The concentration of substrate molecules (the more of them available, the quicker the enzyme molecules collide and bind with them). The concentration of substrate is designated [S] and is expressed in unit of molarity. It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate concentration is then gradually increased, the reaction velocity will increase until it reaches a maximum. After this point, increases in substrate concentration will not increase the velocity (delta A/delta T). This is represented graphically in Figure 5. It is theorized that when this maximum velocity had been reached, all of the available enzyme has been converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax.
The temperature. As the temperature rises, molecular motion - and hence collisions between enzyme and substrate - speed up. But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes and ineffective. However, a ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results.
In the case of enzymatic reactions, this is complicated by the fact that high temperatures adversely affect many enzymes. Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. As shown in Figure 6, the reaction rate increases with temperature to a maximum level, and then abruptly declines with further increase of temperature.
Because most animal enzymes rapidly become denatured at temperatures above 40·C, most enzyme determinations are carried out somewhat below that temperature. Over a period of time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5·C or below is generally the most suitable. Some enzymes lose their activity when frozen.
The presence of inhibitors. Enzyme inhibitors are substances that alter the catalytic action of the enzyme and consequently slow down, or in some cases, stop catalysis. There are three common types of enzyme inhibition - competitive, non-competitive and substrate inhibition. Most theories concerning inhibition mechanisms are based on the existence of the enzyme-substrate complex ES. As mentioned earlier, the existence of temporary ES structures has been verified in the laboratory. Competitive inhibition occurs when the substrate and a substance resembling the substrate are both added to the enzyme.
A theory called the "lock-key theory" of enzyme catalysts can be used to explain why inhibition occurs. The lock and key theory utilizes the concept of an "active site." The concept holds that one particular portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in such a way that its conversion to the reaction products is more favourable. If we consider the enzyme as the lock and the substrate the key is inserted in the lock, is turned, and the door is opened and the reaction proceeds.
However, when an inhibitor that resembles the substrate is present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed down because the inhibitor occupies some of the available enzyme sites. If a dissimilar substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the reaction proceeds normally.
Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the enzyme in a way that it cannot accept the substrate. Figure 7.
Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 8 shows the reaction velocity decreasing after the maximum velocity has been reached.
Additional amounts of substrate added to the reaction mixture after this point actually decreases the reaction rate. This is thought to be due to the fact that there are so many substrate molecules competing for the active sites on the enzyme surfaces that they block the sites (Figure 9) and prevent any other substrate molecules from occupying them. This causes the reaction rate to drop since all of the enzyme present is not being used.
pH. The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its conformation, its activity is likewise affected. The most favourable pH value - the point where the enzyme is most active - is known as the optimum pH. This is graphically illustrated in Figure 10.
Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. The optimum pH value will vary greatly from one enzyme to another, as Table 2 shows:
In addition to temperature and pH there are other factors, such as ionic strength, which can affect the enzymatic reaction. Each of these physical and chemical parameters must be considered and optimised in order for an enzymatic reaction to be accurate and reproducible.
As a rule, enzymes do not attack living cells. As soon as a cell dies, however, enzymes that break down protein rapidly digest it. The resistance of the living cell is due to the enzyme's inability to pass through the membrane of the cell as long as the cell lives. When the cell dies, its membrane becomes permeable, and the enzyme can then enter the cell and destroy the protein within it. Some cells also contain enzyme inhibitors, known as antienzymes, which prevent the action of an enzyme upon a substrate.
Commercial Production and Use of Enzymes
Key Stages in Commercial Production of Enzymes
Fermentation broth is superheated under aseptic conditions to form a completely sterile nutrient medium
The nutrient is converted into a desired enzyme by carefully selected microorganism action in the presence of oxygen. The choice of broth, microorganism, and operating conditions determine the type and yield of enzyme.
Once fermentation is completed, various centrifugal, filtration, and precipitation processes separate the enzyme from the fermentation broth.
Where Enzyme Products Are Used...
Alcohol: starch into sugars, and sugars into alcohol.
Animal Feed: degradation of feed components for improved feed utilization and nutrient digestion. Reduction in nitrogen & phosphorus in waste.
