Starch and diastase with respect to pH

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Starch and diastase with respect to pH

Aim

The aim of this experiment is to assess the effect of pH on enzyme activity. To do this I will use diastase as the enzyme and starch as the substrate, I will then vary the pH and measure the effects.

Hypothesis

There will be an optimum pH, between pH 4 and pH5, at which diastase will hydrolyse starch.

Background information

Enzymes are globular protein molecules that are biological catalysts, so they reduce the energy needed for the reaction but don’t get used up themselves. Being a globular protein, enzymes are formed from a tertiary structure, so they are 3-D molecules. As a result they are able to have an active site, this is where the substrate binds to the enzyme and is broken down into the products. An active site consists of 3-12 amino acids and it is thought that the active sites have specific shapes that correspond to specific substrates. Consequently the enzyme can only break down that particular substrate molecule, this is known as the lock and key theory. However recent research has shown that the enzymes active site is not the right shape to start with, but once it combines with the substrate molecule a small change occurs in the shape of the active site. Subsequently the substrate fits better in the active site, this is known as the induced fit hypothesis.

An enzyme can be affected by different factors. They can be denatured by high temperatures because the particles within the enzymes receive energy as the temperature rises, so a large rise in temperatures results in the molecules vibrating a lot. This disrupts the weak hydrogen bonds holding these particles together the ionic bonds are also broken and so the enzymes active site collapses, this means that the enzyme can no longer break down the substrates as the substrate won’t fit in the active site anymore consequently the rate of reaction is decreased. However small rises of temperature gives the substate and enzyme kinetic energy so they collide more often resulting in an increased rate of reaction. Enzymes are also affected by the concentrations of the substrate and enzyme. As the enzyme concentration increases, the number of acive sites increases resulting in a higher chance of a collision with a substrate molecules. If the substrate concetration increases and the enzyme concentration stays the same the rate of reaction only increases until all the enzymes are being used. After this is doesn’t increase, this is known as Vmax (maximum velocity).

pH also affects enzyme activity. For every enzyme there is an optimum pH, at the optimum pH the enzyme has the most amount of activity. The pH of a solution measures the concentration of hydrogen ions in the solution. If there is a high concentrationof hydrogen ions then the pH is lower. The hydrogen ions affect the charges of the acidic (⎯COOHˉ) and the basic (⎯NO3+) groups of an amino acid. Amino acids make up an enzyme, so when the charges of these groups are altered, the structure of the enzymes is affected. This means that the shape of the acive site changes and so the substratye molecule can no longer bind with the amino acids in the active site. Small changes in the pH do not damage the enzyme irreversably, so if the pH is restored to the optimum pH, the reaction rate will also be restored. However if there is a dramatic change in the pH the enzyme will become denatured permanently.

Diastase is the name given to the group of enzymes that hydrolyse (add a water molecule) to the glycosidic bonds in starch. These enzymes include α-amylase, β-amylase, amyloglucosidase (also called glucoamylase) and pullulanase. They break starch into glucose, maltose, and dextrin (short chains made of glucose units).

Starch is a polymer of glucose units joined together by α-1,4 and α-1,6 glycosidic bonds. Starch is a mixture of two polysaccharides, amylose and amylopectin. Amylopectin makes up approximately 70% of starch. Amylose is an unbranched chain polymer of 200-1,500 glucose residues, linked by α-1,4 glycosidic bonds. Amylopectin is a branched glucose polymer of 2,000-200,000 glucose residues linked by both α-1,4 and α-1,6 glycosidic bonds. The branching occurs because of the α-1,6 glycosidic bonds. These branch points occur about every 20 glucose residues.

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Amylase is found in pancreatic juices released by the pancreas in the small intestine and in the saliva released by the salivary glands in the mouth. The enzymes that make up diastase hydrolyse the different glycosidic bonds that hold the glucose residues together in starch. Amyloglucosidase hydrolyses the α-1,4 and α-1,6 glycosidic bonds, as a result terminal glucose units are removed at the end of the chain. Pullulanase hydrolyses the α-1,6 glycosidic bond at the branching points, giving dextrins. α-amylase hydrolyses the α-1,4 glycosidic bonds, generating dextrins, maltose and glucose. β-amylase hydrolyses alternate α-1,4 glycosidic bonds and accordingly produces ...

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