Experiments with amylases

Enzymes accelerate the course of many chemical reactions, but they do it selectively. Each of them has its own narrow "specialty" - the same process is sometimes accelerated by several enzymes. Let us consider enzymes that destroy starch with the addition of water molecules to its "fragments", i.e. hydrolytic enzymes. To do this, we will carry out experiments with amylases and check how they act on human and animal amylases.

To do this, we will carry out experiments with amylases and check how they act on human and animal amylases

The source of human amylase will be saliva. Amylases of animal origin can be found in bee honey.

First prepare five solutions. The first solution: collect about 0,5 ml of saliva in a test tube and dilute with cold boiled water twenty times. The second solution: a very liquid starch paste (a quarter of a teaspoon of starch in a glass of water). Third solution: pharmacy iodine tincture diluted with water twenty times. Fourth solution: two to three drops of bee honey, diluted ten times with water and thoroughly mixed. Fifth solution: half a teaspoon of baking soda for ten tablespoons of water.

It will take nine tubes. Pour about 5 ml of paste into all tubes. Add 5 drops of vinegar to the tubes 1, 4 and 7, and the same amount of soda solution to the tubes 2, 5 and 8. Add 5 drops of clean water to the remaining tubes. Mix the contents of all tubes and add 10 drops of diluted saliva to each.

After 10 min, add one or two drops of iodine solution to tubes 1, 2, and 3 and mix the mixture. Observe discoloration. After another 15 minutes, add the same portion of iodine to tubes 4, 5 and 6, and after another 10 minutes to the remaining tubes. Starch and dextrins, as you recall, give a different color with iodine, and as the starch is destroyed by amylase, the color changes. Thus, one can judge not only the decay of starch, but also which medium acidic, neutral, or alkaline is more favorable for this process.

The experience with bee honey is set in the same way. The activity of amylases in different samples can vary greatly, therefore, it may be necessary to reduce or increase the hydrolysis time. For example, the saliva of heavy smokers of amylase contains very little.

For the next experiment, you will need barley malt - sprouted grains of barley. Dip the grains in water for several hours and leave them to germinate on a saucer for 4-5 days, adding a little water every day. Separate the seedlings, rinse with water and carefully rub with a wooden pestle or spoon. Dilute the gruel with a double amount of distilled water and squeeze it through a thick cloth into a glass. Such an extract contains two enzymes: alpha-amylase and beta-amylase. Additional processing can destroy one of them in order to observe the action of the other. Alpha amylase is destructible by heating. Add three parts of water to one part of the extract from barley, stir the mixture and heat it for 20 minutes in a water bath at 70 0, mixing thoroughly. The cooled solution contains beta-amylase.

Now, to get a solution of alpha-amylase, you need to destroy beta-amylase with acid. About 5 ml of the extract, cool to 2-3 0C in the refrigerator or on ice, add an incomplete teaspoon of chilled vinegar and add cold water to the tube almost to the top. Mix the mixture and leave for 15-20 minutes, and then neutralize the solution by adding chalk powder until the cessation of bubbles. Stir the mixture again, dilute twice with water, let stand and drain the liquid over the pellet into a clean tube. This completes the preparation for the experiment.

Pour 1 ml of starch solution and 9 ml of water into ten test tubes. In tubes 1-5, add ten drops of an alpha-amylase solution with a pipette, and in the remaining tubes, add the same amount of beta-amylase solution. Mix the contents of all tubes. After 3 min, add one drop of iodine solution to tubes 1 and 6 and mix. Do the same with tubes 2 and 7 after 5 minutes, 3 and 8 after 10 minutes, 4 and 9 after 20 minutes, 5 and 10 after 30 minutes.

You will notice that in the presence of alpha-amylase, the color changes rapidly: blue violet pink yellow: in this case dextrins are formed fragments of starch molecules. Beta-amylase acts differently: it "bites" pieces of starch molecules, as it were, and therefore the color with iodine remains blue, but its brightness decreases as the starch decays.

The results of this experiment clearly show the diversity of properties even in similar amylase enzymes. In living organisms, enzymes usually act together. The transformations occurring in this case are much more complicated than those relatively simple reactions that you observed in test tubes. But knowing the simple is the first step to knowing the complex.