Enzymes
WORK 1. Thermolabile of ferments.
APPARATUSES: support with test-tubes, 50 ml cylinders or conical flasks, different graduated pipettes, eye pipettes, alcohol lamp, thermostat (37oC), glass with ice.
REACTIVES: diluted saliva (take 20-25 ml of distilled water in mouth and collect it through paper filter in cylinder), 0.3% solution of NaCl, 1% solution of starch on the 0.3% solution of 0.3% solution of NaCl, reactive of Lugol (20g of KJ and 10 g of iodine dissolve in 100 ml of water, then this solution dissolves by distilled water as 1:5).
PRACTICE COURSE. Three test-tubes are labeled by numbers and 2-3 ml of diluted saliva is put into the 3 labeled test-tubes. The saliva from the first test-tube is boiling during 1-2 min. Than add 4-5 ml of 1% solution of starch in each of 3 test-tubes. Put test-tubes number 1 and 2 into thermostat (37oC) or water-bath on 10 min. The test-tube number 3 put in glass with ice on 10 min. After that reactive of Lugol is added in all test-tubes by 1 drop. Fill the table of experiment and make summary.
| Number of test-tube | Enzyme | Conditions of experiment | Substrate | Incubation | Colour with iodine |
| Amylase | Denaturated enzyme | Starch | 10 min, 37 | ||
| Amylase | Native enzyme | Starch | 10 min, 37 | ||
| Amylase | Native enzyme | Starch | 10 min, 0 |
Summary:
WORK 2. The influence of activators and inhibitors
APPARATUSES: support with test-tubes, different graduated pipettes, eye pipettes, thermostat (37oC) or water-bath.
REACTIVES: diluted saliva, 1% solution of NaCl, 1% solution of starch on the 0.3% solution of NaCl, 1% solution of CuSO4, distilled water, reactive of Lugol.
PRACTICE COURSE. Three test-tubes are labeled by numbers and 4-5 ml of 1% solution of starch is put into the 3 labeled test-tubes. Than in the test-tube number 1 add 1-2 ml of 1% solution of NaCl, in the test-tube number 2 1-2 ml of 1% solution of CuSO4 and in the test-tube number 3 1-2 ml of distilled water. Each of test-tubes is added by 1-2 ml of diluted saliva. Mix thoroughly each of these test-tubes and incubate them during 10 min into water-bath (37oC). After that add by 1 drop of reactive of Lugol in each test-tube and fill the table of experiment.
| Number of test-tube | Enzyme | Effector | Substrate | Incubation | Colour with iodine |
| Amylase | NaCl | Starch | 10 min,37 | ||
| Amylase | CuSO4 | Starch | 10 min,37 | ||
| Amylase | 2 | Starch | 10 min, 37 |
Summary:
WORK 3. Specific action of yeast enzyme
APPARATUSES: support with test-tubes, different graduated pipettes, eye pipettes, alcohol lamp, thermostat (37oC), porcelain mortar with pestle, funnels, paper filters.
REACTIVES: 0.5 g of dry yeast, 1% solution of starch on the 0.3% solution of NaCl, 1% solution of CuSO4, 10% solution of NaOH, 2% solution of saccharose, distilled water.
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PRACTICE COURSE. First of all take 0.5 g of dry yeast and triturate them in the porcelain mortar with pestle. Than add 5 ml of water and continue triturate yeast together with water. Filtrate the obtain mixture through fluted filter. Filter liquor contain yeast enzyme (saccharase).
Two test-tubes are labeled by numbers and 1 ml of saccharase is put into each of them. Than add 1 ml of 2% solution of saccharose in test-tube number 1 and 1 ml of 1% solution of starch into test-tube number 2. Put all test-tubes into water-bath (37) on 15 minutes. After that do Trommer test. Take two clean test-tube and add in the first of them 5 drops of solution from the test-tube number 1. In the second test-tube fill 5 drops of solution from the test-tube number 2. Than add 10 drops of 10% solution NaOH and 1-3 drops of 1% solution of CuSO4 (II). Mix all solutions and heat them to the boiling. Red colour of solution shows us that glucose is present.
| Number of test-tube | Enzyme (saccharase), ml | Solution of saccharose, ml | Solution of starch, ml | Reaction of Trommer (+ or -) |
| - | ||||
| - |
Summary:
WORK 4. The influence of on the enzymes activity
APPARATUSES: support with test-tubes, different graduated pipettes, eye pipettes, thermostat or water-bath (37oC).
REACTIVES: 0,2 solution of Na2HPO4 (solution A), 0,1 solution of citric acid (C6H8O7.H2O) (solution B). Buffer solutions ( 5,0: 515ml of solution mix with 485 ml of solution B; 6,8: 772,5 ml of solution mix with 227,5 ml of solution B; 8,0: 972,5 ml of solution mix with 27,5 ml of solution B), diluted saliva, 1% solution of starch, reactive of Lugol.
PRACTICE COURSE. Take three test-tubes and labeled them. Fill test-tubes by 2-3 ml of buffer solutions with different values of (5,0; 6,8; 8,0). Add 2-3 ml of diluted saliva and 4-5 ml of 1% solution of starch in each of test-tube. Mix solutions in each test-tube and incubate them during 10 min into water-bath (37). After that add by 1 drop of reactive of Lugol in each test-tube and fill the table of your experiment.
| Number of test-tube | Enzyme | medium | Substrate | Incubation | Colour with iodine |
| Amylase | 5,0 | Starch | 10 min, 37 | ||
| Amylase | 6,8 | Starch | 10 min, 37 | ||
| Amylase | 8,0 | Starch | 10 min, 37 |
Summary:
