How does the concentration of enzymes affect the breakdown of starch by a-amylase in biological washing powders?

Authors Avatar

04/27/07                Exam Number: 9172

How does the concentration of enzymes affect the breakdown of starch by α-amylase in biological washing powders?

        In the cleaning business, it’s important to get a maximum cleaning effect at a minimum cost. This is especially applicable to the washing of clothes (both commercially, before an item of clothing goes on the market, or at home). This means trying to wash clothes at the lowest possible temperature, to keep the amount of electricity used at a minimum, yet trying to make and maintain a low-priced washing product that cleans effectively.

        This is why many washing powders use enzymes: enzymes are biological catalysts that speed up the breakdown of certain substances (in this case the molecules in food stain). Enzymes have a certain optimum temperature (a temperature at which the enzymes function at its best). Optimum temperatures are different for every enzyme, but they tend to be around 45°C. This means that if enzymes are to be used in washing powders, the temperature at which the clothes are washed will have to be at the optimum temperature, in order to achieve maximum enzymatic effect.

This optimum temperature, in the case of the enzymes concerned (the enzymes that break down protein, fats and starch in food stains on clothes), is lower than the normal washing temperature of clothes, 60°C, which means the use of enzymes in washing powders will reduce the washing temperature, thereby making the wash more cost effective. The enzymes break down the food stain deposits much better and much more efficiently than a hot wash with normal soap. Over a longer period of time, using normal hot washings to wash the clothes will wear down the material the clothes are made of, and damage them, making them visibly less attractive. Enzymes on the other hand, do not damage the material. This means that the clothes themselves will be cleaner, less damaged, and washed at lower temperature (not as money spent on electricity), which has a very positive result on the use of enzymes in washing powders. This is the main reason why enzymes are used in washing powders.

        The enzyme I will be focusing on is α-amylase, which is an enzyme present in biological washing powders that breaks down starch deposits in food stains into maltose, which is then further broken down into glucose sub-units. I however, am only looking at the initial breakdown of the starch by α-amylase into maltose.

        Aim:

The aim of my investigation is to investigate the effect that changing enzyme concentration has on the breakdown of starch (i.e. rate of breakdown) by α-amylase. Does concentration affect the rate at which α-amylase breaks down starch? If so, what effect would this have on the concentration of α-amylase used in washing powders? What percentage concentration is most suitable for the breakdown of starch, to be used in washing powders to clean clothes? Hopefully the following investigation will help me in finding the answers to these questions.

        

Hypothesis:

As I increase the concentration solutions of the enzymes, the rate at which the enzyme breaks down the starch in the agar plate will increase (i.e. the rate of breakdown will increase with an increase of enzyme concentration solutions). The investigation below will help to determine this, or to disprove this prediction.

Enzymes are biological catalysts. They differ to inorganic catalysts in that they are specific in that they catalyse if not a few only one specific breakdown/reaction. Enzymes are large globular proteins. This means that they will have a very specific tertiary structure. This also means that the active site of an enzyme (the area on an enzyme where the reaction occurs) will have a very specified structure. This means that the substrate molecule will have to be of that specific shape to be able to fit into the active site of the enzyme. This is why enzymes are seen as so specific; there are usually only one substrate (or very few substrate) molecules that have this specific shape. This also helps to explain why enzymes are so susceptible to factors such as heat: a slight alteration of their overall tertiary shape means a slight change to the shape of the active site. Substrate molecules will therefore not be able to fit into the active site as well, which means the enzyme will not be able to catalyse as efficiently in comparison to their activity at the optimum temperature, which means there is a decrease in the rate of breakdown. When an enzyme’s tertiary shape has been so heavily altered that the substrate molecule will not fit into the active site at all, the enzyme molecule is seen to be ‘denatured’.

Enzymes are thought to work on the idea of a ‘lock-and-key’ mechanism. The idea behind this mechanism is that the substrate molecule fits into the rigid shape of the active site of the enzyme. This helps to explain the specificity of enzymes: a slight alteration in the shape of the active site means a mismatch in the shape of the substrate and the active site. However, this idea is rather illogical, as the idea depends on the random movement of substrate molecules into the active site of the enzyme. This analogy seems to be rather vague for a molecule so important as enzymes. A better theory to explain for the activity of enzymes is what is known as the ‘induced fit’ mechanism. In this mechanism, when a substrate molecule comes into close proximity with an enzyme molecule, the enzyme molecule enfolds the substrate and changes its shape accordingly, so that the enzyme takes up its most effective shape once the substrate has bonded with the active site of the enzyme. Just as the shape of a plastic bag is affected by what is held inside it, the shape of an enzyme is affected by the substrate that it bonds with.

