This is an investigation to determine the effect of concentration on the activity of an enzyme Trypsin on the insoluble protein found in milk known as Casein.

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This is an investigation to determine the effect of concentration on the activity of an enzyme Trypsin on the insoluble protein found in milk known as Casein.


Casein is an insoluble protein, found in milk as a suspension of particles, which are held together by calcium ions. It forms a white or cloudy precipitant.

In the structure of casein, there are no disulphide bridges. Therefore, it has a relatively small secondary and tertiary structure. As a result, it does not denature quite easily. Furthermore, the hydrophobic groups found on the outside of protein make it insoluble in water.

When a suspension of casein is hydrolysed, it becomes cloudy at first and eventually clearer when the product is dissolved. This Hydrolysis Reaction is catalysed by Proteolytic enzyme such as Trypsin.

Trypsin is a digestive protease. It is produced by the pancreas and works in the small intestine, where it breaks down proteins to polypeptides and into amino acids.

  • Dependent Variables

  • Enzyme and Substrate Temperature
  • Buffer Solution
  • pH

  • Independent Variable

  • Enzyme Concentration

  • Confounding Variables

  • Enzyme and Substrate Temperature

The rate of an enzyme controlled reaction is measured upon the substrate used or the products formed over a period time.

Increasing the temperature causes more heat energy, which makes the substrate molecules and enzymes move a lot faster. This increases the kinetic energy; therefore more collisions take place between the substrate and the active site, resulting in more enzyme substrate complexes and formation of more products.

If the temperature is increased even further; above 40°C, the substrate molecules and enzymes will vibrate so energetically that the hydrogen bonds holding the tertiary structure will break. The enzyme will lose its globular shape, which will affect the active site. The substrate will no longer fit into the enzyme’s active site and the enzyme is said to be denatured.

However at lower temperatures; around 4°C, the enzyme is inactivated but not denatured. It will regain its function when the temperature is restored.

Optimum Temperature is the perfect temperature for the enzyme to react with the substrate.

My experiment will be carried out at room temperature, which is around

23 - 24°C therefore every enzyme activity will be affected by the same temperature. This will maintain a steady rate of enzyme activity and keep the temperature constant.

  • Buffer Solution

The buffer solution I will be using is called Sodium Tetraborate.

This buffer solution is used to maintain a constant pH of 9. Combining 0.38g of sodium tetraborate with 100ml of distilled water will produce buffered distilled water, which is then used to make different concentrations of trypsin.

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Throughout this experiment I will be using the same buffer solution provided, and will not make any changes of my own that might affect the enzyme activity.

  • pH

pH is a measure of hydrogen ions in a solution. The greater the concentration of hydrogen ions the lower the pH.

Most enzymes function at a narrow pH range. A change in pH may create a difference in the enzyme activity.

The pH of trypsin is between 8 and 9 and the pH of buffer solution is 9.

The pH of the buffer solution ...

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