Introduction: Enzymes are biological catalysts that regulate and accelerate the rate of biochemical reactions within a cell, but are not used up or changed in those reactions, therefore are accessible for reuse. They allow reactions to occur at a lower temperature than would otherwise be necessary in their absence. However, almost every significant life process is dependent on enzyme activity and without them life would cease. Enzyme activity occurs on chemical substances known as a substrate. Enzymes cannot force chemical reactions to occur, they can only increase the speed of reactions. The roll of an enzyme is to ensure that a reaction can occur without abnormal increases in body temperature.

They require specific conditions in which to work and this is varied depending on the enzyme, they also have optimum levels of temperature and pH. Substrate concentration is another factor that could affect the activity of an enzyme and thus the result of a reaction. Amylase are saccharides (an enzyme that slices complex carbohydrates) that are found in small quantities in human blood and larger quantities in human saliva. It is found in both plants and animals. Salivary amylase is known as ptyalin; although humans have this enzyme in their saliva, some mammals, such as horses, dogs, and cats do not. Amylase is the digestive enzyme needed to digest carbohydrates (one of the three major food groups needed for proper nutrition).

It is an enzyme produced by the salivary glands, released on digestion of food that hydrolyses starches and simple sugars. Amylase digests carbohydrates (polysaccharides) into smaller disaccharide units; eventually converting them into monosaccharides such as glucose, for that reason amylase are found in the salivary glands. It is also found in the pancreas, secreted in active form, but requires the presence of other chemicals for optimal activity. It acts with Protease to stimulate an immune system function. It works in association with lipase to digest fragments of viruses and reduce inflammation and infections. Amylase digests not only carbohydrates but also dead white blood cells (pus).

It is involved in anti-inflammatory reactions such as those caused by the release of histamine and similar substances. Purpose: To plan, choose equipment and resources and perform an investigation to demonstrate the effect of: 1. Increased temperature 2. Change in pH 3. Change in substrate concentration on the activity of amylase THE EFFECT OF INCREASED TEMPERATURE ON THE ACTIVITY OF AMYLASE Risk Assessment: .

The amylase solution and starch solution are not harmful substances, however like any other chemical it is not recommended to swallow them, unless they are made specifically for ingestion. All glassware should be thoroughly cleaned before being used in the experiment. Whilst the Bunsen burner is in use, care must be taken at all times and should not be left unattended. Loose hair and clothing should be tied back to prevent the risk of incidents occurring.

Safety goggles are to be worn at all times. The teacher should be informed in the event of an accident. At the conclusion of the experiment liquid waste can be disposed of down the sink and other waste to the bin. All spillages and broken glass are to be cleaned up immediately and disposed of in an appropriate manner Hypothesis: The activity of amylase will increase with increased temperature.

When the temperature reaches 36-37^0 C the reaction will be most effective. Purpose: Test the effect of increased temperature on the activity of the activity of amylase Materials: – Amylase solution – Beakers – Bunsen burner – Gauze mat – Gloves – Ice – Iodine – Matches – Measuring cylinders – Safety goggles – Starch solution – Stop watch – Test tube rack – Test tubes – Thermometer – Tripod – Water Method: At 0^0 C 1.2 test tubes were placed into the test tube rack 2.10 mL of starch solution was measured our and 5 mL of that was poured into each test tube 3.2 drops of iodine to both of the test tubes were added 4.1 of the test tubes was marked as a control 5. The beaker was filled with icy water 6.2 test tubes were placed into the beaker and their temperatures were measured until they reached 0^0 C 7.2 mL of the amylase solution was added only to the experimental test tube 8. Timing was started with the stopwatch 9. Changes in colour were observed 10. Times were noted down when any colour changes ceased 11.

This method was repeated At 18^0 C 1.2 test tubes were placed into the test tube rack 2.10 mL of starch solution was measured our and 5 mL of that was poured into each test tube 3.2 drops of iodine to both of the test tubes were added 4.1 of the test tubes was marked as a control 5. The beaker was filled with tap water 6. The temperature of the water was measured 7. If the water went: – above 18^0 C a little bit was empted and a little bit of colder water was added until it reached 18^0 C – below 18^0 C a little bit was empted and a little bit of hotter water was added until it reached 18^0 C 8.2 test tubes were placed into the beaker and their temperatures were measured until they reached 18^0 C 9.2 mL of the amylase solution was added to only the experimental test tube 10. Timing was started with the stopwatch 11.

Changes in colour were observed 12. Times were noted down when any colour changes ceased 13. This method was repeated At 36^0 C 1. A heat mat was placed on the desk 2. The Bunsen burner was placed in the centre of the heat mat and its tube was attached to the gas outlet 3. The tripod was placed on top of the Bunsen burner 4.