Enzyme solutions are a mixture of enzyme and other substances (in this case, it is enzyme and distilled water). Due to this mixture, a ratio is created of enzyme-water molecules. In a pure sample of enzyme, no water is present, and therefore only enzyme molecules are present. If water is added, however, there will be a certain number of water molecules for every enzyme molecule. As this ratio increases, the concentration of the enzyme solution decreases, as there are more and more water molecules for each enzyme molecule. Increasing the concentration of enzyme means increasing the number of enzyme molecules in the solution. This has the same type of effect upon considering the addition of substrate into the solution. Taking for example a pure enzyme solution, adding a substrate into this solution means the ratio between the enzyme molecules and the substrate molecules is fairly small, which means there are a small number of substrate molecules for each enzyme molecule. This means that there is a quick breakdown of substrate, as each enzyme molecule has less substrate to break down. However, decreasing the concentration of the enzyme solution means decreasing the number of enzyme molecules present, which means the ratio between the number of enzyme and substrate molecules gets bigger and bigger. This means that there are an increasing number of substrate molecules for each enzyme molecule, which implies that each enzyme molecule gets an increased workload, and will take longer to break down the substrate molecules. This suggests that if the enzyme solution concentration decreases, the period of time needed to break down the substrate increases (i.e. gets longer).

        Chosen techniques:

At the beginning of this experiment, there were a number of different ways I could go about conducting the experiment.

One way of doing this would be mixing enzyme solutions to starch suspension in test tubes and adding the iodine solution afterwards to test for the breakdown of starch. In a number of ways, this is a good method of conducting the method. As the experiment is done in test tubes, the test tubes could then be placed in a water bath, in order to keep the solutions at a constant 37°C, which is the optimum temperature. This would mean the enzyme would be working at its most efficient point, which would be very applicable to the idea of using enzymes in washing powder (37°C being close to the normal washing temperature using biological washing powders). In a number of ways this is also a bad way of conducting the experiment. It would take a great deal of time to set all of the equipment up (i.e. test tubes, water baths). Once the equipment has been set up, the actual obtaining of the results would be quite time consuming as well. Firstly, only one concentration can be done at one time. This means in order to test all of the enzyme concentration solutions and to then repeat them twice to get a reliable set of result would take an extremely large period of time. This method would also not very realistic; comparing the time needed to conduct the experiment this way to the time given to conduct the experiment. Secondly, once testing for the breakdown of starch, what would be done is the iodine solution would be added into the test tubes, to show how far the starch has been broken down. The results obtained from this, however, are only observations by the human eye. There is nothing there that can actually be measured using equipment of any sort. This means there is a lack of solid evidence to prove or disprove my hypothesis.

        A second way of conducting this experiment would be to use starch agar plates, and to make wells in the agar plates using a cork bored in which the enzyme concentrations are put. The agar plates would then be put in an incubator/oven, and left for a certain period of time. When taken out of the incubator/oven, iodine solution would be added to the starch agar to show how far around the wells the α-amylase has broken down the starch, which would be measure using Vernier calipers. In some ways this would be a very good method to conduct the experiment. Firstly, many different concentrations of enzyme solution can be tested at once, as more than one well can be bored in one agar plate (the enzyme will not react so far as that one concentration will take up one agar plate). This means a fairly small number of agar plates will be need to conduct the experiment, let alone do two repeats of the experiment. This means that a lot less time will be spent on doing the experiment. As the plates are left in an incubator/oven for a certain period of time, most of the time will be spent preparing the plates to go into the incubator/oven and obtaining results (which is comparatively still a very small period of time, if compared to the first method). Secondly, upon obtaining the results, when adding the iodine solution, a circle of faded colour will be seen where the starch has been broken down. This circle can be measured in diameter, and this measurement can be used as values for the rate of breakdown. This means there is solid concrete evidence on which I can base my analysis and with which I can prove or disprove my hypothesis. In some ways, however, this would be a bad method for conducting the experiment. The optimum temperature of α-amylase is 37°C, yet the highest temperature at which a successful experiment can be carried out on the agar plates at is 26°C. This means the temperature at which the experiment is carried out is a considerably lower temperature than the optimum temperature of the enzyme. This means that in order to obtain the same kind of results when conducting the experiment at the optimum temperature, the agar plates will need to be left in the incubator/oven for a much longer period of time.

