Investigating the effect of temperature on the activity of free and immobilised enzymes.

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Investigating the effect of temperature on the activity of free and immobilised enzymes

Aim

To investigate how temperature affects the rate of reaction of the enzyme lactase in free and immobilized states.  I will investigate this using immobilised enzyme beads and free enzyme solution.

Scientific background

Enzymes are biological catalysts which increase the rate of reactions by lowering the activation energy needed for the reaction to tale place. The activation energy is the amount of energy needed for molecules to react when they collide. Molecules need to collide in order to react, this is known as the collision theory. When they collide they may not react, as a certain amount of energy is required to break bonds, this energy is the activation energy.

Enzymes are made of a long amino acid chain; within this some molecules are attracted to each other, so the chain folds in on itself to form a 3D shape.

An area on the surface of the enzyme is known as the active site. This is where reactions take place to form or break down substances. Enzymes are specific which means a particular enzyme only works on one substance known as its substrate. For example, the substrate of amylase is starch and the substrate of lipase is fats. The bonds which bind the substrate to the active site have to be weak so that the binding can be readily reversed when the products need to leave the active site after the reaction.  The bonds are usually hydrogen bonds.  The binding may also cause other bonds in the substrate to weaken or its shape may change so that the atoms are brought into contact to help them react.

After the reaction the product leaves the enzyme and the enzyme to free to start again with another substrate molecule.  They are generally more stable in concentrated, rather than dilute, solutions.

 “Lock and Key” hypothesis

They only have one substrate because the active site is formed in a different shape for each enzyme, where only one substance can fit. The ‘lock and key’ hypothesis states that the enzyme is like a lock, which will only have one key.

Induced fit model

However, it has been discovered that competitors for an active site (similar in shape to the substrate) could fit even though they are larger than the substrate. This means that the substrate and active site are a little flexible.  This has lead to the induced fit model.   

When the enzyme and substrate form a complex, structural changes occur so that the active site fits precisely around the substrate (the substrate induces the active site to change shape).  The reaction will take place and the product, being a different shape to the substrate, moves away from the active site. The active site then returns to its original shape.

The substrate shown is the only substance that fits the enzyme. An enzyme substrate complex is the compound formed when the substrate is attached to the active site; it is only in this form for a short time while the substrate is being broken down.

Lactase

Lactase (β-Galactosidase) breaks down lactose, the sugar in milk, to galactose and glucose.  Commercially produced microbial lactases are obtained from Aspergillus spp. and the yeast Kluyveromyces spp. An important application of lactase is to hydrolyse the lactose in milk to make it suitable for people who are intolerant to lactose.  As glucose and galactose both taste sweeter than lactose, lactase can be used to increase the sweetness of products such as ice cream and to produce a sweet syrup from whey, which otherwise would be discarded as a waste product from the cheese industry.  In ice cream manufacture, use of lactase also prevents crystallising of lactose which happens at low temperature causing a “sandy” texture.

Enzymes can break substances, known as catabolism, or can join substances together, known as anabolism. Together they form metabolism which is every chemical reaction in the body.

The reaction I’m investigating is a catabolic reaction as:

                            β-Galactosidase

Lactose + Water                             Glucose + Galactose

                

  


Immobilised enzymes

The picture shows an immobilised enzyme bead.  There are several different ways to immobilise enzymes and there are advantages to immobilising enzymes.

Advantages

  • Solve handling problems, particularly if the enzyme is noticeably toxic or antigenic
  • Productivity can be greatly increased as it may be used at higher substrate concentrations for longer periods than the free enzyme
  • Easier to separate the enzyme from the product than free enzymes which contaminate the end product.
  • Cheaper over time as they can be used for longer than free enzymes
  • Prevents end-product inhibition of lactase caused by galactose, which decreases activity.

Disadvantages

  • Immobilised enzymes have an additional cost.
  • Some immobilised methods such as covalent bonded are still open to attack by inhibitors.

Immobilised enzymes systems

There are 4 different ways to immobilise enzymes:

  • Adsorption (a)
  • Covalent binding (b)
  • Entrapment (c)
  • Membrane confinement (d)

Adsorption - enzyme non-covalently adsorbed to an insoluble particle, such as glass beads, dextran micro beads or DEAE-cellulose.

Covalent bonding - enzyme covalently attached to an Insoluble particle.

Entrapment - enzyme entrapped within an insoluble particle by a cross-linked polymer.

Membrane confinement - enzyme confined within a semi permeable membrane.

I’m going to use entrapment (using sodium alginate solution) to create my immobilised enzyme beads because the cost is moderate and entrapment provides protection against microbes.

Variables

Enzymes are affected by four factors which are

1. Temperature

2. pH

3. Enzyme concentration

4. Substrate concentration

Temperature

A temperature increase gives more energy to the substrate and the enzyme so they are more likely to collide and react. The frequency of the collisions with the right activation energy will increase so the rate of reaction will increase. The rate of increase is shown by a mathematical coefficient known as Q10, which states that a ten-degree rise in temperature will cause the rate of reaction to approximately double. However at high temperatures enzymes will begin to denature. This is because the attractions holding together the shape of the enzyme will begin to break so the active site loses its unique shape and is unable to react with its substrate to form enzyme-substrate complex.  The attractions or bonds which form the tertiary structure are: ionic bonds

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                disulphide bonding

                hydrogen bonding

The optimum temperature for most enzymes is 37°C. The enzymes in the body have this optimum temperature and the body has adapted to control its temperature so the enzymes are working at their best.  

pH levels

Enzymes also have an optimum pH level, where they work best, any changes to this level will cause the enzymes to begin to denature.  Denaturing of the enzyme is caused because changes in pH affect the ionic bonding which determines the tertiary structure of an enzyme.  This change in the globular protein causes changes in the shape ...

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