Enzyme investigation

Wild mushrooms grow in grassy meadows in all parts of the world. Warm and damp weather suits them best, hence the reason why they grow more rapidly in autumn, than in any other season. Fungi, for example, mushrooms, fulfil important ecological functions. Mushrooms are saprophytic, decomposing fungi. Saprophytic fungi are basically recyclers of organic material. Mushrooms secrete enzymes and acids which break down large organic molecular complexes into simpler compounds which are then absorbed. All ecosystems depend partly upon fungi’s ability to decompose organic plant matter, such as, leaves falling from trees, other organic debris and excretory products from animal and plant sources. The result of the decomposition of the organic matter is the return of carbon, hydrogen, nitrogen, and minerals back into the soil in forms which can be useful to plants, insects and other organisms.

Fungi are one of the most important groups of organisms on the planet. Fungi are eukaryotes, which are organisms with a distinct nucleus. Fungi comprise the moulds of yeast, mildews, mushrooms, puffballs and rusts. They can be saprophytic, feeding on dead organic matter or parasitic.

The main body of most fungi is made up of fine, branching, and usually colourless threads called hyphae, which are 5-10 um in diameter. Each fungus will have vast numbers of these hyphae, all intertwining to make up a tangled web called the mycelium. In a specialised part of the mycelium, spores are produced in vast numbers and dispersed. The mycelium is generally too fine to be seen by the naked eye, except where the hyphae are very closely packed together. The tangled mycelial mass is usually hidden deep within its food sources, such as rotting matter in the soil, leaf litter, rotting wood or dead animals. The mycelium remains undetected until it develops one or more fruiting bodies, containing the reproductive spores, called sporangiophores.

The hyphae secrete enzymes, which diffuse through the cell wall, into the food. Here, they break down big, insoluble molecules into small, soluble ones that can be absorbed into the hyphae by facilitated diffusion and active transport. This is known as extracellular digestion because digestion happens outside of the mould’s cell.

Hypothesis

Temperature has an affect on the denaturation rate of fungal amylase.

Prediction

I predict that as the temperature increases the rate at which organic materials, i.e. starch, are broken down increases. At high temperatures the optimum temperature would have been exceeded therefore the enzyme will be denatured and the rate will decrease.

Background Information

Enzymes are biological catalysts; they increase the rate of a reaction without being used up. The rate of a reaction is speeded up because the enzyme provides an alternative route for the reaction with a lower activation energy. The enzyme provides an alternative route by holding substrate molecules in such a way that breaking bonds (catabolism) or making bonds (anabolism) requires less energy input.

Enzymes are globular proteins with a precise three dimensional (tertiary) structure. Their tertiary structure produces an active site, which is a depression in the globular protein shape that is complementary to the shape of the substrate. This means that enzymes will only bind with one specific substrate. This is described as the ‘lock and key’ hypothesis. Once the enzyme has bound to a substrate molecule bond making or bond breaking will occur in the region of the active site. The enzyme then releases the substrate and it is free to catalyse another reaction.

Some enzymes do not have a permanent active site; they only develop in the presence of their substrate. This is known as the induced fit theory.

Enzymes, being proteins are sensitive to changes is their environment. Changes in temperature, pH and concentration can alter the shape of the enzyme molecule and will therefore affect its activity.

Raising the temperature often increases the rate of a reaction. This is because molecules have more kinetic energy and so move faster at higher temperatures compared to lower ones. They are therefore more likely to collide into each other and react. Since they have more energy, when they do collide they are more likely to overcome the activation energy barrier and form a product.

In enzyme controlled reactions, the same is also true. As the temperature increases the kinetic energy of the molecules increases, causing more collisions between the substrate and enzyme active site, therefore increasing the rate of the reaction.

Until the optimum temperature is exceeded for every 100C rise in temperature the rate of an enzyme controlled reaction the rate doubles. This is known as the temperature coefficient (Q10).

Q10 = Rate of the reaction at T0C+10

Rate of reaction at T0C

The Q10 for enzyme controlled reactions will be 2, whereas the Q10 for a reaction where no reaction is present is closer to 1.3.

However, after the initial rise in the rate of the reaction, as the temperature continues to increase, due to enzymes being made of protein they are affected by high temperatures, usually above 400C many enzymes are denatured. As temperature is increased the intramolecular kinetic energy and increased vibration within the enzyme causes hydrogen bonds to break. The breaking of the hydrogen bonds and eventually disulphide bridges causes the tertiary structure of the enzyme to change and the active site can no longer bind to the substrate and the rate of reaction will decrease. The process of denaturation is the irreversible destruction of the precise three dimensional tertiary structure of the enzyme. The highest temperature at which the enzyme is still active is called the optimum temperature.

Any change in pH affects the ionic and hydrogen bonding in an enzyme and so alters it shape. Each enzyme has an optimum pH at which its active site best fits the substrate. Variation either side of pH results in denaturation of the enzyme and a slower rate of reaction.

When there is an excess of enzyme molecules, an increase in the substrate concentration, produces a corresponding increase in the rate of reaction. If there are sufficient substrate molecules to occupy all of the enzymes’ active sites, the rate of reaction is unaffected by further increases in substrate concentration as the enzymes are unable to break down the greater quantity of substrate any quicker.

Inhibitors compete with the substrate for the active sites of the enzyme (competitive inhibitors) or attach themselves to the enzyme, altering the shape of the active site so that the substrate is unable to occupy it and the enzyme cannot function (non-competitive inhibitors). Inhibitors therefore slow the rate of reaction. They should not have affected this investigation, as none were added.

Enzyme Concentration – Provided there is an excess substrate, an increase in enzyme concentration will lead to a corresponding increase in rate of reaction. Where the substrate is in short supply (i.e. it is limiting) an increase in enzyme concentration has no effect.

Starch is a polymer of glucose monomers and is a mixture of amylose and amylopectin. Amylose makes up about 30% of starch and consist of unbranched chains in which the monomers are joined by 1,4 glycosidic bonds. Amylopectin makes up the remaining 70% of the starch molecule and consists of chains of glucose monomers linked with 1,4 glycosidic bonds and branches which are due to the formation of 1,6 glycosidic bonds at various points along the chain. Starch is designed particularly for its function as a storage compound. Starch is insoluble and therefore cannot move out of the cells in which it is stored. Starch is compact and does not take up much space. Starch does not become involved in chemical reactions inside the cell. The presence of starch can be detected by a solution of iodine. This reddish brown solution turns blue-black when starch is present. I know from previous experiments that this is an obvious result but it is not always easy to time the exact point when the entire blue-black colour has disappeared.

Amylases, which are enzymes, hydrolyse glycosidic bonds in polysaccharides such as starch as glycogen, converting them to dextrins (shorter length chains of glucose units) or to maltose. Amyloglucosidase hydrolyses the internal -1,4 glcosidic links forming glucose. Pullulanase, which is also known as the debranching enzyme, hydrolyses the -1,6 glycosidic links at the branching points of the polysaccharide forming dextrins.

Variables

Variables are parameters that can change. There are three different types of variables. The dependent variable is one that is measured. The independent variable is one that is manipulated. Control variables are kept constant. The independent variable was temperature, controlled during the experiment. The variable dependant on this was starch concentration. All other variables therefore had to be kept constant to ensure that the experiment was a fair test. These controlled variables were concentration of amylase and starch in the solution, time period over which the experiment was conducted and the volume of amylase solution and starch solution. The apparatus was also kept the same throughout.

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