The specific steps of amylase activity determination are as follows:
1. Materials, equipment and reagents
1. Materials: germinated wheat seeds (bud length is about 1cm).
2. Instruments and equipment: spectrophotometer; Centrifuge; Constant temperature water bath; Calibration test tube with plug; Graduated pipette; A volumetric flask.
3. reagents (all analytically pure)
standard maltose solution; 3.5- dinitrosalicylic acid reagent; .1 mol/l PH5.6 citric acid buffer and 1% starch solution.
Second, experimental steps
1. Making maltose standard curve: Take seven clean calibrated test tubes with plugs, number them, and add reagents according to the table. Shake well and boil 5min boiling water bath for 5 minutes. Take it out, cool it with running water, and add distilled water to constant volume to 2ml. Using No.1 tube as blank zero point, colorimetric determination was carried out at 54nm wavelength. Draw a standard curve with maltose content as horizontal coordinate and absorbance value as vertical coordinate.
2. Preparation of enzyme solution: Weigh 1g of wheat seeds germinated for 3 days, put them in a mortar, add a small amount of quartz sand and 2ml of distilled water, and grind them into homogenate. Pour the homogenate into a centrifuge tube, and wash the residue into the centrifuge tube with 6ml of distilled water.
The extraction solution was left at room temperature for 15 ~ 2min, and stirred every few minutes to make it fully extracted. Then centrifuge at 3rpm for 1min, pour the supernatant into a 1ml volumetric flask, add distilled water to constant volume to the scale, and shake well to obtain the amylase stock solution.
absorb 1ml of the above-mentioned amylase stock solution, put it into a 5ml volumetric flask, adjust the volume to the scale with distilled water, and shake well to obtain the amylase diluent.
3. Determination of enzyme activity: Take 6 clean calibrated test tubes with plugs, number them and operate according to the table.
4. calculation of results: amylase activity =C×VT/(W×Vs×T)(mg/g/min).