Enzyme Lab

Factors Affecting the Rate of Enzyme Activity

Malt amylase (enzyme) was tested in different enzyme concentration, substrate concentration and environmental factors such as pH level, temperature and inhibitor (ZnCl2) in order to see the effect of those factors to the ability of enzyme breaking down substrate. In the experiments for effects of changes in enzyme concentration, substrate concentration and inhibitor on enzyme’s function, one drop of iodine was added to three controls and nine variables and each variables contained different concentration of enzyme, substrate or inhibitor depending on the experiment. In the experiments for effects of changes in temperature and pH on enzyme’s function, one drop of iodine was added to each test tube. The experiments for temperature and pH contained 3 different parts: test tubes with water at room temperature (25в„Ñ"), 7в„Ñ" and 100в„Ñ" were tested and test tubes with pH 7, pH 4 and pH 11 were tested. Since this lab was performed for two days, the concentrations of enzymes were different between two days. Although measuring standard time curve for enzyme was performed for both day, the different concentration of enzyme could have brought variations to the results by not having same controls for two days. This might have cause wrong interpretation of results as variables were needed to be compared with controls to interpret the results. This experiment should have been performed in one day so that the same concentration of enzyme could be used to create consistent enzymes to observe the enzyme activity and see results by comparing with consistent controls.

Introduction

Enzymes are functional proteins that catalyze specific reactions in living organisms. It is often referred as biological catalysts because of its ability to help endothermic and exothermic reaction in living organisms. Enzymes work by lowering the activation energy of reactions, which must be overcame in order for the reaction to complete. For example, human body has amylase which is used in this experiment to break down starch (polysaccharide) into maltose (disaccharide). Then there is another enzyme such as maltase that breaks down maltose into two alpha glucose (monosaccharide). Without these enzymes, it will take forever for the starch to be broken down to alpha glucose which is the primary source of energy.

Enzymes have active sites that substrates can fit into. These active sites are consisted of charged amino acids which hold the substrate with its charges. There are two models that explain how enzymes work: one is key-lock model and the other is induced fit model. Enzymes with lock-key model have fixed 3-D shape and they can only work with substrates that can fit into the fixed shape of active sites of the enzyme. Due to their inability to break down every substrate, those enzymes are called substrate-specific. The other, induced fit model, states that enzymes can mould around the substrate to be fit into the shape of substrate. In this way, the enzymes can work with almost any substrate. Lock-key model applies to more enzymes than induced fit model; thus enzymes are mostly substrate specific.

In this experiment, substrate concentration, enzyme concentration, temperature, pH and inhibitor (ZnCl2) was the factors that affect enzymes ability to help reactions. As concentration of substrate increases, the activity rate of enzyme increase up to a point. As all the enzymes are occupied by the starch (substrate), the rate of reaction cannot increase any more. As concentration of enzyme increases, the activity rate of enzyme could increase without plateau. Since amount of enzyme is much smaller than substrates, the increase of enzyme concentration mean increases activity rate without plateau. For temperature and pH level, enzymes have optimum temperature and Ph where they can work at the maximum rate. As temperature and pH go above or below, the rate of enzyme activity decreases. Malt amylase and starch are used as enzyme and substrate in this experiment, respectively.

Procedure

Standard Curve (determining the time for amylase to catalyze the breakdown of starch):

1. Prepare a spot plate with 12 spots.

2. Add 18 drops of water and 2 drops of the enzyme solution in first spot. This is control 1, which has not starch. Add 1 drop of iodine and stir.

3. Add 19 drops of water and 1 drop of starch in second spot. Then, add 1 drop of iodine. This is control 2, which shows the colour of solution that has no enzyme. Stir.

4. Add 20 drops of water only for third spot. Then, add 1 drop of iodine. This is control 3 which show the colour of the solution when starch is completely broken down.

5. Fill the remaining 9 spot holes with 17 drops of water, 1 drop of starch and 2 drops of the enzyme. Stir each spot hole. They will be labeled from time zero to time eight.

6. In every one minute, add 1 drop of iodine to holes from time zero to time eight. (i.e. Add iodine to hole labeled time zero as soon as the enzyme is added, one minute later add iodine to hole labeled time one and so on)

7. Compare the colour of each spot with control 2 and look for a spot which matches to the colour of control 2.

8. The time of adding iodine to the spot will be the standard time and this timing will be used for the rest of the investigation after adding enzyme.

The purpose of this was to find how long does it take for the enzyme to break down all substrate (starch).

Part I: the effect that a change in temperature will have on the rate of amylase activity

1. Add 19 drops of room temperature water and 1 drop of starch to the first of three test tubes. This is control 1. This control ensures that the iodine will still stain the starch despite the presence of a different temperature.

2. Add 20 drops of room temperature water to the second test tube. This is control 2, showing the colour of the solution when starch is completely broken down.

3. Add 17 drops of room temperature water, 1 drop of starch and 2 drops of enzyme to the third test tube.

4. Add 1 drop of iodine to all of these solutions after 3 minutes (the number of minutes the enzyme reaction to occur; from determining standard curve) after having added the enzyme.

5. Heat water in a beaker to 100в„Ñ".

6. Repeat steps

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