        Comparing the two methods, I feel that the second method would be a much better option for conducting this experiment, as the results obtained are much more concrete and accurate than the results obtained in the first method. Taking time into account, the second method would be a much more logical option, as this would leave me more time in the emergency if something did happen to go wrong during the investigation, and the investigation would have to be repeated all over again. The second method would also be much easier to repeat.

        

Apparatus:

  • Termamyl® 60T α-amylase granules
  • Distilled water (used to make concentration solutions)
  • Beaker (250cm³)
  • Measuring cylinder, 50cm³ (accurate to 1cm³, ±0.5cm³)
  • Balance (accurate to 2d.p.)
  • Starch agar plates, pre-prepared (filled to a depth of 6mm, using 1% starch agar)
  • Cork borer, ∅ 5mm
  • Vernier calipers (accurate to 0.1mm, ±0.05mm error)
  • Stirring rod
  • Iodine solution of concentration 0.01M (used to test for rate of breakdown of starch)
  • Pipette

Brief method:

        Concentration solutions at concentrations shown in ‘Variables’ section below will be made up. On one agar plate, three equidistantly placed holes of ∅5mm will be bored into the starch agar plates, using a cork borer. Two starch agar plates will be produced like this, to provide enough holes for the concentration solutions. Using a pipette, drop 4/5 drops of each concentration solution into the wells. The agar plates are then placed in an incubator/oven at 26°C for a period of 24 hrs. When the plates are taken out again, the plates are flooded with iodine solution, to show where the enzyme has broken the starch agar down. Iodine solution in the presence of starch is blue/black; iodine solution itself is orange. This means where the enzyme has broken the starch down, there will be a circle of orange inside a circle of blue/black. The diameter of this circle is measured using Vernier calipers, and written down on a results table. The experiment is repeated twice.

Constants:

The volume of distilled water used to make up the solutions for the enzyme concentrations will be kept the same throughout the whole experiment: 100cm³. If this volume of water is not kept the same throughout the experiment, there will be an imbalance between the concentrations. Some concentrations will be more dilute than necessary, as more water is being used, and some concentrations will be more concentrated, which would result in an unfair testing of the concentrations, and the results obtained would be false. The volume of distilled water used to make up the concentration solutions is therefore kept a constant.

The depth at which the agar plates are filled will be kept the same: 6mm. If the agar plates are not filled to an equal depth, some agar plates will contain a larger volume of starch agar that others. This means that comparing one concentration on two different agar plates, on one agar plate the enzyme will have much more starch agar to break down than on the other plate. This means the two results obtained for the same concentration of enzyme will be very varied, making the results false and unusable. The depth to which the agar plates are filled with starch agar is therefore kept a constant.

Join now!

The same cork borer will be used to bore all the wells in the agar plates in this investigation, to ensure the holes are the same size. If the wells bored with the cork borer to store the enzyme concentration solutions are different sizes, different surface areas over which the enzyme reacts are provided, which means the test will not be a fair test, leaving the results obtained false. The diameter of the wells is therefore kept a constant.

The temperature at which the starch agar plates are stored will be kept at a constant. This will be done by ...

This is a preview of the whole essay

Here's what a teacher thought of this essay

Avatar

**** A very thoroughly planned and documented report which shows the student has considered the way in which the variables are controlled and sources of error in depth. The project could be improved by giving more thought to the way the data was analysed. Research and rationale The rationale for the investigation is clearly justified in terms of its scope and appropriate biological principles are discussed. Additional sources were selected and used effectively. Planning There is evidence of thought and ingenuity in the deign of the experiment. A testable hypothesis was formulated and biological knowledge used to explain the prediction. A quantitative prediction would gain more marks. The important variables to be considered were described but not all were controlled. The apparatus was listed and justified. Safety has been considered but the recommendations could have been presented in a more concise way. Implementing It appears the apparatus was used competently in an organized manner. The data was recorded well but the organization of the tables could be improved. Analysis and Evaluation Graphs were not available to view but summary tables were presented and the results discussed at length. Results were interpreted using biological principles. It would have been better to discuss the summary of the overall findings rather than each individual set of results which did not help to make the key findings clear. Statistical tests were not included. The anomalies were discussed at length and some possible causes of errors evaluated. Communicating The layout of the report was acceptable although the additions of more subheadings would have made the organization a little better. Data could have been recorded more effectively to aid the comparison of the figures. Spelling, punctuation and grammar are acceptable with scientific terms used correctly throughout.