Specificity of amylase and sucrase.

Laboratory work 4. Enzymes

Detection of peroxidase in potatoes.

Grate some potatoes and put 2 ml of pulp into a test tube. Add 1 ml of pyrogallol 1% solution and 2 drops of hydrogen peroxide 2% solution.

Numerous dehydrogenation (oxidation) of pyrogallol takes place in participation with peroxidase, which transfers atoms of hydrogen from the substrate to hydrogen peroxide. A number of intermediate products and purpurogallin are formed.

3. Hydrolysis of starch by α-amylase.

Rinse the mouth 2–3 times with water to remove the scraps of food. Measure 30 ml of distilled water and use it to rinse the mouth for 2 minutes. Filter the liquid through cotton wool. Use the filtrate for experiments. Prepare solutions into the test tubes according to the table.

Prepare two water baths with temperatures of 37-40°С and 100°С. Place enumerated test tubes into the water bath. Place a drop of Lugol’s solution on the glass plate with a glass stick every 2 minutes. Then place а drop of the solution from the test tube is with a clean stick. The mixture of two drops gives coloring. The experiment lasts for 10 minutes. Put results of the experiment and the conclusions of hydrolysis passing (full, partial or does not pass) are given in the table.

The scheme of hydrolysis of starch by α-amylase:

(С6Н10О5)n starch		(С6Н10О5)m amylodextrines
(С6Н10О5)k erythrodextrines		(С6Н10О5)l achrodextrines
(С6Н10О5)x maltodextrines		С12Н22О22 maltose

Compare activities of inorganic catalyst and enzyme; make a conclusion about pH and T influence on amylase activity.

Specificity of amylase and sucrase.

The polysaccharides starch and glycogen are specific substrates for amylase. The sucrose disaccharide is a substrate for sucrase enzyme. The hydrolysis process in the presence of enzymes can be detected by Trommer’s test. This test is not positive with initial carbohydrates.

Enumerate four test tubes. Pour 2 ml of starch solution into test tubes number 1 and 2; and 2 ml of sucrose solution into test tubes number 3 and 4. Add 0.5 ml. of saliva solution into test tubes number 1 and 3, and 0.5 ml. of 1% yeast sucrase solution into test tubes number 2 and 4. Mix and place in the water bath at the temperature of 38-40°С for 10 minutes. After cooling make test with iodine in test tubes number 1 and 2; then use Trommer’s test in test tubes number 3 and 4. The final step is making a conclusion about the specificity of the learned enzymes.

sucrose glucose glycoside hydroxyls fructose

Table. The results of starch hydrolysis.

№	The contents of the test tube	Т, °С	Time, min.	The color in the reaction with iodine	The presence of hydrolysis
	3 ml of starch solution + + 2 ml of 10% H2SO4
	3 ml of starch solution + + 2 ml of 10% H2SO4
	3 ml of starch solution + + 3 ml of amylase solution
	3 ml of starch solution + + 3 ml of amylase solution + + 1 ml of 2 n. НС1
	3 ml of starch solution + + 3 ml of the boiled amylase solution

Tasks

1. What classes are enzymes used in the lab belong to?

2. Wright the equation of sucrose hydrolysis by sucrase.

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