Baking: modification of flour for improved baking properties, anti-staling.
Brewing: faster maturation of beer, chill proofing, removal of carbohydrates for light beers, breaks down beta-glucanases.
Dairy: chymosin for cheese making, removal/conversion of lactose in milk.
Detergent: an active biological component of washing powders or liquids. Granulated proteases, amylases, and lipases break down starch and fatty stains. Celluloses are included for the depilling, colour brightening, and softening of cotton garments being washed.
Fats & Oils: modification of lecithin’s and syntheses of specialty fats and oils.
Leather: soaking of hides and skins, unhairing, bating, and defatting.
Personal Care: biotechnology ingredients for personal care products.
Protein: improvement of nutritional and functional properties of animal and vegetable proteins. Development of flavour bases based on proteins.
Pulp & Paper: control of pitch problems. Reduction of chlorine consumption in pulp, bleaching, viscosity control in starch-based coatings, de-inking for recycling programs.
Starch: production of dextrose, fructose, and special syrups for the baking, confectionery, and soft drink industries
Textiles: stone washing of denim (in combination with pumice stones), bio-polishing and softening of cotton and defibrillation of Tencel® fabrics, degumming of silk, bleaching, clean up, removal of starch from woven materials.
Wine & Juice: degradation of pectin for clarification and increase in juice yields.
Global Markets
Industrial Enzymes
1997 (US $ 1.45 Billion)
Alcoholic fermentation and other important industrial processes such as the making of chocolates, syrups and other food products and extracting fruit juices depend on the action of enzymes and bacteria used in the production process. Alcoholic fermentation is the action of zymase secreted by yeast converts simple sugars, such as glucose and fructose, into ethyl alcohol and carbon dioxide. Many other kinds of fermentation occur naturally, as in the formation of butanoic acid when butter becomes rancid and of ethanoic (acetic) acid when wine turns to vinegar.
Generally, fermentation results in the breakdown of complex organic substances into simpler ones through the action of catalysis. For example, by the action of diastase, zymase, and invertase, starch is broken down (hydrolysed) into complex sugars, then simple sugars, and finally alcohol.
Biological Washing Powders, use proteases, because stains like grime and sweat contain protein. These washing powders work at quite low temperatures. Proteases are also used to tenderise meat and to remove hairs from skins. Enzymes from microbes are used in fermentation to make beer, wine and vinegar (as discussed above) and for extracting fruit juices; the enzyme pectinases is used to help break down plant cell walls. They can be used for softening vegetables too. Finally, Carbohydrases are used in the making of chocolates, syrups and other food products.
Glycerine, propanone, butanoic alcohol, and butyric acid are now produced on a large commercial scale by special fermentation processes. Various fermentation productions of milk, such as acidophilus milk, Bulgarian milk, and yoghurt, are widely consumed for their nutritive properties.
The action of certain bacteria on undigested carbohydrates causes fermentation in the human intestine. As a result, gases such as hydrogen sulphide and carbon dioxide may form in amounts large enough to cause distention and pain. Acids such as lactic acid and ethanoic acid may also form in the intestines of infants, causing diarrhoea.
A number of enzymes are used for medical purposes. Some have been useful in treating areas of local inflammation; trypsin is employed in removing foreign matter and dead tissue from wounds and burns. The medical uses of enzymes are illustrated by research into L-asparaginase, which is thought to be a potent weapon for treatment of leukaemia; into dextrinases, which may prevent tooth decay; and into the malfunctions of enzymes that may be linked to such diseases as phenylketonuria, diabetes, and anaemia and other blood disorders.
