Investigate the effect of enzyme temperature on the activity of the enzyme Trypsin on the substrate Casein.

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The Activity of the Enzyme Trypsin on the Insoluble Milk Protein Casein

Planning (skills A and B): -

Aim: -

To investigate the effect of enzyme temperature on the activity of the enzyme Trypsin on the substrate Casein.

Introduction: -

        Casein is an insoluble protein found in powdered milk, such as Marvel, which forms a white or cloudy suspension (not dissolved). A white suspension of Marvel milk in water clears if a protein-digesting enzyme is added. The enzyme used in this experiment is trypsin. When the suspension is hydrolysed (broken down), it starts off cloudy, but becomes clearer as the products (peptides and amino acids) dissolve. This reaction is catalysed by proteolytic enzymes (proteinases) such as trypsin. Trypsin is released into the small intestine in alkaline conditions. It is one of the main digestive proteinases, as well as pepsin and chymotrypsin. Trypsin acts with other proteinases to break down proteins to peptides and amino acids. It just continues the digestion process that was begun in the stomach in acidic conditions. Trypsin is produced in an inactive form by the pancreas.

Its chemical composition and structure is extremely similar to chymotrypsin. Both enzymes appear to have similar mechanisms of action as they have residues of histidine and serine in their active sites. The difference between them is their specificity. Each is active only against the peptide bonds in protein molecules that have carboxyl groups donated by certain amino acids. For trypsin these amino acids are arginine and lysine. Trypsin is the most discriminating of all the proteolytic enzymes in terms of the restricted number of chemical bonds that it will attack. This means it is widely used to determine the sequence of amino acids in proteins and as a reagent. Trypsin is an enzyme that acts to degrade proteins. It breaks down the casein into polypeptides which are soluble. That’s why the solution goes clear.

Enzymes like trypsin are biological catalysts that speed up a reaction without being used up. They are proteins and therefore have a specific shape. This means they are specific in the reactions that they catalyse. One type of enzyme will react with only one type of substrate. The protein has an area on the surface where the reaction occurs called the active site. This is where the substrate binds. The active site of one type of enzyme has the same arrangement of amino acids; therefore it has a highly specific shape. Usually there is only one active site on each enzyme and only one type of substrate will fit into it.

Trypsin catalyses the hydrolysis of peptide bonds but due to its shape, the active site of trypsin only splits bonds after a basic or straight chain amino acid. This specificity leads to the lock and key hypothesis. Reactions require energy to initiate them as they are not spontaneous. This is called the activation energy. When the substrate reacts, it needs to form a complex called the transition state before the reaction actually occurs. This transition state has a higher energy level than either the substrates or the product. Outside the body, a lot of heat is used to initiate the reaction. However, this is dangerous inside the body, so we have enzymes that an alternative way with a different transition state and lower activation energy. 

      

A number of factors affect the rate of activity of trypsin on casein:

  • pH
  • Temperature of enzyme and substrate
  • Concentration of enzyme and substrate

*surface area is usually a factor, however, in this investigation both the substrate and enzyme are liquids.

        Enzymes work best at temperatures of 40oC, the optimum temperature. This is because the heat provides the enzyme and substrate with kinetic energy, therefore producing more collisions. The more collisions there are the more substrate will bind to the active sites of more enzymes. This increases the reaction rate. Above the optimum temperature, the enzyme is denatured as it is a protein and its structure begins to break down due to excessive heat. The bonds holding its structure will break and the active site loses its shape meaning it won’t work.

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        Enzymes also have an optimum pH level. If it changes from the optimum pH level, the chemical nature of the amino acid can change. The tertiary structure will break down as the bonds will change. The active site will be disrupted if the tertiary structure breaks down and the enzyme will be denatured.

        If the enzyme concentration is too low there is great competition for active sites and there are not enough to bind to, making the reaction rate extremely slow. If there are more enzymes, there ...

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