An experiment to investigate the effect of enzyme concentration on the rate of milk lipid digestion by lipase.

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Adam Hussein 6B1 28-12-02

An experiment to investigate the effect of enzyme concentration on the rate of milk lipid digestion by lipase.

Biology Coursework Plan:

Aim:

This investigation aims to find out how enzyme concentration affects the rate of milk fat digestion by lipase enzyme. As a result of the experiment I aim to have quantitative results which I can then use to plot graphs illustrating the effect of lipase.

Prediction:

I predict that as the concentration of lipase enzyme increases, so to will the rate of reaction. The two will be directly proportional to each other up to the point where the substrate (milk fat) becomes rate limiting. This is the point where there are so many enzyme molecules and not enough substrate molecules for them to catalyse. The rate will therefore remain the same past this point as shown in the below graph.

Variables:

These are factors that will affect the reaction in some way. In order for the experiment to be successful in supplying reliably accurate results all variables must be kept constant apart from the enzyme concentration will be varied by known amounts to show the effect it has on the rate of lipid digestion by lipase. Below are the variables and explanations on how they will be kept constant in order to keep this experiment a fair test, and what affect them not being kept constant will have on the results.

- Bile concentration:

==> What is it:

Bile is an alkaline substance due to it containing sodium hydrogen carbonate. It is this that neutralises the stomach acid before entering the duodenum through a sphincter. It is very important because without it the enzymes secreted by the pancreas (in pancreatic juice) would be denatured by such extremes in pH. What assists the digestion of fat are the bile salts contained in bile. These help by emulsifying the large fat droplets into tiny droplets called micelles, thus increasing the surface area that the enzyme lipase has to work on therefore increasing the rate of reaction. Bile is produced in the liver and stored in the gall bladder near by. It is transported to the stomach through the bile duct. Below is an emulsification diagram, showing first, a large lipid droplet with only a small number of enzymes able to work on it. The second diagram shows small lipid micelles after being emulsified by bile. The effect that bile has on lipids can be clearly seen. Many more enzymes can work on the same amount of lipid once emulsified, resulting in a quicker reaction.

==> How it affects the reaction:

Bile concentration can greatly affect the reaction. It is not only altered when more or less bile is added but when more or less milk fat is added. The affect of increased concentration should results in a quicker rate of reaction. This would be due to more collisions between the bile salts and milk fat droplets, either down to there being more bile, or less milk fat consequently resulting in their being more bile available at one time to emulsify the milk fat droplets. The increased collisions will mean more surface area for the lipase to digest the fat from quicker resulting in a quicker, yet equal pH change. However there is a limit to this variable having an affect. This limit will be reached when the bile concentration gets so high that there is simply not enough lipid for it to emulsify all at once.

==> How bile concentration will be kept constant.

Most obviously the same type of measuring instruments will be used each time and the same measures (2cm3 ) will be taken, using the same graduated pipette.

- Lipase concentration:

==> What is it:

Lipase is an enzyme, a biological catalyst, which as the name suggests digests lipids. One lipid molecule is digested into three fatty acid chains and one glycerol molecule. All enzymes act in a way to reduce the activation energy thus speeding up the rate of a chemical reaction, while also not being consumed during the reaction.

==> How it affects the reaction:

The rate of the reaction between enzyme (lipase) and substrate (milk fat) is directly proportional to the enzyme concentration up to the point where substrate concentration exceeds enzyme concentration. This is simply due to more enzyme molecules being present to react and catalyse the reaction. Also as the enzyme concentration increases, the faster rate of reaction due to the fatty acids (the product of the reaction) lowering the pH of the solution closer to lipase' optimum pH.

==> How enzyme concentration must be kept accurate:

We are using different enzyme concentrations in this reaction so even though they are not being kept constant they must be accurate. The source of the lipase enzyme must remain constant due to possibly more or less impurities possibly acting as inhibitors therefore causing anomalies. The sample of lipase must be kept in suitable conditions (example; the same temperature throughout) so as not to cause denaturation of enzymes. Also, the same graduated pipette must be used each time.

- Fat content of milk:

==> What is it:

Triglycerides can be solid or liquid at room temperature. The solids are called fats while the liquids are called oils. Fats are solid due to the high proportion of saturated fatty acids (those with no double bonds).

==> How it affects the reaction:

The fat content of the milk must remain constant throughout all the tests. We will be using the same measures of milk for each experiment, however if the fat concentration of the milk alters, the same measures will contain different amounts of lipid. This will have the opposite effects to changing enzyme concentration in the way that increase in milk lipid concentration will result in a faster rate of reaction up until the point where lipase becomes rate limiting and milk lipid molecules are hugely in excess of the lipase enzyme. In this case, there would simply be too few enzymes to catalyse much of the substrate (milk lipid) at once.

==> How fat content of milk will be kept constant:

To ensure that the fat concentration of milk is kept constant, we will use milk from the same sample. Homogenised milk also must be used. This is milk where the fat is equally distributed throughout. This is different to normal milk as no fat is suspended at the top of the milk, which would mean the concentration would differ with each sample we take. Although homogenised milk is being used it must still be stirred before taking a measurement as a precautionary step to increase accuracy.

- Temperature:

==>How it affects the reaction:

This reaction using enzymes will be greatly affected by differences in temperature if they arise. The temperature will have to be kept constant throughout. The higher the temperature is, the higher the rate of reaction will be up to the enzymes optimum temperature. For lipase this is around 37º ideal for the body's internal temperature also at 37º. Beyond this temperature, the enzyme will begin to get denatured by the heat. What this is, is the change in shape of the enzyme, by altering the specific tertiary structure of the enzyme so the substrate no longer fits the active site and the enzyme is completely useless. This process is irreversible so enzyme cannot be used after becoming denatured. Increasing temperatures up to this point increase the rate of the reaction by giving the enzyme and the substrate increased kinetic energy so they move faster and collisions between enzyme and substrate are more often and also stronger ensuring more successful collisions.
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==> How temperature will be kept constant:

The experiment is being carried out at room temperature so everything used in the test will be kept at room temperature before being used. By doing this all tests should be carried out at the same temperatures. It must be insured that samples are not placed next to radiators etc which will cause anomalous results.

- pH (sodium carbonate):

==> What is it:

pH is a measure of the concentration of hydrogen ions in a solution. Acidic solutions contain a high proportion of hydrogen ions and alkaline ...

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