Historical Review
Alcoholic fermentation is undoubtedly the oldest known enzyme reaction. This and similar phenomena were believed to be spontaneous reactions until 1857, when the French chemist Louis Pasteur proved that fermentation occurs only in the presence of living cells. Subsequently, however, the German chemist Eduard Buchner discovered (1897) that a cell-free extract of yeast could cause alcoholic fermentation. The ancient puzzle was then solved; the yeast cell produces the enzyme, and the enzyme brings about the fermentation. As early as 1783 the Italian biologist Lazzaro Spallanzani had observed that meat could be digested by gastric juices extracted from hawks. This experiment was probably the first in which a vital reaction was performed outside the living organism. After Buchner's discovery scientists assumed that fermentations and vital reactions in general were caused by enzymes. Nevertheless, all attempts to isolate and identify their chemical nature were unsuccessful. In 1926, however, the American biochemist James B. Sumner succeeded in isolating and crystallizing urease. Four years later pepsin and trypsin were isolated and crystallized by the American biochemist John H. Northrop.
Enzymes were found to be proteins and Northrop proved that the protein was actually the enzyme and not simply a carrier for another compound.
Research in enzyme chemistry in recent years has shed new light on some of the most basic functions of life. American researchers synthesized ribonuclease, a simple three-dimensional enzyme discovered in 1938 by the American bacteriologist René Dubos and isolated in 1946 by the American chemist Moses Kunitz, in 1969.
The synthesis involves hooking together 124 molecules in a very specific sequence to form the macromolecule. Such syntheses led to the probability of identifying those areas of the molecule that carry out its chemical functions, and opened up the possibility of creating specialized enzymes with properties not possessed by the natural substances. This potential has been greatly expanded in recent years by genetic engineering techniques that have made it possible to produce some enzymes in great quantity.
Amylase
Amylase, like other enzymes, works as a catalyst, i.e. it is unchanged by the reaction, but makes the reaction easier by reducing the energy required for it to happen. Catalysts speed up the reaction. The theory behind the working is called the "lock and key" theory: the enzyme is shaped so that the products fit into them, react and are released (as discussed previously). Amylase digests starch by catalysing hydrolysis, which is splitting by the addition of a water molecule. Therefore starch plus water becomes maltose (which is equivalent to two joined glucose molecules).
There are two kinds of amylase enzymes. Alpha-amylase is found in saliva and is called ptyalin. This can carry on working in the stomach for several hours (and can digest up to 40% of starch under correct conditions of stomach acidity and food solidity). The other kind is called pancreatic amylase and is secreted in pancreatic juice, into the small intestine or ileum. Other enzymes then further digest the maltose to glucose and this is then absorbed through the wall of the small intestine by the body to be used as energy after being taken to the liver.
Body temperature is optimal for the best reaction of amylase (as with other enzymes) - if the temperature is too high, it comes apart, and if too low, the reaction slows to a stop.
Amylase Enzyme
Alpha and Beta Amylase
International Union of Biochemistry and Molecular Biology (IUBMB) Enzyme Nomenclature:
EC 3.2.1.1 Recommended name: α - amylase
Other name(s): Glycogenase; α Amylase, Alpha – Amylase; Endoamylase; Taka-Amylase A.
EC 3.2.1.2 Recommended name: β - amylase
Other name(s): Saccharogen Amylase; Gylcogenas; β Amylase, Beta-Amylase.
These enzymes break down starch, which is the main constituent of foods like bread, rice and potatoes. Starch consists of long chains of a sugar called glucose. Amylase breaks down starch into smaller units consisting of several glucose molecules. These small breakdown chains are called oligosaccharides. These oligosaccharides from starch are broken down to glucose by other enzymes in the membranes of the cells lining the intestine. The glucose can then enter the bloodstream to be sent as an energy source to the various organs of the body. Amylase is either produced in the saliva (so it gets mixed food as it is chewed) or produced by your pancreas and secreted into the intestine after the food has passed through the stomach.
Alpha amylase is resistant to comparatively high temperatures; its temperature optimum is 70°C (in mash to make beer) and it is destroyed at 80°C. It functions best at pH is 5.8. Beta-amylase is destroyed at 75°C and its optimum operating temperature is 60-65°C (in mash to make beer) and optimum pH is 5.4.
A typical example of industrial usage of Amylase is in bread making. Many food product designers consider enzyme use new and innovative. While this is true for many categories, the baking industry actually has a long history of enzyme study and application. In fact, some references to the use of added enzymes in bakery foods are over 100 years old.
