Effect of environmental conditions on amylase activity
First, the purpose
Amylase (in two types, α-, β-) is present in plant tissues, which can hydrolyze stored starch into maltose. Thereby affecting the germination of the seed or the growth of the plant. The purpose of this experiment was to determine amylase activity and observe environmental conditions (such as temperature, pH) and the effects of certain chemicals on amylase activity.
Second, the principle
Amylase activity, like other enzymes, is very sensitive to changes in temperature and pH, especially by temperature and pH. In the environment where zui is suitable for temperature and pH is suitable for pH, the activity of the enzyme is strong, and the enzymatic reaction rate is high; below or above the suitable temperature of zui and the pH of zui, the enzyme activity is weak, and the enzymatic reaction rate is lowered.
In addition to temperature and pH, certain chemicals can inhibit or promote the activity of the enzyme. A chemical substance that inhibits enzyme activity, called an enzyme inhibitor, a chemical substance that promotes enzyme activity, and is called an activator of an enzyme.
After being affected by environmental conditions, the strength of amylase activity can be checked by iodine test.
Third, materials, equipment and reagents
1. Materials: wheat seeds; rice seeds.
2. Apparatus: constant temperature water bath ; constant temperature incubator; test tube ; 150ml triangle bottle; mortar ; beaker ; pipette ; plastic dropper; test tube holder; thermometer .
3. Reagents: series phosphate-citrate buffer; pH 5.0 acetate buffer (prepared according to Appendix II).
I- KI solution: 2 g of KI was weighed and dissolved in distilled water, and then 1 g of I 2 was added. After all dissolved, it was made up to 300 ml with distilled water and stored in a brown bottle for use.
0.5 % starch solution.
1 % CuSO 4 solution.
1 % KI solution.
0.1 % HgCl 2 solution.
1 % NaCl solution.
0.1% AgNO 3 solution.
Fourth, the experimental steps
1. Extraction of amylase
Take 20 grains of germinated rice or wheat seeds (3 to 4 cm in length), grind in a mortar , rinse the homogenate with 100 ml of water, concentrate in a 150 ml flask and stir well for 1 hour. Filter into another flask, the amylase is contained in the filtrate. If it is to be stored for a short period of time, add a few drops of toluene to the antiseptic and put it in the refrigerator.
2. Effect of temperature on activity Amylase
Take 5 test tubes, respectively, for 15, 30, 45, 60, 75 ° C temperature mark, and indicate "enzyme liquid tube", each added enzyme extract 5ml; another 5 test tubes, also for 15, 30, 45 60, 75 ° C temperature mark, and indicate "starch liquid tube", each added 0.5% starch solution 5ml; then each tube is placed in a water bath of the corresponding temperature, about 5min, after the tube temperature is consistent, the enzyme Pour the solution into the starch tube at the same temperature, shake it up and record the time. Take it after 10 minutes (the time should be the same between different treatments), put it in ice water for 1 min, add 5 drops of I-KI solution to each tube. Shake well. Observe the difference in color, compare the effects of different temperatures on amylase activity, and judge the appropriate enzymatic temperature of Zui.
3. Effect of pH on amylase activity:
Four medium-sized tubes were taken and 3 ml of the following series of pH phosphate-citrate buffers were added and labeled: pH 3.6, 4.8, 6.0, 7.2. Add 3 ml of starch solution and 2 ml of amylase extract to each tube, shake well, record the time, place it at a temperature of about 30 ° C for 20 min, add 5 drops of I-KI solution to each tube, shake well, and compare according to the color produced. Amylase activity.
4. Effect of chemical substances on amylase activity
Take 6 tubes and weave them from 1 to 6. Add 1 ml of amylase extract and 1 ml of pH 5.0 acetate buffer to each tube, and then add 1 ml of distilled water to the No. 1 tube (control). 2 tube plus 1% NaCl 1ml, tube 3 plus 1% potassium iodide 1ml, tube 4 added 1% CuSO 4 1ml, tube 5 plus 0.1% AgNO 3 1ml, tube 6 added 0.1% HgCl 2 (note, Toxic!) 1ml, add 0.5ml of 0.5% starch solution to each tube, shake it, place it in a constant temperature water bath at 40 °C, record the start time, keep it for 8-10 minutes, take out the test tube, and add I-KI solution to each tube after cooling. Drip, shake, and observe the difference in color of each tube.
V. Recording and analysis of experimental results
1. Use text to indicate the color of each tube after treatment with different temperatures, pH and various chemicals.
2. Briefly analyze the effects of environmental conditions on amylase activity using text.
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