Measurement experiment of amylase activity
Measurement experiment of amylase activity
Amylase in plants hydrolyzes stored starch into maltose. Amylase is present in almost all plants, with the strongest amylase activity in cereal seeds. There are α-amylase and β-amylase in plants, and their activities vary according to the growth and development period of plants. The purpose of this experiment is to master the method of determining these two amylases separately.
Operation method
Measurement experiment of amylase activity
Principle
α-amylase and β-amylase, each has its own certain characteristics, such as β-amylase is not heat-resistant, easy to passivate at high temperatures, and α-amylase is not acid-resistant, below pH 3.6 passivation occurs, usually extracts at the same time there are two amylases exist, the determination of their characteristics can be processed separately, passivation of one of them, you can measure the activity of the other enzyme. The extract is heated to 70 ℃ for 15 minutes to passivate β-amylase, then the activity of α-amylase can be measured. Alternatively, the extract can be treated with acetic acid at pH 3.6 at 0°C to passivate the activity of α-amylase and measure the activity of β-amylase. The maltose produced by the hydrolysis of starch by amylase can be measured with the 3,5-dinitrosalicylic acid reagent. As maltose can reduce the latter to generate 3-amino-5-nitrosalicylic acid chromogenic group, within a certain range of the depth of its color is proportional to the depth of the sugar, so the content of maltose can be found to the milligrams of maltose to indicate the size of amylase activity. Move I. Experimental materials, instruments and reagents For more product details, please visit Aladdin Scientific website.
1, experimental materials: sprouting wheat (bud length of about 1 cm)
2. Apparatus: (1) small scale; (2) mortar; (3) volumetric flask: 100 ml × 1; (4) stoppered graduated test tube 25 ml × 13; (5) test tubes: 8; (6) graduated pipettes: 1 ml × 2, 2 ml × 2; (7) centrifuge; (8) constant temperature water bath; (9) spectrophotometer.
3, Reagents:
(1) 1% starch solution;
(2) 0.4 N NaOH;
(3) pH5.6 citric acid buffer: A, weigh 20.01 g of citric acid, dissolve and dilute to 1 liter; B, weigh 29.41 g of sodium citrate, dissolve and dilute to 1 liter. Take 13.7 ml of liquid A and 26.3 ml of liquid B and mix well, i.e. pH5.6 buffer.
(4) 3,5-dinitrosalicylic acid: accurately weighed 3,5-dinitrosalicylic acid 1 gram dissolved in 20 ml of 1N sodium hydroxide, add 50 ml of distilled water, then add 30 grams of sodium tartrate, to be dissolved, diluted to 100 ml with distilled water, corked, do not allow carbon dioxide into the bottle.
(5) maltose standard solution: weigh 0.100 g of maltose dissolved in a small amount of distilled water, carefully moved into a 100 ml volumetric flask, diluted with distilled water to the scale.
Second, the operation steps:
1, the extraction of enzyme solution:
Weigh 2 grams of sprouted wheat seeds (bud length of about 1 cm), to the research bowl with one gram of quartz sand, ground into a homogenate poured into 25 ml plugged graduated test tube, diluted with distilled water to the scale, mixed at room temperature (20 ℃) at room temperature (20 ℃), shaking every few minutes, placed 15-20 minutes, centrifugation, take the supernatant for backup.
2, Determination of α-amylase activity:
(1) Take 4 test tubes, indicating two as the assay tubes.
(2) Add 1 ml of enzyme extract in each tube, and heat it accurately in a constant temperature water bath at 70℃ (the change of water temperature should not be more than ±0.5℃) for 15 minutes, during which the β-amylase is blunted by the heat, and then quickly cool it in tap water after removing it.
(3) Add 1 ml of pH 5.6 citrate buffer to each test tube.
(4) 4 ml of 0.4N sodium hydroxide was added to the control tube to passivate the enzyme activity.
(5) Place the assay tube and the control tube in a constant temperature water bath at 40°C (±0.5°C) for 15 min, then add 2 ml of starch solution preheated at 40°C to each tube, shake well, and immediately place them in a 40°C water bath for 5 min of accurate holding time before removing them, and add 4 ml of 0.4N sodium hydroxide to each assay tube quickly to terminate enzyme activity, and then prepare for the next step of the sugar assay.
3、Determination of total activity of α-amylase and β-amylase:
Take 2-6 ml of the above enzyme solution, put it into a volumetric flask, dilute it with distilled water to 100 ml (the degree of dilution depends on the size of enzyme activity) and mix it well, then take 4 test tubes, add 1 ml of diluted enzyme solution and 1 ml of citrate buffer of pH 5.6 to each tube. Repeat the following steps for α-amylase (4) and (5) to prepare for the sugar assay.
4. Determination of maltose:
(1) Take seven 25 ml graduated test tubes, number them, add maltose standard solution (1 mg/ml) 0, 0.2, 0.6, 1.0, 1.4, 1.8, 2.0 ml respectively, put them into a boiling water bath to boil accurately for 5 minutes, take them out and cool them down, dilute them to 25 ml with distilled water, and then use a spectrophotometer to make a colorimetric comparison at a wavelength of 520 nm, record the extinction value, and use the extinction value as the vertical coordinate and the maltose content as the horizontal coordinate. maltose content as the horizontal coordinate to draw the standard curve.
(2) Determination of samples: Take 2 ml each of the solution after enzyme action in the above tubes and the solution in the control tube, put them into 25 ml stoppered graduated test tubes respectively, add 2 ml of 3,5-dinitrosalicylic acid reagent, mix well and put them into boiling water to boil accurately for 5 minutes, take them out to cool down, dilute them to 25 ml with distilled water and mix well. The colorimetry was carried out with a spectrophotometer at 520 nm, the extinction value was recorded, and the maltose content was found out from the maltose standard curve, and then the results were calculated.