The alpha-amylase enzyme hydrolyzes starch into soluble dextrins. These dextrins may subsequently be hydrolyzed by beta-amylase to yield maltose, and/or amyloglucosidase to yield glucose. Because starch exists as a tightly packed granule, amylases must act upon starch granules that are damaged (as many are during flour milling) or on granules that have been gelatinized by moisture and heat (such as when a dough is mixed and baked).
The sugars resulting from amylase activity act as food for yeast in yeast-raised products. As a result, the presence of these enzymes in the proper proportions is critical to carbon dioxide generation. Most flour naturally contains both alpha- and beta-amylase. The beta-amylase is, however, the only one naturally present in sufficient quantities. Thus, controlling the gassing power of the dough requires added alpha-amylase.
Amylases also can affect the consistency of dough. Damaged starch granules absorb more water than intact granules. This ability is reduced when the damaged granules are acted upon by amylases. With their ability to immobilize water reduced, the damaged granules release free water that softens the dough and makes it more mobile.
A third function of amylases is their ability to retard staling. Over time, the crumb of baked products firms due to a complex set of changes that includes recrystallization (or retrogradation) of amylopectin in the starch. By hydrolyzing the amylopectin into smaller units, bacterial alpha-amylase can maintain softness and extend shelf life.
One theory behind this suggests that amylopectin still crystallizes at the same rate with added enzymes, but that the shortened chain length maintains greater flexibility and softness when crystallized. Another theory is that the shortened amylopectin chains have a lesser tendency to retrograde. Either way, the enzyme must continue to hydrolyze starch after baking is completed. The fact that bacterial alpha-amylase is more thermally stable than other alpha-amylase sources is the reason it is used.
Because the enzyme is active in the finished baked product, it is possible for the enzyme activity to go too far. Rather than maintaining softness, the crumb can actually become gummy. The starting enzyme dosage is critical to preventing this. For even greater assurance against overdosing, amyloglucosidase or pullulanase may be added along with the alpha-amylase. These enzymes don't contribute to anti-staling when used alone, but help prevent gumminess when combined with the amylase.
A final use for amylases in bakery products is for replacing potassium bromate, an oxidizing agent that strengthens gluten strands. Strengthened gluten produces a dough with improved gas retention and, consequently, higher volume in the finished product.
Based on various health studies, bromate use is on a sharp decline. Other oxidants -- such as ascorbic acid -- can promote comparable volume, but they don't provide a direct match for bromate. To compensate, alpha-amylase can be added with ascorbic acid to improve the volume and increase the quality of the crumb. Bakeries may either add alpha-amylase and ascorbic acid separately or select a custom blend featuring an optimized mixture of the two components.
Hypothesis
I predict that the rate of reaction will increase as the temperature increases (having a positive correlation) until the reaction reaches an optimum temperature. Above this optimum temperature, the rate of reaction will fall to zero very quickly as the enzyme denatures.
The breakdown of starch will be quicker when the temperature is increased until it exceeds 40° C. Then the amylase will no longer catalyse the breakdown of starch.
Prediction
Using the my background information about enzymes and Amylase together with my knowledge of kinetic theory, it is possible to understand how temperature affects the rate of reaction. The Kinetic theory states that when a substance is heated, energy is given to the particles and they speed up. Therefore, when heat is applied to an enzyme and substrate, the particles speed up, increasing the rate at which they bind with each other. This would suggest that a rise in temperature increases the rate of most chemical reactions and a fall in temperature will slow them down (the rate of reaction is proportional to the temperature). In many cases a rise in 10° C will double the rate of reaction in a cell. Since the kinetic energy increases, the velocity of particles will also increase. As the speed of particles increases, they should collide more often and therefore the speed of reaction increases. The particles will also have more energy thereby speeding up the reaction even more.
At low temperatures particles of reacting substances do not have much energy. However, when the substances are heated, the particles take in energy. This causes them to move faster and collide more often. The collisions have more energy, so more of them are successful. Therefore the rate of reaction increases.
