the effect of bile concentration on the activity of the enzyme lipase during the break down of milk

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Planning

Aim

To investigate the effect of the concentration of bile salts on the activity of lipase on the breakdown of milk.

Introduction

From prior AS knowledge involving biological molecule I know that lipids are broken down into fatty acids and glycerol by the action of the enzyme lipase. Subsequently using this information in the experiment we will be measuring the fall in pH using a pH probe to find the rate of reaction of the experiment. This is because if lipase is breaking down the lipids then the pH should fall and become more acidic as fatty acids which are acidic are being produced. By keeping the variables the same except for the concentration of bile salts we can be able to see what affect, if any the bile salts have on the activity of the enzyme lipase on the breakdown of lipids, with our source of lipids being milk. In addition to this by using accurate apparatus and by keeping the conditions the experiment is done in the same I will be able to make my results both precise and accurate.

Background

Enzymes

An enzyme is a biological catalyst that speeds up the rate of a chemical reaction by lowering the activation energy needed by the reactants to react, thus allowing the reaction to precede much faster by a factor of millions as seen in the graph below. Like all catalysts, enzymes remain unaltered by the completed reaction and can therefore continue to function, although unlike other catalysts enzymes are very specific to the substrates they bind to and therefore the reactions they catalyse. Because most of the reactions in biological cells would occur too slowly without the use of enzymes, enzymes prove to be essential to sustain life. Subsequently they are so important that a malfunction (mutation, overproduction, underproduction or deletion) of a single critical enzyme can lead to a severe disease.

Enzymes are globular proteins and like all proteins their structure determines their function. Consequently all enzymes have a three dimensional structure caused by the folding of sequences of amino acids called polypeptide chains. However despite there relatively large size there is only a small region that is functional in enzyme called an active site, which only a few amino acids of the enzyme molecule make, leaving the remainder of the enzyme molecule to maintain the overall three dimensional shape of the enzyme. Because the size and shape the active site varies from enzyme to enzyme, enzymes are very specific to which substrate molecule the bind to, as they would only bind to substrate molecules which complement the size and shape of the active site forming an enzyme substrate complex. Subsequently the substrate molecule is held in place with the active site by bonds formed temporarily between the R groups of the amino acids of the active site and the groups on the substrate molecule until the products are made from the substrate molecule.

Because we are dealing with enzyme the above information is relevant to the investigation we are carrying out. This is because it informs us about how specific an enzyme can be because of its active site which tells us that the lipase in our experiment will only break down the fat molecules in the milk and not the protein molecules.

How enzymes work

Lock and key modal – Suggested by Emil Fischer in the 1890s this theory states that in the same way a key is very specific shape which, on the wholes, fits and operates only one lock, enzymes are also have a specific shape which substrate molecules bind to, called an active site. Consequently only certain substrate molecules that complement the shape of this active site will be able to bind to the enzyme to form an enzyme substrate complex. Enzymes are therefore specific in the reaction they catalyse as they are specific to the substrate molecules they bind to.

Induced fit modal – Suggested in 1958 by Daniel Koshland as a modification to the lock and key theory this theory states that because enzymes are rather flexible structures, the active site of an enzyme can be modified as the substrate interacts with the enzyme. Subsequently even though the enzyme has a certain basic shape it becomes slightly different as it alters its shape in the presence of the substrate to put strain on the substrate molecule and thereby lower its activation energy.

By telling us how enzyme works we can be able to get a clearer understanding of what is occurring during our experiment. The information also reinforces the fact that enzymes are very specific to the substrate they bind to due to the 3 dimensional shape of active site. Consequently this information is relevant to the investigation.

Factors affecting rate of reaction in enzyme-catalysed reactions

Presence of Inhibitors – An inhibitor is a type of effector that prevents or decreases the rate of chemical reactions. Inhibitors that permanently prevent an enzyme functioning are called non-reversible inhibitors and do so by binding strongly to the active site of an enzyme so that they cannot be removed examples of these inhibitors are heavy metal ions such as mercury and silver. On the other hand inhibitors that temporary attach themselves to the active sites of enzymes are called reversible inhibitor and there are two type of this competitive and non-competitive.

A competitive inhibitor is a molecule which has a similar shape to that of the substrate molecule which allows them to also occupy the active site of an enzyme. They therefore compete with the substrate molecule for available active site and as a result hinder the ability of an enzyme to catalyse a reaction because the difference between the concentration of the inhibitor and the concentration of the substrate determines this catalysing ability. Eventually all the substrate molecules will find an active site although the greater the concentration of the inhibitor the longer this will take.

A non-competitive inhibitor is an inhibitor that disrupts an enzyme catalysing ability be attaching itself not to the active site of the enzyme molecule but to the allosteric site. Subsequently upon attachment the inhibitors alters the shape of the active site of the enzyme in such a way that the substrate molecule will no longer be able to bind to the enzyme at the place. Because the inhibitor does not compete for the same site increasing the substrate concentration does not decrease the effect of the inhibitor. Cyanide is an example of a non-competitive inhibitor which inhibits the respiratory enzyme, cytochrome oxidase.