The gauze mat was placed on top of the tripod 5.2 test tubes were placed into the test tube rack 6.10 mL of starch solution was measured our and 5 mL of that was poured into each test tube 7.2 drops of iodine to both of the test tubes were added 8.1 of the test tubes was marked as a control 9. The beaker was filled with tap water 10. The beaker was placed on top of the gauze mat 11. Whilst lighting the Bunsen burner the gas was turned on 12. The temperature was raised to 36^0 C 13. If the water went: – above 36^0 C a little bit was empted and a little bit of colder water was added until it reached 36^0 C – below 36^0 C a little bit was empted and a little bit of hotter water was added until it reached 36^0 C 14.2 test tubes were placed into the beaker and their temperatures were measured until they reached 36^0 C 15.2 mL of the amylase solution was added only to the experimental test tube 16.

Timing was started with the stopwatch 17. Changes in colour were observed 18. Times were noted down when any colour changes ceased 19. This method was repeated At 54^0 C 1. Place beaker on top of gauze mat 11. The beaker was placed on top of the gauze mat 12.

Whilst lighting the Bunsen burner the gas was turned on 13. The temperature was raised to 54^0 C 14. If the water went: – above 54^0 C a little bit was empted and a little bit of colder water was added until it reached 54^0 C – below 54^0 C a little bit was empted and a little bit of hotter water was added until it reached 54^0 C 15.2 test tubes were placed into the beaker and their temperatures were measured until they reached 54^0 C 16.2 mL of the amylase solution was added only to the experimental test tube 17. Timing was started with the stopwatch 18. Changes in colour were observed 19. Times were noted down when any colour changes ceased 20.

This method was repeated At 72^0 C 1. The temperature was raised to 72^0 C 13. If the water went: – above 72^0 C a little bit was empted and a little bit of colder water was added until it reached 72^0 C – below 72^0 C a little bit was empted and a little bit of hotter water was added until it reached 72^0 C 14.2 test tubes were placed into the beaker and their temperatures were measured until they reached 72^0 C 15.2 mL of the amylase solution was added only to the experimental test tube 16. This method was repeated Results: Test Tube Starch Solution (mL) Iodine (Drops) Amylase (mL) Temperature (^0 C) Time Taken For Starch To Disappear (Mins) 1 5 2 2 0 – 2 5 2 2 0 – 3 5 2 2 18 16.25 4 5 2 2 18 15.33 5 5 2 2 36 4.55 6 5 2 2 36 5.04 7 5 2 2 54 11.43 8 5 2 2 54 13.05 9 5 2 2 72 – 10 5 2 2 72 – Average Results: Starch Solution (mL) Iodine (Drops) Amylase (mL) Temperature (^0 C) Time Taken For Starch To Disappear (Mins) 10 2 2 0 – 10 2 2 18 15.59 10 2 2 36 5.00 10 2 2 54 12.24 10 2 2 72 – Explanation of Results: As predicted in the original hypothesis, the activity of amylase increased with increased temperature, therefore proving that average body temperature of 36-37^0 C is the optimal temperature for starch to disappear, as at that temperature the reaction was the fastest than the other varied temperatures. However, this is not to say that the experiments didn’t work at other temperatures, because as the results show the disappearance of starch happened at 18^0 C and 54^0 C only at a slower rate than the optimal temperature. The reason for this is that 18^0 C is colder and thus the molecules were moving at a slower rate and delaying the reaction.

And at 54^0 C the enzymes were on their way to becoming denatured and cannot be fixed thus the enzymes become waste and are not accessible for reuse. Graph 1: The Effect of Increased Temperature on the Activity of Amylase THE EFFECT OF CHANGE IN PH ON THE ACTIVITY OF AMYLASE Risk Assessment: . The amylase solution and starch solution are not harmful substances, however like any other chemical it is not recommended to swallow them, unless they are made specifically for ingestion. pH buffers can be dangerous to the skin, eyes and if swallowed could poison. Therefore gloves and safety goggles are to be worn at all times while handling the buffers. The pH buffers are flammable and so are to be kept away from any heated area and / or flame. Safety goggles should be worn at all times.