The more successful the collisions are the faster the reaction. The same can be said for reactions controlled by enzymes. However, there is a limit to the temperature at which an enzyme can work because excessive heat causes an enzyme to become denatured and stop working (as they are made of protein which makes enzymes sensitive to heat making it lose it’s shape and no longer able to combine with the starch). As you go over the optimum temperature of the enzyme, although chemically you are increasing the chances of starch breakdown, you are also increasing the chances of the breakdown of the three-dimensional structure of the enzyme. As the heat in the system increases, the vibrational energy of the entire alpha-amylase molecule also increases. This puts a strain on the weak interactions that hold the enzyme together. At temperatures just above optima, there may be a situation where the enzyme is in a sort of equilibrium where it temporarily loses some of its structure and then regains it to work again. At higher temperatures these bonds literally get shaken apart and the three -dimensional structure of the protein destabilises. This is called denaturation.
Also, there is a minimum temperature at which an enzyme can function. Every chemical reaction requires activation energy in order to get started. Although enzyme catalysis greatly reduces this, some energy is still required. Because of this the reaction is still unable to happen below a given temperature (this varies depending on the type of enzyme and reaction, as does the maximum temperature). If warmed to above the activation temperature, an enzyme will work again as normal. A denatured enzyme, however, is damaged and will not work again even if cooled below the optimum temperature.
To investigate the effect of temperature on enzymes I will conduct an experiment.
Apparatus
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Starch (5cm3 for each experiment) and amylase (1cm3 for each experiment) solution
- Bunsen burner (bench mat, gauze, tripod).
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Beaker full of 200 cm3 of water.
This apparatus was used in order to accurately measure substances and time to gain a reliable set of results. For example, the syringe will allow me to measure exactly how much starch and amylase to use and the thermometer will allow me to measure the exact temperature of the water bath. This will all help me gain an accurate and reliable set of results.
Safety when Conducting Experiments
Before conducting any experiments, safety procedures and precautions should be taken to minimize hazards and the risks of any accidents taking place.
In this experiment I will be handling an enzyme – amylase. I will be using a solution of amylase (not solid form i.e. powdered enzyme). The control measures to reduce the risks are:
- Use the lowest concentration possible.
- Reduce risk of skin contact by wearing disposable gloves.
- Wear eye protection.
In the case of an emergency:
- If in the eye – Wash out the eye for at least 10 minutes.
- If swallowed – Wash out mouth. Drink a glass of water also.
- Spilt on skin or clothing – Remove contaminated clothing. Wash off the skin with soap and water. Also rinse the clothing.
- If spilt on floor/bench – Wipe up solution spills with a damp cloth.
I will also be using Iodine, which is a chemical (again I will be using a solution of Iodine). The nature of the hazards are:
- Harmful if in contact with skin. Causes burns to the skin if left for some time.
- Easily vaporised if heated. Produces violet vapour, which is dangerous to eyes.
The control measures to reduce the risks are:
- Use the lowest concentration and the smallest volume.
- Wear eye protection for all but the most dilute solutions.
- Wear protective gloves.
- Avoid breathing iodine vapour, for example, by using the fume cupboard if necessary.
In the case of an emergency:
- If in the eye - Wash out with water for 10 minutes.
- If vapour is breathed in – Remove victim to fresh air.
- If swallowed – Wash out mouth. Drink a glass of water.
- If spilt on skin or clothing – Brush off. Remove contaminated clothing. Drench skin with plenty of water.
- If split on floor/bench - Wipe off any spillage. Add Sodium thiosulphate solution (20%, 1M) to remaining spill and leave for an hour. Mop up and then rinse with water.
During the experiment, heating will be done of substances and therefore burns are a possible hazard. To reduce the risks:
- Wear protective eyewear.
- Keep bunsen burner on yellow flame when it is not heating substances.
- Tie back long hair and remove coats and jumpers.
In the case of an emergency:
- If burn on skin – Place under a cold tap and leave for approximately 10-15 minutes depending on the severity of the burn. Inform a member of staff immediately and seek medical attention, if necessary.
- If clothing or hair catches fire – Extinguish the fire using a suitable* fire extinguisher. Inform a member of staff immediately and in case of burn carry out the above instruction.
