Effect of Temperature on Activity of Alcalase and Savinase Essay Example

Effect of Temperature on Activity of Alcalase and Savinase Paper Hypothesis The optimum temperatures of Alcalase and Savinase will be different. Above and below their optimum temperatures activity will decrease. Biological explanation This investigation is designed to look at the effect of temperature on the activity of the proteases Alcalase and Savinase. By the end of it I hope to know the optimum temperature of both proteases. The substrate I am going to use during the experiments is the protein gelatin, which is a translucent, colourless, brittle solid substance found in the collagen inside an animals’ connective tissues. In my experiments it is going to be in the form of a single, thin layer, used on the surface of photographic film. It is useful in photography because it acts as protein glue, sticking the silver halide crystals to the surface of the plastic film. I am using it in this form, as it is easy to see when the enzyme has digested the gelatin. This is because normally the surface of the gelatine-silver halide layer turns black when exposed to light. However, when the enzyme has removed the gelatin the black colouring will disappear and only the clear plastic will be visible. We will write a custom essay sample on Effect of Temperature on Activity of Alcalase and Savinase specifically for you for only $16.38 $13.9/page Order now We will write a custom essay sample on Effect of Temperature on Activity of Alcalase and Savinase specifically for you FOR ONLY $16.38 $13.9/page Hire Writer We will write a custom essay sample on Effect of Temperature on Activity of Alcalase and Savinase specifically for you FOR ONLY $16.38 $13.9/page Hire Writer Therefore, it can be easily identified when the reaction between the enzyme and the gelatin is complete, so this form of gelatin is very appropriate. Alcalase is a high temperature protease, meaning it works best at high temperatures, so its optimum temperature must be fairly high in relative terms, taking into account that most biological enzymes have an optimum temperature of 37. 5Â °C. It is commonly found in soil. Due to it being a high temperature protease I would expect its activity to increase with the temperature up to its optimum temperature, which I think may be about 50Â °C. I predict its optimum temperature to be around this figure because the enzyme is used in washing powders and this is a reasonable temperature to washing clothes at. Savinase is a low temperature protease, meaning it works best at low temperatures, so its optimum temperature must be fairly low in relative terms, taking into account that most biological enzymes have an optimum temperature of 37. 5Â °C. It also is found in soil. Due to it being a low temperature protease I would expect its activity to decrease as the temperature increases once the temperature is above its optimum temperature. I think the optimum temperature will be about 30Â °C because this enzyme is also used in washing powder, but in special energy saving washing powder, which operates at 30Â °C. The proteases are able to break down the protein gelatin because they are specific to the reaction needing to take place. They are specific in that their active sites on the surface of the enzyme fit the gelatin substrate, fulfilling the lock and key hypothesis and forming an enzyme-substrate complex. The optimum temperature is the temperature at which these formations occur most efficiently, due to the enzymes active site being the most accurate shape to fit the substrate. Therefore, temperature affects the activity of enzymes by changing the shape of the active site, which means it is changing the tertiary structure of the enzyme. The tertiary structure is changed because the weak hydrogen bonds that hold the protein in its 3D helical shape are broken due to the heat. As well as the enzymes active site being the correct shape at the optimum temperature there is a better balance of kinetic energy, causing more collisions between enzyme and substrate and therefore more enzyme-substrate complexes are formed, increasing activity. At high temperatures in comparison with the optimum temperature the enzymes tertiary structure may change completely, disabling all activity, as the substrate won’t fit the active site. This is known as denaturation. However, at temperatures below the optimum, the tertiary structure of the enzyme isn’t altered and denaturation does not occur, it is simply a slower rate of reaction due to less kinetic energy and therefore reduced collisions between the enzymes and substrates. Apparatus *2 200cm3 Volumetric Flask – to hold the enzyme solutions *2 Stirring rods – to assist in covering film strips in solution *3 Boiling tubes – to hold strips of photographic film in water bath *Scissors – to cut photographic film *Ruler – to measure a length of photographic film *Stop clock – to time incubation period Balance accurate to 2d. p. – to weigh out mass of enzyme needed *Exposed, developed photographic film – as substrate *4g Encapsulated Alcalase – as high temperature protease enzyme *4g Encapsulated Savinase – as low temperature protease enzyme *Water bath – to incubate boiling tubes holding photographic film at temperatu res 30Â °C -100Â °C at 10Â °C intervals *400cm3 pH8. 