All spillages and broken glass are to be cleaned up immediately and disposed of in an appropriate manner. At the conclusion of the experiment liquid waste and chemicals may be disposed of down the sink, and other waste to the bin Hypothesis: The activity of amylase will be most effective at neutral (pH 7) Purpose: Test the effect of change in pH on the activity of amylase Materials: – Amylase solution – Beakers – Bunsen burner – Gauze mat – Gloves – Iodine – Matches – Measuring cylinders – pH 2 buffer: hydrochloric acid – pH 7 buffer: distilled water – pH 10 buffer: sodium hydroxide – Safety goggles – Starch solution – Stop watch – Test tube rack – Test tubes – Thermometer – Tripod – Water Method: 1. The gauze mat was placed on top of the tripod 5.2 test tubes were placed into the test tube rack 6.10 mL of starch solution was measured our and 5 mL of that was poured into each test tube 7.2 drops of iodine to both of the test tubes were added 8.1 mL of the pH 2 buffer was added both of the test tubes 9.1 of the test tubes was marked as a control 10. The beaker was filled with tap water 11. The temperature was raised to 36^0 C 14. If the water went: – above 36^0 C a little bit was empted and a little bit of colder water was added until it reached 36^0 C – below 36^0 C a little bit was empted and a little bit of hotter water was added until it reached 36^0 C 15.2 test tubes were placed into the beaker and their temperatures were measured until they reached 36^0 C 16.2 mL of the amylase solution was added only to the experimental test tube 17.

This method was repeated This experiment was followed exactly the same only in step 8 the pH 2 was replaced by pH 7 and pH 10 Results: Test Tube Starch Solution (mL) Iodine (Drops) Amylase (mL) Temperature (^0 C) pH Buffers (mL / pH) Time Taken For Starch To Disappear (Mins) 1 5 2 2 36 1 / 2 19.25 2 5 2 2 36 1 / 2 18.46 3 5 2 2 36 1 / 7 5.40 4 5 2 2 36 1 / 7 6.05 5 5 2 2 36 1 / 10 – 6 5 2 2 36 1 / 10 – Average Results: Starch Solution (mL) Iodine (Drops) Amylase (mL) Temperature (^0 C) pH Buffers (mL / pH) Time Taken For Starch To Disappear (Mins) 5 2 2 36 1 / 2 19.06 5 2 2 36 1 / 7 5.52 5 2 2 36 1 / 10 – Explanation of Results: Amylase is found in saliva that has a pH of 6.5 (slightly acidic) and pancreatic juices that have a pH of 8 (slightly alkaline), thus making pH 7 its optimum level and supporting the original hypothesis that the activity of amylase would be most effective at a neutral pH of 7. The experiment was still successful at a pH of 2, however time was exceedingly long and not as effective. A pH of 10 was far too alkaline for the amylase to remain active, and the active site becoming distorted creating the enzymes to become denatured. Graph 2: The Effect of Change in pH on the Activity of Amylase THE EFFECT OF CHANGE IN SUBSTRATE CONCENTRATION ON THE ACTIVITY OF AMYLASE Risk Assessment: . At the conclusion of the experiment liquid waste may be disposed of down the sink, and other waste to the bin Hypothesis: The activity of amylase will increase with increased starch concentration.

When the starch concentration reaches its maximum the reaction will be most effective. Purpose: Test the effect of change in starch concentration on the activity of amylase Materials: – Amylase solution – Beakers – Bunsen burner – Gauze mat – Gloves – Iodine – Matches – Measuring cylinders – pH 7 buffer: distilled water – Safety goggles – Starch – Stop watch – Test tube rack – Test tubes – Thermometer – Tripod – Water Method: 1. The gauze mat was placed on top of the tripod 5.2 test tubes were placed into the test tube rack 6. No starch solution was added either of the test tubes 7.2 drops of iodine were added to both the test tubes 8.5 mL of distilled water was added to both test tubes 9.1 of the test tubes was marked as a control 10. This method was repeated This experiment was carried out a further 5 times but changing: – step 6 to: 1 mL of starch solution was added to both test tubes step 8 to: 4 mL of the distilled water was added to both test tubes – step 6 to: 2 mL of starch solution was added to both test tubes step 8 to: 3 mL of the distilled water was added to both test tubes – step 6 to: 3 mL of starch solution was added to both test tubes step 8 to: 2 mL of the distilled water was added to both test tubes – step 6 to: 4 mL of starch solution was added to both test tubes step 8 to: 1 mL of the distilled water was added to both test tubes – step 6 to: 5 mL of starch solution was added to both test tubes step 8 to: None of the distilled water was added either of the test tubes Results: Test Tube Iodine (Drops) Amylase (mL) Temperature (^0 C) Starch Solution (mL) Distilled Water (mL) Time Taken For Starch To Disappear (Mins) 1 2 2 36 0 5 – 2 2 2 36 0 5 – 3 2 2 36 1 4 – 4 2 2 36 1 4 – 5 2 2 36 2 3 40.41 6 2 2 36 2 3 38.49 7 2 2 36 3 2 25.03 8 2 2 36 3 2 22.55 9 2 2 36 4 1 11.06 10 2 2 36 4 1 12.24 11 2 2 36 5 0 4.15 12 2 2 36 5 0 4.20 Average Results: Iodine (Drops) Amylase (mL) Temperature (^0 C) Starch Solution (mL) Distilled Water (mL) Time Taken For Starch To Disappear (Mins) 2 2 36 0 5 – 2 2 36 1 4 – 2 2 36 2 3 39.45 2 2 36 3 2 23.59 2 2 36 4 1 11.45 2 2 36 5 0 4.18 Explanation of Results: The original hypothesis supported the activity of the amylase on starch increasing with the substrate concentration.