- In the case of fire appearing to get out of control, inform a member of staff so that the gas and the electricity supply can cut off at the mains. Sound the fire alarm.
Finally, the experiment deals with heating and using glass wear. The control measures to reduce the risks of breakage are:
- Place glass wear such as beakers away from edges of tables. Place test tubes in test tube racks and pipettes and thermometers in a book to prevent it rolling on the table.
- Wear protective eyewear in case of any breakage happening.
In the case of an emergency:
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If broken glass on floor/bench – scoop up any pieces using a dustpan and brush. Inform a member of staff about the breakage and dispose of the broken glass.
- If broken in sink – Pick up any big pieces of glass and inform a member of staff about the breakage. Again, dispose of the glass.
- If there is a broken thermometer – Inform a member of staff immediately. Remove the broken thermometer from the spillage area and dispose of it.
- If a student is hurt due to a glass breakage – Immediately inform a member of staff and seek medical advice.
REMEMBER: Always wear protective eyewear when conducting any experiment in the lab. You may also wear gloves and a protective lab coat, as they will minimise the hazards of any chemical spillages on your cloths and skin.
Preliminary Test
By conducting a preliminary test, I will have the opportunity to check my method is right and to observe how I can make the test more accurate by having right amounts of substances. This will ensure that my results are accurate and reliable.
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Warm up 250cm3 of water in a beaker to about 150C. This will act as a water bath
- for the test tubes during the experiment. Check the temperature accurately using a thermometer.
- Label two test tubes A and B.
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Using a syringe, place 0.5cm3 of amylase solution into test tube A.
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Using a separate syringe, place 5cm3 of 5% starch solution in test tube B.
- Place both test tubes in the beaker making sure that the temperature is at a constant. Allow both test tubes to be warmed in the water for 10 minutes.
- Whilst waiting for the contents of the test tube to be warmed up, set up a dimple tile with 2 drops of iodine in each dimple using a pipette. Place some starch into one dimple full of iodine. A blue/black colour should appear. This will act as a control.
- Pour the amylase solution (test tube A) into the test tube of starch (test tube B) after both have been in the water bath for 10 minutes. Start the stop clock as soon as both contents of the test tubes have been mixed.
- Test the solution to see if there is any starch left every 15 seconds by taking a sample with a pipette from the solution and placing it in the dimple tray full of iodine. Using the control dimple, keep testing the solution every 15 seconds till there is a negative result and the colour of the solution is no longer black.
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Repeat steps 3-8 raising the temperature of the water bath by 50C each time until the enzyme is denatured. Try till about 60-700C to get a good set of results.
- Collect the data and record the results in a table.
After conducting this preliminary test, I realised that there are some changes to make in order to improve the experiment.
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It would be better to just warm up 200cm3 of water rather than 250cm3 as it will reduce the time needed to heat the water, thus allowing time for the conduct of more experiments done at different temperatures.
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Start the temperature experimenting at 200C rather than 150C as the preliminary test showed that 200C was the lowest temperature at which the enzyme would function at all. Also, increase the temperature every time by 100C. This will ensure that there is enough time to go back and repeat the experiment in order to get an average time for the digestion of starch for each temperature.
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Instead of using 0.5cm3 of amylase, use 1cm3. This will speed up the experiment slightly because before the amylase was mixed with 5cm3 of starch and it took longer to digest the starch. Also other preliminary test on the effect of pH on amylase activity suggests that 1cm3 of amylase is a better amount to use with 5cm3 of starch.
- Leave both test tubes in the water bath for 5 minutes rather than 10 minutes. This is because 10 minutes is slightly too long. Other preliminary work done on the effect of pH on amylase activity also indicates that 5 minutes is a better time to leave the test tubes in the water bath.
- It would be better to test the amylase-starch solution every 30 seconds once it has been mixed as the preliminary test showed that 15-second intervals were too short for any significant change to take place.
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Finally, test until about 700C rather than 60-700C as the preliminary test showed that the enzyme activity still continued slightly at 700C.
