How temperature affects the activity of lipase

Jide Adeoye

LBIO5

Biology Coursework

Title: How temperature affects the activity of lipase

Aim: To investigate the effect that temperature has on lipase with increasing and decreasing temperatures and if it changes the rate of reaction?

Hypothesis: I think that the reaction will work best at 37oC, and the rate of enzyme action will increase as temperature increases, and decreases when temperature decreases but only to certain temperatures, because if the temperature is too high the enzyme actions may stop, because of the braking of H-bonds in the enzymes (denatured), and if too low the enzymes may freeze and may not move, or move but really slowly, so their will be no or may be a slow action from enzymes.

Enzymes are biological catalysts made out of proteins the blood test for lipase (enzymes) is used to help diagnose the swelling of the pancreas and other . It is also used in lesser extent in the diagnosis and follow-up of , , and their function is determined by their complex structure and there are around 40,000 different enzymes in human cells, with different functions and structure. They are said to increase rate reaction by 106 to 1012 times such as in metabolic reactions (rate of respiration, digestion and photosynthesis etc is increased). They may also act as motors, membrane pumps and receptors.

The active site is found in a small part of the enzyme and is where reactions take place, while the rest of the protein acts as “scaffolding”. As I said before, each enzyme is specific which means only one molecule (substrate) will bind to its active site, this is because amino acids around the active site attach to the substrate molecule and hold it in position while the reaction take place. The substrate molecule fits into the active site of the enzyme molecule like a key fitting into a lock (in fact it is sometimes called a lock and key mechanism). Once there, the enzyme changes shape slightly, distorting the molecule in the active site, and making it more likely to change into the product. The enzyme is roughly unchanged in the reaction and can be used again and again and can react with a lager number of substrates within a second to produces products e.g.

The end results are the same, but a different route is taken, so that the reaction can be made to happen much more quickly. Alternatively the enzyme can make the local conditions inside the active site quite different from those outside (such as pH, water concentration, charge), so as well increasing the rate of reaction. But before it can change into a product, the substrate must overcome an "energy barrier" called the activation energy (EA). The larger the activation energy, the slower the reaction will be because only a few substrate molecules will by chance have sufficient energy to overcome the activation energy barrier.

Enzymes are affected by in many different ways like, temperature, pH, enzyme concentration, inhibitors and substrate concentration. Optimum temperature is what enzymes work best at. For mammalian enzymes this is about 40°C (fig 1.1), but there are enzymes that work best at very different temperatures, e.g. enzymes from the arctic snow flea work at -10°C or at 0°C enzymes still work in the fridge (and food still goes off), but they work slowly and enzymes from hot (thermophilic) places containing bacteria work at 90°C.

Up to the Optimum temperature the rate or reaction increases steeply (it’s a curve).the rate of ...

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Up to the Optimum temperature the rate or reaction increases steeply (it’s a curve).the rate of reaction increases because more enzymes and substrate has the right (sufficient) K.E. to over come the Ea. Above the optimum temperature the rate slows down because more enzymes denature. Due to the breaking of the weak H-bonds holing the 2nd and tertiary structure of the enzyme together by the thermal energy, so the enzyme losses its shape change the active site, so substrates are unable to bind to enzymes, so the rate of reaction decreases or even stops.

Enzymes also have an Optimum pH and are where they work fastest. In most enzymes their pH is roughly around 7-8(fig 1.2) which is the pH for most animal cells. Some enzymes can work in extreme acidity (pH 1) like protease, where they are found in animal stomachs.

Fig 1.1 fig 1.1 Fig 1.1 Fig 1.2

Some enzyme reaction may be effected by Inhibitors. Inhibitors inhibit the activity of enzymes, reducing the rate of their reactions. They are found naturally, and there are two kinds of inhibitors, competitive inhibitor and non-competitive inhibitor. Competitive inhibitors are molecules which have the same shape (structure) as the substrate molecule, which means that they can fit into the enzymes active site. So you could say that it competes with the substrate to get to the active site, hence slowing down the reaction (Fig 1.3). The rate of reaction can be increase if the concentration of the substrate is greater than before, so there will be a greater chance of the substrates combining to the enzymes active site. Non-competitive inhibitors are quite different in structure from the substrates, and they do not fit into the active site. It binds to another part of the enzymes molecule, which changes the shape of the whole enzyme (including the active site) hence the enzymes can no longer bind to the substrate molecules (Fig 1.4). So non-competitive inhibitors basically decrease the activity of enzymes actions or decreases concentration of enzymes, so decreasing vmax, but have no effect on Km. The types of inhibitors that bind very weakly and that are able to wash off are said to be called reversible inhibitors, and those that bind tightly and cannot be washed or removed are said to be called irreversible inhibitors. Non-competitive inhibitors are things like metal ions and some insecticides.

Fig 1.3 Fig 1.4

Apparatus: I will need 5-6 measuring beakers to measure the amount of NaCa, Phenolphthalein and marvel powdered milk, and also we need it to contain these items. A pipette to make sure that drops of NaHCO3 and Phenolphthalein are drop to the nearest 0.2mm3, a 15 cm3 syringe to collect the right amount of marvel powdered milk accurately, a 250cm3 measuring cylinder to also measure the amount of NaCa, Phenolphthalein and marvel powdered milk in the experiment, 6 test/boiling tubes to do the experiment in and to use in the water bath, a test tube rack to hold the test tubes in after being taken out of the water bath, a stop clock to measure the time spent in the water bath, in order to keep the experiment fair, marvel milk because it contains fatty acids and glycerol’s joined by ester bonds in condensation, to form triglycerides and can be broken down by hydrolysis(with help from lipase), water can be used as a solvent in the reaction, some ice can be used to cool the reaction to -0oC, a control is needed in the reaction (NaCa, Phenolphthalein and lipase), pH Indicator will be needed to find out the acidity of the products, a water bath is needed to increase the temperature in order to see were the enzymes will work best at.

