Following the Progress of an Enzyme Controlled Reaction

Enzymes are a widely used source of biological catalyst; they are used in widely in industry as in the biological aspects. Enzymes are biological catalyst; this means that they will speed up a reaction with out becoming used up. The enzymes for this by not actually interfering with the reaction its self but basically align the two substrates on the active site of the enzyme.

Amylases are widely spread enzymes that hydrolyse starch to maltose. They are often found in two forms, a-amylase, which degrades starch molecules into, fragments 10 glucose residues long and b-amylase, which breaks down these into maltose, made up of two glucose molecules. Both work by hydrolysis adding one molecule of water across glycosidic link.


My hypothesis is that as the time of the enzyme reaction goes on the amount of substrate reacted by time goes in a proportional relationship.

In theory time Vs substrate concentration should have a proportional relationship as the relative enzymes will all have plenty of the substrate molecules to align with, therefore creating a constant time for the reaction. The substrate being used is starch, starch is widely found inn various substances. It is greatly found in bread, the starch is a very useful nutrient for human life forms as it is the source for sugars, which produce energy for the human body.

Starch begins to be broken down as soon as it enters the human body, first salivary Amylase produced in the mouth and bucal cavity. In this part of the digestion the starch is broken down to Maltose by the Amylase, the reaction of this is increase by the Amylase and by the chewing mechanism. From this the maltose is in the form of a disaccharide, from here it is then broken down by Maltase to form a monosaccharide, Glucose. The Amylase is most efficient at an optimum rate at pH 7 in the mouth this is neutral.

Amylase, any member of a class of enzymes that catalyze the hydrolysis (splitting of a compound by addition of a water molecule) of starch into smaller carbohydrate molecules such as maltose (a molecule composed of two glucose molecules). Two categories of Amylases, denoted alpha and beta, differ in the way they attack the bonds of the starch molecules.

Alpha-Amylase is widespread among living organisms. In the digestive systems of humans and many other mammals, an alpha-Amylase called ptyalin is produced by the salivary glands, whereas the pancreas into the small intestine secretes pancreatic Amylase.

Ptyalin is mixed with food in the mouth, where it acts upon starches. Although the food remains in the mouth for only a short time, the action of ptyalin continues for up to several hours in the stomach–until the food is mixed with the stomach secretions, the high acidity of which inactivates ptyalin. Ptyalin’s digestive action depends upon how much acid is in the stomach, how rapidly the stomach contents empty, and how thoroughly the food has mixed with the acid. Under optimal conditions as much as 30 to 40 percent of ingested starches can be broken down to maltose by ptyalin during digestion in the stomach.

When food passes to the small intestine, the remainder of the starch molecules is catalyzed mainly to maltose by pancreatic Amylase. This step in starch digestion occurs in the first section of the small intestine (the duodenum), the region into which the pancreatic juices empty. Other enzymes into molecules of glucose, which are rapidly absorbed through the intestinal wall, ultimately break down the by-products of Amylase hydrolysis.

Beta-Amylases are present in yeasts, molds, bacteria, and plants, particularly in the seeds. They are the principal components of a mixture called diastase that is used in the removal of starchy sizing agents from textiles and in the conversion of cereal grains to fermentable sugars.

Enzymes are also change very much to their conditions they are in and are very sensitive to change. For example temperature will change the rate at which the enzymes work at as it increase the rate at which they work and the rate of motion that they incur. The general optimum temperature at which enzyme are most suited to is at around 40-50 degrees c. However this varies from different enzymes and this optimum temperature bay fluctuate or is reduced. If the temperature conditions increase to a certain extent the enzyme will become denatured and will there fore be inactive and useless.

Enzymes also vary their rate of reaction when it comes to the concentration of the enzyme its self or the substrate around it. For example is the substrate concentration is increased the rate of reaction will increase to an extant however if to much substrate present it will slowly reduce the rate of reaction as there will be to many substrate molecules to the amount of enzymes. Other conditions that effect the rate at which a reaction is complete by the influence of an enzyme is the concentration of the enzyme and pH.

Outline Method

Prepare one boiling tube with the reacting substrate and enzyme in (starch and Amylase). Makes sure the content in this is kept mixed so that the reaction will not stop due to the factors of slowing the experiment down, such as surface area. Make sure your 10 test tubes are at hand and then withdraw 1ml from the reacting solution. Put this into one of the 10 test tubes and add the dilute iodine to the solution. Shake the solution to ensure that it is fully mixed, this maybe done by inverting the apparatus. Now with draw 1mls of the solution and put into the tube for the colorimeter test.

This will then give you a reading of how much starch has been broken down by the Amylase; this will be in light transmission. The higher the light transmission the less starch present and the reverse, I will not be repeating the experiment individually though, however there are several groups taking part in the experiment and therefore I will be able to find the averages of the groups. This will enable me to highlight any anomalies and errors as I can compare the different groups data as well as my own.

Key Variables

The volumes with drawn from the reacting boiling tube must be the same for all 10 of the test tubes.

The same colorimeter has to be used through out the experiment as well as the same blank. Also to check the colorimeter does not vary its starting point, therefore the transmission should be checked that it is at 0 at the start of each reading.

The time intervals at which samples from the test tubes are taken have to be at relative intervals to enable the results to be easily conveyed whilst analyzing.

The temperature of the test tubes which the enzymes lay in has to be kept at a constant temperature, in this case room temperature. This has to be kept the same as a rise or decrease in temperature will affect the temperature greatly.

All measurements are taken at the same part of the meniscus, for example if you have measured from the bottom (the incorrect place) carry on doing so until the end of the experiment.

Keeping these various variables the same will hopefully make the experiment accurate and fair. If these variables are broken it should be highlighted in the results and there fore can be stated as an anomaly.

To ensure that these variables are met there must be a simple case of careful measurement of the variables above. These must all be kept to the specific volumes and concentrations, which will be stated, in the detailed method to follow. It is very important that these variables are kept as the accuracy of the experiment depends on these features also to keep a fair test. I will be making a control for the experiment; this will be in the form of a blank. The blank compensates the colorimeter for the colour which is naturally present in the iodine. It will now register changes in opacity relative to the blank reference solution. The colorimeter shall have a red slide used, as this is the colour at the other end of the spectrum to the colour of iodine.

The measurements in the experiment that have to be kept the same are mainly the amount of the dilute iodine solution added to the timed interval extractions from the reacting mixture. Also the amount of the reacting solution withdrawn from the reacting boiling tube has to be kept the same for each of the 10 test tubes at the various timed intervals.

The colorimeter should give the reading of 100% transmission; this means that it is 0% absorbance. The change in reaction stage will be picked up by the change in light transmission, as the starch broken down by the Amylase the more light transmission will be read of the colorimeter. This will be the opposite at the start of the experiment as in the first few minutes I don’t think there will be must starch broken down. There fore the light transmission will be very low. In comparison with the blank (which has 100% light transmission) it will be very different until the later stages.

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