Final Method
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Warm up 200cm3 of water in a beaker to about 200C. This will act as a water bath for the test tubes during the experiment. Check the temperature accurately using a thermometer.
- Label two test tubes A and B.
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Using a syringe place 1cm3 of amylase solution into test tube A.
-
Using a separate syringe, place 5cm3 of 5% starch solution in test tube B.
- Place both test tubes into the beaker making sure that the temperature is at a constant. Allow both test tubes to be warmed in the water bath for 5 minutes.
- Whilst waiting for the contents of the test tube to be warmed up, set up a dimple tile with 2 drops of iodine in each dimple using a pipette. Place some starch into one dimple full of iodine. A blue/black colour should appear. This will act as a control.
- Pour the amylase solution (test tube A) into the test tube of starch (test tube B) after both have been in the bath for 5 minutes. Start the stop clock as soon as both contents of the test tubes have been mixed.
- Test the solution to see if there is any starch left every 30 seconds by taking a sample with a pipette from the solution and placing it in the dimple tray full of iodine. Using the control dimple, keep testing the solution every 30 seconds till there is a negative result and the colour of the solution is no longer black.
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Repeat steps 3 - 8 raising the temperature of the water bath by 100C each time until the enzyme is denatured (try till about 700C).
- Collect the data and record the results in a table.
- Repeat the entire experiment again if there is extra time as this will help gain an average of the results. Also, repeat any experiments that show anomalous results.
Fair Testing and Variables
To ensure that the test is fair, I will measure all quantities carefully and keep them the same for each experiment since small variations in the amount of enzyme or solution used can make significant variations in the results. The concentration of the amylase and the starch has to be kept the same too because if it isn’t the reaction will be effected (discussed in the background information). Also, when the test tubes are to be heated in the water bath, the temperature must be kept at a constant as a change in the temperature can increase (in the early stages of the test), the rate of reactivity. The apparatus will also be kept the same throughout the experiment. Also the amount of iodine used in each dimple to test whether there is any starch left or not is to be kept the same too. These are my control variables.
The independent variable in this experiment will be the temperature because I am trying to understand the effect temperature has on enzyme activity. The dependent variable will be the breakdown of the starch and the time it takes. All other variables will be kept constant. Changing anything else in the experiment would not make the test fair.
Results
The following results were obtained:
The amylase was also tested around its optimum temperature (350C and 450C) in order to gain a more accurate result of which temperature range the enzyme worked best at and to get more definite results. Ideally further experimentation should have been done for 310C, 320C 330C etc till 450C as this will have shown exactly what temperature the enzyme worked best at and this would have been able to show whether it is in fact most effective at body temperature or not (370C) but it would have been too difficult to keep the temperature at a constant for each temperature and it would also have been quite time consuming to do.
Analysis of Results
The graph shows that, between temperatures of 20oC and 40oC, the efficiency of the enzyme increases with temperature. However, the graph between these points is a curve so the efficiency of the enzyme is not proportional to the temperature. With enough results (from a very large range of temperatures), it would be possible to find a mathematical formula for the construction of the curve on the graph and hence find the relationship between the temperature and the rate of reaction. Because the curve is not perfectly symmetrical, it may have to be investigated between individual points in order to find a relationship between the temperature and the rate of reaction. Between 40oC and 60oC, the efficiency of the enzyme decreases with temperature, mirroring the first part of the graph. The graph shows that the optimum temperature of the amylase tested was 40oC.
The graph supports my prediction that the optimum temperature of the enzyme would be around 40oC, and would have decreasing efficiency towards 0oC, at which the amylase would be unable to break down the starch at all.
The reason for the behaviour of the enzyme shown in the graph involves the kinetic theory mentioned in my prediction. In this experiment, as the temperature increased, the enzyme and starch molecules collided more frequently (Brownian motion) and with more energy, which caused them to react more efficiently. At low temperatures, i.e. at 200C, the molecules did not collide as frequently and the starch was therefore not broken down as quickly. This is true of any reaction, whether or not it involves catalysts, biological or otherwise.
