To determine if the rate of reaction of free enzymes is higher that the rate of reaction in immobilised enzymes in different pH concentrations of solution.

AS Level Human Biology

Investigation into the effects of pH on free and immobilised amylase

By:

Laura Carolan-Munslow

Aim: To determine if the rate of reaction of free enzymes is higher that the rate of reaction in immobilised enzymes in different pH concentrations of solution.

Introduction:

Enzymes are biological catalysts that help biological reactions, which normally occur very slowly at biological temperatures, to occur faster. Enzymes do this by lowering the activation energy (the energy required to get the reaction started, and break some bonds initially). By lowering the activation energy, the reactions can occur much faster.

Most enzymes are globular proteins, (though some are RNA enzymes). Protein enzymes have a 3D formation, which has to remain intact for the enzyme to function correctly. If this 3D shape is destroyed, or even altered, the enzyme cannot function. When this happens the enzyme is said to be denatured.

An enzyme can be denatured in various ways; the main factors that affect the activity of the enzyme are pH, temperature, and substrate concentration. If the pH is higher or lower than the optimum level, then the enzyme will denature. If the temperature is too high, the bonds of the 3D shape will break, and thus the enzyme will denature.

Every enzyme has an optimum pH, (the pH at which the enzyme will function best) at this pH the highest reaction rate will be recorded. This will depend on the 3D bonds of the enzyme. Above the optimum pH of the enzyme, the conditions will cause ionization of the enzyme, which results in the decrease of the number of reactions, due to the breaking of the bonds holding the 3D shape together.

Below the optimum pH of the enzyme, the number of reactions will decrease because the pH affects the enzyme, ionization occurs, and the hydrogen bonds holding the 3D shape together are broken. Thus the enzyme again, denatures.

The enzyme is usually active over a restricted pH range, and usually the optimum pH is quite obvious. The pH optimum for most enzymes is around pH7, though some enzymes are very low or very high. e.g. pepsin pH 2.

There are four commonly used immobilisation methods, adsorption, covalent coupling, membrane incorporation and entrapment. The immobilisation process used for this experiment will be entrapment.

The most common entrapment method involves the formation of calcium alginate beads. The enzyme (in this case, amylase) is mixed with sodium alginate, (an acidic polysaccharide) and the mixture is dropped into a solution of calcium chloride. The calcium ions replace the sodium ions and “cross-link” the polysaccharide. This results in the formation of insoluble calcium alginate beads, which contain the trapped enzymes.

Enzymes are usually very expensive, and this is why enzyme immobilsation occurs. Immobilisation makes it easier to remove the enzyme, from the product (in this case the starch/pH buffer solution), the enzyme can then be ...