0 buffer – to maintain a constant pH *2 200 cm3 Volumetric Flask – to measure the volume of buffer needed *Thermometer – to check temperature of solution when in water bath *Volumetric Pipette – to measure out the volume of enzyme needed Variables *Temperature – This is the only variable I will purposely change. I will do this by using a water bath at several different temperatures. These temperatures are 30Â °C, 40Â °C, 50Â °C, 60Â °C, 70Â °C, 80Â °C, 90Â °C and 100Â °C. Temperature must be controlled because to find the optimum temperature I need to try the above exact temperatures and if it wasn’t controlled to the exact temperature I couldn’t specify the exact optimum temperature. *pH – Must be kept constant. I will keep the pH optimised throughout using 200cm3 of pH8. 0 buffer. It must be kept constant to ensure fair results. *Enzyme concentration – Must be kept constant. I will use 4g of the encapsulated enzyme, made up to 200 cm3 of solution, where there will be a 2% concentration of the enzyme in all my experiments using a balance, accurate to 2d. p. Enzyme concentration needs to be kept constant because if there was a higher concentration in one experiment than in the other the rate of reaction may be increased or decreased in comparison to what it should have been, therefore the results will be affected and it will be an unfair test. Substrate concentration – Must be kept constant. I will use the same length and width of photographic film, measured using a ruler, in all my experiments. Substrate Concentration needs to be kept constant because if there was a higher concentration in one experiment than in the other the rate of reaction may be increased or decreased in comparison to what it should have been, therefore the results will be aff ected and it will be an unfair test. *Incubation period – This will change depending on how fast the rate of reaction is. The period will end as soon as the photographic film turns clear. The times are recorded and will form the basis of my results. *Reaction temperature – Will not be a constant time that it takes to heat the solution to the correct temperature before the film is added, but check must be made to see that it is at the correct temperature before the film is added. If it isn’t thoroughly heated through before the film is added then the results will be inaccurate, in that they will be lower than would be expected. I will check the temperature of the solution using a thermometer. *Volume of enzyme used – This will remain the same at 2cm3 throughout the whole investigation. I will keep it exactly the same using a 1cm3 volumetric pipette. It needs to be kept constant because if there is more enzyme solution in some experiments and less in others the rate of reaction and therefore the results will be affected, in that they may turn out to be lower than expected and become inaccurate. Exposure of film – All the photographic film used will be exposed in full sunlight prior to the investigation. The amount of light received needs to be the same for all the film used because if some is exposed to brighter light than others it will be more black in colour and therefore will need a longer or more vigorous reaction to make it totally clear, which could make results unreliable and inaccurate. Procedures 1. Set the water bath at 30Â °C. . Weigh out 4g of each enzyme and place in two 200cm3 v olumetric flasks. 3. Make up to the 200cm3 line on the flask with pH8. 0 buffer. 4. Add a lid to each flask and invert in turn to mix the substances thoroughly until enzymes are completely dissolved. 5. Cut off 3 strips of photographic film at 1cm in length and width. 6. Add 2cm3 of Alcalase and buffer solution to one boiling tube and 2cm3 of Savinase and buffer solution to the other. 7. Place the 2 boiling tubes in the water bath, along with an empty one for the control. 8. Leave them for 5 minutes and check the temperature with a thermometer to make sure the solutions are at the right temperature before adding the photographic film. 9. When the solutions are at the right temperature add a strip of photographic film to each boiling tube, making sure the strips have solution on the whole of them by using different stirring rods for the separate boiling tubes, to prod the strips down. 0. Start the stop clock and time how long it takes before the strip of photographic film has turned clear. 11. Record the time it took on the stop clock for the gelatin to be completely digested and the film to become clear. 12. Repeat steps 5 to 9 twice for the same temperature and decide on an average colour change for each enzyme and the control. 13. Set the water bath at 40Â °C, 50Â °C, 60Â °C, 70Â °C, 80Â °C, 90Â °C and 100Â °C repeating steps 5 to 10 for each temperature.