The enzyme was most effective at around 40oC because this is near to body temperature, at which is it most used to working. In order for it to function most efficiently in the body, amylase must have an optimum temperature of around 40oC. The graph shows that there is less difference between the average time taken for the starch to disappear at 350C and 400C than at 400C and 450C, which means that, the exact optimum temperature lies in between 350C and 400C.
The reason why the amylase was less effective at higher temperatures was that it had started to denature. All enzymes start to denature at temperatures above their optimum temperatures, which renders them unable of catalysing reactions.
The Rate of Amylase Activity in Relation to Temperature
Evaluating the Evidence
The experiment worked well overall, proving beyond reasonable doubt that the optimum temperature of the amylase used in the experiment was around 40oC. Despite the erratic nature of the experiment, the results were sufficiently accurate that they fit the general trend, and were taken at intervals far enough apart that the readings were clearly distinguishable from each other.
However, the results were not totally accurate. For example, the first time the experiment was carried out at 70oC, the starch disappeared in 3 minutes (180 seconds), while the second time the enzyme denatured quickly and the third time the starch disappeared after 6 minutes (400 seconds) which was roughly twice as much as the first experiment. At 60oC, the second time the experiment was conducted at this temperature, the starch took twice as long (6minutes or 360 seconds) to disappear than the time it took in the first experiment. Also, in the first experiment, you would expect the time taken for the starch to disappear to be slight more at 600C and 700C than at 500C but in fact they all showed to take 3 minutes (180 seconds). The graph was not a perfectly smooth curve, and this was due to several different factors.
The apparatus used could have been improved in many ways. The water baths used were not all at the exact temperatures required, and each contained a different amount of water (due to the addition of cold water to cool down to the right temperature). If better quality water baths had been used, and there was time to ensure that each had exactly the same amount of water and was at the exact temperature required, anomalous results could have been eliminated. This could also have been achieved by repeating the experiment for each temperature more than three times, and also by performing the experiment at intervals smaller than 10oC. Another problem with the experiment was the use of iodine. Although iodine is a good indicator of whether or not starch is present, it does not provide accurate information about the concentration of starch present. It would have been more useful to obtain this information so that it could be plotted, to analyse how the concentration changes over time rather than at what single time there is no more starch. This quantitative approach could have been achieved by using a colorimeter. This device provides an indication of how deep a colour is, and could have been used to measure the index of concentration of the samples throughout the experiment. Values for concentration of starch in the samples could have been obtained by first recording a reading for known concentrations, and then comparing these readings with those obtained with the samples collected during the experiment.
Additionally, the pipettes used were another area of error. More accurate results could have been obtained by cleaning the pipette between each reading, or using a new pipette each time, but this could not practically happen. There was always some solution left over in the pipette from the previous reason. Another problem with the pipettes was that there was time for the amylase to act on the starch while the solution was in the pipette, making the timings recorded slightly too small. However, this effect was lessened with most of the temperatures as the mixture was cooling down to room temperature in the pipette.
Finally, the volumes of each solution could have been made more accurate by measuring the solutions using a narrower gauge measuring tube.
An ideal solution would have been to automate the whole system, with a sample of the mixture being automatically taken every minute, or preferably more frequently, and the concentration of starch stored on computer. This would have overcome the inaccuracies of the timing, which could not always be exact using a stop clock and someone watching it, and would have eliminated the effect of human error from the experiment.
In conclusion, the accuracy of the results was certainly good enough to make a sensible conclusion. If the experiment had been conducted under more strict conditions, with more advanced instruments and with sufficient time, the conclusion would not have been different although the individual results might have been more accurate and the graph might have looked very slightly different.
Additional work, which could have been carried out, is to test a range of starch solution concentrations or using different enzymes such as protease with protein to see the effect temperature has on all enzymes. Temperatures around the optimum of each enzyme could have been tested going up in 10C to obtain immaculately accurate results that would show exactly where the optimum temperature of the enzyme lay. Different amylases could also have been tested to see what conditions they require in order to work efficiently.
Bibliography
Microsoft Encarta 95
Dorling Kindersley Encyclopaedia
Biology –A Modern Introduction (GCSE Education) By B.S.Beckett
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