Procedure:

First I will measure out 3-5 cm3 of marvel milk using a syringe

Secondly I will put the 3-5 cm3 of marvel powdered milk into a test tube and put 6 drops (0.2-0.5mm3) of Phenolphthalein, which should be taken from a 250 cm3 measuring beaker using a drop pipette and confirm the pH, using pH indicator.

Add 6-10 drops of NaCa, which should be taken from a 250 cm3 measuring beaker using a drop pipette and drop 6-10 NaCa into the powdered milk solution.

Then change the temperature to 0oC using ice and 10 oC, 20 oC, 30 oC, 40 oC and 50 oC using a water bath and record your results.

Control variables:

In the experiment, I will have to keep the concentration of enzymes the same, because if the concentration of enzymes increases, it will affect the enzymes action, hence speeding up the reaction, but if decreased, the reaction will also decrease because, the substrate have less chance of binding to an enzyme because the enzymes concentration has been reduced. So I will have to keep the concentration of enzymes the same for all the reactions, or if not I will have to change the reaction, for all the other reactions to be the same, so that the experiment will remain fair.

In the experiment I will have to keep the concentration of substrate the same, because if the concentration of substrate increases, it will affect the reaction, hence making the substrate queue up for the enzymes, but if decreased, the reaction will increase because there is less substrate in the solution than there is enzymes, so the substrate will have a better chance of binding to the enzymes. So I will have to keep the concentration of substrate the same for all the reactions, or if not I will have to change the reaction, for all the other reactions to be the same, so that the experiment will remain fair.

In the experiment I will have to keep the concentration of acid the same, because if the concentration of acid increases, it will affect the enzymes action, hence stopping and destroying the reaction, but if decreased, the reaction may also decrease. So I will have to keep the concentration of acid the same for all the reactions, or if not I will have to change the reaction, for all the other reactions to be the same, so that the experiment will remain fair.

In the experiment I will have to keep the pH the same, because this will have an effect on the enzymes, if increased, because it may destroy the enzyme, and if the ph was to change, I will have to change the pH in all the other reactions to keep the experiment fair.

In the experiment, I will have to keep the concentration and the amount of powdered marvel milk the same, because if the concentration and the amount of powdered marvel milk increases, it will affect the reaction, hence slowing down the reaction, but if decreased, the reaction will increase because, the substrate have more chance of binding to an enzyme because the enzymes concentration is higher. So I will have to keep the concentration and the amount of powdered marvel milk the same for all the reactions, or if not I will have to change the reaction, for all the other reactions to be the same, so that the experiment will remain fair.

When repeating the experiment, the water bath must be heated (or cooled) to six different temperatures: 0oC, 20˚C, 30˚C, 40˚C and 50˚C, I will make this also fair experiment, by ensuring that all the solutions will be heated for an equal amount of time, because if not the experiment will be unfair, because the solution will have heated, longer in the water bath, hence a longer time for reaction. And the amount of acid added, will be accurately measured from the meniscus in the burette, so that each solution has the same accurate pH, to maintain a fair result.

I have chosen this choice of equipment because it is reliable and relatively easy and quick to set up. I decided for the use of a water bath, to heat the solutions, as it is easy to use, and far more accurate. I will also use the same apparatus, to keep the experiment fair, and to minimize error results.

Variables that I need to change:

Temperature - I will use different temperatures each time e.g. 0oC, 20˚C, 30˚C, 40˚C and 50˚C.

Safety:

When doing the experiment, I will use plastic glasses, to protect my eyes from any sort of acid. I will also wear a lab coat, to protect me from any chemicals which may spill onto my clothes. I will also work on a table and ensure that my chair is pushed in, because this may result in an accident. I will also wear hand protection (plastic gloves), to protect me against any sort of acid pouring onto my hands.

Results:

Analysis of results:

This trend can be explained through the theory of denature, of enzymes as temperature increases As temperature increases molecules and ions are gaining more kinetic energy, so there will be a increase in collision frequency and the rate of reaction will increases as a result. However, at temperatures, or above the optimum, (which we know is about 45oC); the delicate tertiary structure of the active site on the lipase molecule will be destroyed. This is because there is an increase in kinetic energy and so the vigorous vibration causes the H-bonds to break and denature, and the substrate molecule will no longer be able to fit into the active site. Consequently, the high temperatures will have an effect on lipases active site.

This comparison indicates that the rate of reaction increases with an increased in temperature, although it is at a slower rate at the higher temperature. As a summary, although the time of clarification and the rate of reaction increase with an increase in temperature, these two factors are lower in general as the temperature increases, in comparison to the lowest temperature in the range.

* this website was very useful, because it give the Info I needed.

* this website was very useful, because it give the info I needed.

*Biological science 1 & 2, ISBN 0-521-56178-7, Chapter 4, Pg 116 - 125. This book was very useful, because it talk about enzymes