The amino acid side chains on some of the enzymes are only used for activity while cofactors utilize additional chemical components. Enzymes work best at body temperature and they also have to have the correct pH. A catalyst is any substance which makes a chemical reaction go faster, without itself being changed. It can also be used over again in a chemical reaction as it does not get used up. Enzymes are the same, however, they are easily denatured, which means they are destroyed, by heat. If this happens the catalytic activity is destroyed.
An enzyme only works for one specific reaction, however, a catalyst may be used for several different chemical reactions. Enzymes also have an active site which consists of a three-dimensional pocket or surface on which the substrate is attached, and then broken up or joined. The active site is made up of amino acid residues into which the substrate can fit. The amino acids within the active site take part in the catalytic reaction. This happens by directly reacting with the substrate or stabilising reaction intermediates. In relation to the practical, amylase is an enzyme which is contained in human saliva.
This enzyme is what helps starch turn into sugar called maltose. Amylase works best in neutral or slightly alkaline conditions, for example, at about pH7. The amylase stops working when there is too much acid in the stomach, this tends to happen when a person swallows a mouthful of food. Food then enters the small intestine where more amylase is made by the pancreas. By doing this it turns the remaining starch into maltose. An enzyme known as maltase turns maltose into glucose which is then absorbed into the blood. The aim of the practical was to determine the optimum temperature for ? -amylase.
Method There was two parts to this practical, the first being to determine the construction of a standard curve for maltose. Seven test tubes were set up and accurately measured solutions were put into them. Each tube contained 0. 5cm? sodium hydroxide, 0. 5cm? DNS EXPLAIN reagent and varying volumes of 5mM maltose to make each concentration standard. Phosphate buffer was then added to make a total volume of 5cm?. Tube 1 contained 0. 25ml of 5mM maltose making a 0. 25mM concentration and 3. 75ml of phosphate buffer to make a volume of 5ml. Tube 2 contained 0. 5ml of 5mM maltose making a 0.
The test tubes were then heated in a boiling water bath for 10 minutes and were then left to cool. A spectrometer was used and the wavelength was set to ? to 540nm. Each test tube was then measured against the BLANK. The second part of the practical was to determine the effect of temperature on the activity of ? -amylase. Seven test tubes were set up and accurately measured solutions were put into them. All the 7 test tubes had the same volume of solutions which were, 2. 0ml of phosphate buffer, 1. 0ml of 1% (w/v) starch solution and 0. 5ml of 20mM of sodium chloride.
The solution in which was then added to the BLANK was 0. 5ml of sodium hydroxide. The sodium hydroxide acted as a stop which ensured that no reaction occurred within this tube. The solution was also used throughout the practical to stop the reactions as required. 0. 5ml of ? -amylase solution was added to the BLANK test tube. The tube was then covered with cling film and the contents were mixed. The BLANK test tube was then left at room temperature. 0. 5ml of ? -amylase solution was added to one tube and a record of the time was made. Again, cling film was put on top; however, a small hole was made in the top.
The cling film is there to reduce the evaporation from the tube. The test tube was then placed immediately into the water bath/incubator at one of the temperatures. After 20 minutes, the reaction was stopped by adding 0. 5ml of sodium hydroxide. When the reaction stopped, the time was recorded. After the reaction was stopped, 0. 5ml of DNS reagent to each tube, including the blank one, and the water was heated to boiling point for 10 minutes. The tubes were then allowed to cool and the absorbance for each experiment was read against the BLANK in a spectrometer set to ? 540nm.