The results of this experiment prove that. By steadily increasing the starch concentration to distilled water the rate of reaction increased as all the active sites gradually became occupied whilst the starch concentration reached its maximum of 100%. Once the maximum concentration is reached the rate of reaction stabilizes and can go no faster. Graph 3: The Effect of Change in Substrate Concentration on the Activity of Amylase Justification of Method: The methods were chosen in order to achieve the most reliable results and have as few variables as possible that made the experiments easier and more accurate. The methods were repeated to ensure accuracy.

Several factors of the method were controlled that certified precise and successful experiments. The dependent variables in all the experiments were the times taken for the substrate (starch) to disappear from the solution through a change in colour of the solution. The independent variables were the temperatures, pH and substrate concentrations. The methods supported the control of the variables by giving easy-to-follow instructions with accurate measurements. Along with this, controls were used to ensure that it was in fact the enzyme that caused the reaction and not any other factors. However, before the actual methods were planned, ‘mini experiments’ took place (that are not documented) to determine correct ratios and amounts of solutions to be used.

Limitations of Equipment and Methodology and Suggested Improvements: Human error plays the primary role in these experiments, as the basis of the results is determined by the judgement of sight. The ability to accurately predict by sight could improve the reliability of results. Because of the long periods that the starch takes to disappear from the solution, it becomes confusing to decide if it is actually disappearing at all, as the change in colour is not dramatic, but a more gradual process. Another aspect of human error is the prior knowledge of enzymes and the experience in practical experiments the scientist has. This could be improved by further research and investigation taken before the commencement of the experiments. Equipment such as measuring cylinders use the judgement of the human eye to decide if amounts are correct, therefore there is always going to be leeway in accuracy because of this.

The only way to remove the effects of human error in an experiment would be to take them out of the equation all together and use machines; although, due to the limited amount of facilities available in the labs this was not possible. Reliability and Accuracy: The experiment was repeated to provide two sets of results to achieve accuracy in measurement and reliability with method. The results were compared with recommended and endorsed textbooks and websites. The results differed at the most 2.08 minutes.

A control was used to ensure the results were from the enzyme activity. All equipment was thoroughly cleaned prior to commencement of the experiment to prevent contamination. Time was measured precisely with a stopwatch. Measuring cylinders were identical. Consideration was given for human error when estimating colour change.

Internal environment: The normal and most desirable condition of body functioning is homeostasis. Its loss or destruction always leads to a temporary or permanent condition. Homeostasis is described as a dynamic state of equilibrium or a balance in which internal conditions change and vary, but always within relatively narrow ranges. Most homeostatic control mechanisms are negative feedback mechanisms. In such systems an increase in the output of the system feeds back and decreases the input into the system. The net effect is to decrease the original stimulus or reduce its effects, slowing the activity or shifting it off entirely.

These mechanisms cause the variable to change in a direction opposite to that of the initial change, thus the name ‘negative’ feedback mechanism. Drastic changes in the body temperature are life threatening. At normal body temperature most reactions would proceed very slowly – far too slow to maintain life – were it not for the presence of catalysts. Conclusion: The experiment procedures are complete.

By comparing all the results, it can be determined that the hypotheses are correct. The enzyme amylase has its own optimum temperature and pH at which it catalyses reactions at the fastest rate. This is related to the conditions within the living organism. Amylase has been proven to be most effective at 37^0 C with a pH of 7 and with a maximum substrate concentration. Dreher, Christine.

“Primal Defense. net”. [Online] web “Effects of amylase on starch”. [Online] web Enzyme Essentials. “The importance of carbohydrates”. [Online] web Google.

“Amylase”. [Online] web Instant Essays. “Salivary amylase lab”. [Online] web “Investigating the effect of temperature on the breakdown of starch by amylase” [Online] web November 9, 2003 Kinnear, J. and Martin, M. 2001 Biology 2: HSC course, John Wiley & Sons, Milton. Marie, E.N. 1989. Human anatomy and physiology, Benjamin Cumming, Redwood City.

Science Line. “Biology and medicine”. [Online] web November 9, 2003 Sterne, Richard G. “Factors effecting enzyme action”. [Online] web Wang, Nam Sun. “Experiment no. 5 starch hydrolysis by amylase”.

[Online] web Wikipedia. “Amylase”. [Online] web.

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