WORK № 4. The influence of рН on the enzymes activity

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 (37^oC), 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 (37^oC) 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.

Summary:

WORK № 2. The influence of activators and inhibitors

APPARATUSES: support with test-tubes, different graduated pipettes, eye pipettes, thermostat (37^oC) 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 (37^oC). After that add by 1 drop of reactive of Lugol in each test-tube and fill the table of experiment.

Summary:

WORK № 3. Specific action of yeast enzyme

APPARATUSES: support with test-tubes, different graduated pipettes, eye pipettes, alcohol lamp, thermostat (37^oC), 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.

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^оС) 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.

Summary:

WORK № 4. The influence of рН on the enzymes activity

APPARATUSES: support with test-tubes, different graduated pipettes, eye pipettes, thermostat or water-bath (37^oC).

REACTIVES:0,2М solution of Na₂HPO₄ (solution A), 0,1М solution of citric acid (C₆H₈O₇^.H₂O) (solution B). Buffer solutions (рН 5,0 : 515ml of solution А mix with 485 ml of solution B; рН 6,8 : 772,5 ml of solution А mix with 227,5 ml of solution B; рН 8,0 : 972,5 ml of solution А 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 рН (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^оС). After that add by 1 drop of reactive of Lugol in each test-tube and fill the table of your experiment.

Summary:

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