced by starch decomposition for titration to calculate the enzyme activity.
Spectrophotometry: Quantify the enzyme activity by measuring the absorbance change of starch decomposition products (such as glucose).
Gel electrophoresis: Evaluate the thermal or pH stability of the enzyme by the change of its electrophoretic mobility.
Isothermal titration calorimetry: Measure the heat change during the amylase-catalyzed reaction to evaluate the thermal stability.
Fourier transform infrared spectroscopy: Analyze the changes in the molecular structure of amylase to evaluate its stability and activity.

7. Precautions

Sample processing: Select appropriate pretreatment methods according to sample type (such as serum, urine, food samples, etc.).
Instrument calibration: Ensure that the instruments used (such as spectrophotometer, HPLC, etc.) are accurately calibrated to ensure the accuracy of the measurement results.
Reagent quality: Use high-quality reagents to avoid the influence of reagent contamination or deterioration on the experimental results.
Operation specifications: Strictly follow the experimental steps to avoid errors caused by improper operation.
In summary, there are many methods for detecting amylase activity, and the appropriate method should be selected according to specific needs and experimental conditions. At the same time, pay attention to details such as sample processing, instrument calibration, reagent quality, and operation specifications to ensure the accuracy and reliability of the experimental results.