Temperature – In general, chemical reactions speed up as the temperature is raised. When the temperature increases, more of the reacting molecules have the kinetic energy required to undergo the reaction. Enzyme catalyzed reactions also tend to go faster with increasing temperature until a temperature optimum is reached. This is because the enzyme and substrate molecules will collide together more frequently resulting in more successful collision where an enzyme substrate complex is formed and the substrate molecule can be converted into the products. However when you increase the temperature beyond the optimum temperature enzyme molecule is disrupted because the atoms that make up the enzyme begin to vibrate distorting the active site of the enzyme making it much harder for the substrate to fit into it because of the specific shape that it needed to be to fit into the specific shape of the enzymes active site. Eventually the hydrogen bond and other bonds that hold the enzyme in its shape will break resulting in the enzyme being denatured as the distortion of the active site is so bad that the substrate is no longer able to bind to the enzyme, consequently even if the temperature is reduced the damage done to the enzymes shape and active site is irreversible and it will cease to function and be unable to catalyse any reactions. Different enzymes have different optimum temperatures as they are suited to the condition they work in with the organism. The lipase enzyme I will be using from a pig has an optimum temperature of 37°C since this about the body temperature of a sheep.

pH – pH is a measure of the acidity or hydrogen ion concentration of a solution. It is measured on a scale of 0-14 with pH values below 7 being acidic, values above 7 being basic and a value around 7 is neutral. As the pH drops into the acidic range an enzyme tends to gain hydrogen ions from the solution. As the pH moves into the basic range the enzyme tends to lose hydrogen ions to the solution. In both cases the changes produced in the chemical bonds of the enzyme molecule result in a change in conformation that decreases enzyme activity. The lipase enzyme I will be using in my experiment has an optimum pH of 8 as this is the pH of the small intestine where it is released.

Concentration – If all other conditions are held constant, the rate of the reaction should increase with increasing concentrations of substrate. At very low values of substrate the reaction rate will increase very rapidly. At higher substrate concentrations the rate begins to level off. Eventually the maximum rate for that reaction will be achieved and further increases in substrate concentration will have no effect.

The information above is crucial to the investigation and because of this is very relevant as it tells us factor affecting enzyme activity. Consequently since this information tells us these factors it makes us aware of the variables we need to control in order to carry out a fair test which will produce precise and accurate results.

Lipase

Lipase is a digestive enzyme found in the small intestines of animals; it is produced by the pancreas and transported into the duodenum of the small intestine via the pancreatic duct. Its fundamental use in the body is to catalyse the hydrolysis of the ester bond in fats therefore breaking them down into their constituent parts of glycerol and fatty acids. For lipase that is produced by the pancreas to work at its optimum it requires a pH of that of the small intestine which is pH 8 making it slightly alkaline. The body neutralises the acidic conditions produced by stomach to this pH by secreting bile into the duodenum of the small intestine which contain hydrogencarbonate ions. This is important for this investigation as to maintain lipases optimum working conditions not only do we have to monitor and control the temperature but we have to do the same for the pH as this is just as crucial to the preciseness of our results as the temperature.

The background information above about lipase is very relevant to the investigation since we are going to be using this enzyme and the information also tells us its optimum pH which we will need to control in order to keep the conditions the enzyme works in optimum.

Bile salts

Bile salts are a constituent of bile which is produced by the liver. They help in the digestion of fats by emulsifying large fat molecules into smaller ones in order to allow lipase to catalyse the hydrolysis of the fats. They are produced by the hepatocytes which are liver cells from cholesterol and stored in the gall bladder until they are required where they are transported into the duodenum of the small intestine via the bile duct. Their importance in digestion of fats is significant because lipase is only capable on working on small droplets of fats and the bile salts help emulsify these fat globules by lowering their surface tension causing large drops to break up into tiny droplets. After they have been for emulsifying the fat globules they are reabsorbed by transporters in the intestine and are returned to the liver via the portal vein. This recycling pathway is called the entero-hepatic circulation.

Fats

Lipids are large molecules containing carbon, hydrogen and oxygen. They are grouped into several main groups including triglycerides, phospholipids and waxes according to their non-polar nature making them all hydrophobic. Because of this they do not mix with water and form layers on top of the water. “Triglycerides which occur in animal fats and vegetable oils are the major storage form energy needed to drive reaction in plants and animals”. A triglyceride molecule is made from a glycerol which is bonded to three fatty acid tails in an ester bond and during chemical digestion of fats it is these ester bonds which are broken down by hydrolysis using the enzyme lipase as a biological catalyst which speed the rate of reaction. During chemical digestion the “detergent action of bile salts, particularly lecithin is required to disperse the fat into small globules for efficient lipase digestion” as pancreatic lipase is a water soluble enzyme and can therefore only act on the surface of fat globules.

The information above is important to the investigation as it explain what happens what fats are broken down into. Consequently by using this information I can be able to see that when a fat molecule is broken down it will produce fatty acids which will lower the pH as they are acidic and by measuring the rate at which the pH drops you can see the rate of reaction.

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Milk

Milk is made up of butterfat globules in a water based fluid as an emulsion. These fat globules are surrounded by a membrane consisting of phospholipids and proteins which act as emulsifiers and keep the individual globules from joining together into larger grains of butterfat. Consequently this protects the fat globules from the fat digesting activity of enzymes and therefore resistant to the pancreatic lipase we will be using. For lipolysis to occur these fat globules need to be disrupted by the work of bile salts to allow lipase to act on them.

Hypothesis

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