Investigation - enzymes and food tests

• The enzyme used is AMYLASE to react with starch solution and by using iodine solution (changes from brown-orange to blue-black in the presence of starch), to observe the results.

  
• Amylase Activity

  If the enzyme does it’s job, the starch will turn into maltose and the iodine solution will be BROWN-ORANGE.

  • If the enzyme DENATURE, starch will remain and the iodine solution will be BLUE-BLACK.

  
• Amylase Activity


  A BUFFER solution is used to change the pH of the reaction
• buffer solution

  a solution that's used to control the pH, since pH are always changing.

  • a good buffer will keep it stable.
• Understanding the Results for amylase activity

  • The sooner the colour of the iodine solution stops turning BLUE-BLACK, the FASTER the rate of reaction, and the better the enzyme works.
  • The pH at which the iodine stops turning blue-black the FASTEST is the optimum pH.
  • If the iodine remained BLUE-BLACK at a pH, the amylase is denatured, (the starch hasn't been broken down).
  • Comparing rates will highlight the pH sensitivity of amylase.

  CONTROLLED VARIABLES: amylase concentration and temperature.

  
• Testing for Sugars: BENEDICTS solution

  • Grind up food
  • Place benedict's solution with food sample in a tube, into a 75°C water bath for 5 mins.
  • A colour change shows the presence of reducing sugars, with the colour going from BLUE to GREEN to YELLOW or BRICK-RED (depending on sugar concentration).

  
• Testing for Starch: IODINE SOLUTION

  • Place the food sample in a test tube and add iodine solution.
  • A colour change from BROWN-ORANGE to BLUE-BLACK signifies the presence of starch.

  
• Testing for Proteins: BIURET solution

  • Grind up food
  • Combine the food sample with Biuret solution in a test tube.
  • Gently shake; a change to PURPLE indicates protein. If no protein, the solution remains BLUE.

  
• Testing for Lipids: SUDAN III TEST:

  • Add the food sample and Sudan III stain solution to a test tube without filtering.
  • Gently shake the tube. Lipid presence will show as a separate bright red layer.

  
• Rate = 1000 / time
• rate = volume / time
• we repeat the amylase experiment with pH 6, 7, 8 to get a slight change, since if we used strong acids / alkalis, the amylase would've denatured.
• we repeat the amylase experiment with pH 6, 7, 8 to get a slight change, since if we used strong acids / alkalis, the amylase would've denatured.

  • amylase optimum pH is pH 7.
  • pH 6 - weak acid
  • pH 7 - neutral
  • pH 8 - weak alkali
• problems with amylase experiment



  We are only taking samples every 30s, so we only have an approximate time for the reaction to complete.

  • we could solve this by taking samples every 10s.
• problems with amylase experiment


  the colour change can be difficult to see, when the reaction has finished, the solution is to ask several people to look at the spotting tile and decide when the reaction has completed.

  
• The results are more precise, when the results are closer to the mean.
• increments


  An increase in quantity, by a factor of one unit.
• determine the rate of sugar production per min at 40sec using the picture.


  rate = 2.25 arbitrary units per minute

  
• explain how the structure of enzyme molecules is related to the effect of pH on the activity of amylase.


  enzymes are protein molecules, so it has a 3D structure and has an active site, which has a specific shape that will only match shape of substrate. Starch is substrate for amylase.

  • at pH values above / below the optimum, the shape of active site is changed, so substrate can no longer fit the active site, since it's denatured.

  so amylase can no longer digest starch and rate of digestion decreases.
• it would of taken 10 minutes until the iodine solution and amylase-starch solution mixture was yellow-brown.

  
• To get a more accurate time is to test the mixture with iodine solution every 30 seconds.

  
• Preparation for Food sample:

  • Start by breaking down the food using a PESTLE AND MORTAR.
  • Transfer the ground food into a BEAKER with distilled water.
  • STIR the mixture to dissolve some food particles.
  • FILTER the mixture to remove solid residues
  • This leaves the sample to use for the food tests.

  
• Emulsion test for lipids:

  • Get food sample and add ethanol.
  • Shake vigorously
  • Add distilled water
  • Lipid is present if there's a cloudy white colour.

  
• safety precautions when using benedict's solution:

  • wear goggles
  • use a water bath to heat the solution / mixture
  • wash spills from bench / skin
  • wash hands
  • wear gloves
• A account of the Effect of pH on Amylase:

  • Place a drop of iodine into each well of a spotting tile
  • Get solutions in tubes of:
  • starch
  • amylase
  • pH buffer
  • Place all test tubes in a 30.C water bath, and leave it, till its the right temperature
  • Combine the solutions into one test tube and mix with a stirring rod
  • Return the water bath to place the tube and start a stopwatch
  • Every 30s, place a drop of THE solution (in the water bath) in a well, which contains iodine
  • Do it till the iodine is orange - starch is not present
  • Repeat the experiment with other pH buffers: pH 6, 7 and 8

  
• Data can be shown as repeatable and reproducible because a experiment is repeated with the same equipment and got similar results.
• Describe how to investigate the effect of pH on the breakdown of starch by amylase:

  • Do a range of 3 pH values.
  • keep amount or concentration of starch and amylase the same.
  • keep temperature the same using water bath.
  • use iodine test to make observations
  • observe colour changes at different temperatures
  • do repeats at each pH
• The starch amylase solution does not become yellow-brown at 5 °C and at 80 °C because:

  • The amylase hasn't digested the starch, so the iodine still has starch present and won't turn yellow-brown.
  • At 5 °C, there's low kinetic energy, therefore there are fewer enzyme-substrate collisions.
  • At 80 °C, It's too hot for the enzyme and the bonds holding the enzyme together would be broken.
  • that the active site changed shape enough, that the enzyme won't be able to bind to the substrate and there's no breakdown.
  • The enzyme has been denatured.
• Explain how amylase breaks down starch using lock and key theory


  Amylase has an active site that binds perfectly to the shape of the starch molecule, like a key fits into a lock, as it's complementary to starch.

  • It breaks the bonds of starch, turning it into simpler sugars and the products are released.
  • The amylase enzyme is not changed.
• Control variables used in the investigation:


  • time before mixing starch and amylase solution
  • volume of:
  • amylase solution
  • iodine solution
  • mixture added to spotting tile

  
• The student leave the starch solution and amylase solution for 5 minutes before mixing them, to allow the solutions to reach the same temperature as the water.

  
• What conclusion can be made about the effect of temperature on amylase activity between 20 and 65?


  As the temperature increases, enzyme activity increases until 35 reached, after which activity decreases.