Chapter 9: Enzymes

1
Chapter 9 Enzymes

• Leaving Certificate Biology
• Higher Level

2
Cell Metabolism

• Metabolism is the sum of all the chemical
  reactions occurring in a living organism
• Organisms ultimately get their energy from the
  sun via photosynthesis
• Plants ? Animals ? Humans
• ATP and enzymes drive metabolism

3
Enzymes

• Enzymes are folded proteins, that are globular in
  shape, and catalyse biochemical reactions without
  being used up in the reaction
• Due to the protein nature and globular, folded
  shape of enzymes they are affected by two
  important environmental factors
• pH
• Temperature

4
Enzyme Function

• Active Site Theory
• All enzymes have an active site where the enzyme
  combines with its specific substrate
• When the substrate enters the active site of the
  enzyme the enzyme changes shape very slightly to
  accommodate the substrate better this is called
  the induced fit model of enzyme action

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Induced Fit Model

• Substrate enters active site
• The enzyme changes its shape slightly to accept
  substrate
• An enzyme-substrate complex is formed
• Substrate is changed into product(s)
• Product(s) exit the active site upon which point
  the enzyme returns to its original shape

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Induced Fit Model
Products
1.
4.
Substrate
Enzyme
Enzyme
2.
3.
Substrate
Enzyme shape changes slightly
Enzyme-substrate complex
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Optimal Activity of an Enzyme

• Each enzyme has conditions under which it works
  best
• Generally, most human enzymes work best at 37 ºC
  and pH 7
• Exceptions would be enzymes in the stomach that
  work at a pH of 1 2
• Plant enzymes work best between the temperatures
  of 10 30 ºC depending on their natural habitat

8
Heat Denaturation of Enzymes

• Enzymes become denatured at extreme temperatures
• Denaturation involves a permanent change in the
  shape of an enzyme so that it does not act on its
  substrate
• For example human enzymes will begin to denature
  at around 40 ºC
• During infections the temperature of the human
  body can reach 42 ºC the bodys cells produce
  heat shock proteins which protect the folded
  shape of important enzymes

9
Bioprocessing

• Bioprocessing is the use of living cells or their
  components, such as enzymes to make useful
  products
• Examples
• Production of beer using yeast
• Production of insulin using genetically-modified
  E coli bacteria
• Production of cheese using the enzyme rennin
• Production of fructose from glucose using glucose
  isomerase

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Bioprocessing with Immobilised Enzymes

• Immobilised enzymes are enzymes that are attached
  to or trapped in an inert insoluble material
• Three ways in which enzymes are immobilised
• Carrier-binding method
• Physical adsorption
• Ionic binding
• Covalent binding
• Cross-linking method
• Entrapment method

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Uses of Immobilised Enzymes

• Immobilised lactase breaks down lactose in milk
  for lactose-intolerant people
• Immobilised rennin is used in the cheese-making
  process
• Immobilised glucose isomerase is used in sweet
  manufacture as fructose is sweeter than glucose

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Advantages of Immobilised Enzymes

• Immobilised enzymes have advantages over free
  enzyme (enzyme in solution)
• Immobilised enzyme is more stable
• Efficiency of the enzyme is unaffected
• Easy recovery of product and enzyme at end of
  reaction
• Immobilised enzymes can be reused many times
  reducing costs to manufacturers

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Bioprocessing with Immobilised Enzymes

• Bioprocessing is carried out in bioreactors
• A bioreactor is a vessel in which a product is
  formed by a cell or cell component, such as an
  enzyme
• Immobilised enzyme and substrate are placed in
  the bioreactor and the bioreactor is kept very
  carefully at the correct temperature and pH in
  order to achieve the maximum amount of product

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Bioprocessing

• Bioprocessing is carried out using one of two
  general procedures
• Batch culture
• Continuous-flow culture
• A fixed amount of substrate is placed in
  bioreactor
• Reaction is allowed to proceed
• Product is collected at end of reaction
• Bioreactor is then cleaned out for the next batch
• Continuous-flow culture

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Batch Culture

• A fixed amount of substrate is placed in
  bioreactor
• Reaction is allowed to proceed
• Product is collected at end of reaction
• Bioreactor is then cleaned out for the next batch

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Continuous-flow Culture

• Substrate is continually infused into the
  bioreactor
• Reaction proceeds and conditions within are
  strictly controlled using sensors
• Product is continually collected

17
Experiment to investigate effect of pH on enzyme
action

• Three graduated cylinders with celery (catalase
  enzyme), pH buffer (4, 7, 13) and 1 drop
  washing-up liquid set up in 25C water bath.
• Hydrogen peroxide added to all three.
• Volumes in graduated cylinders noted at 0 min, 1
  min and 2 min.
• Rate of enzyme action calculated by 2 min volume
  minus 1 min volume.
• Result pH 7 graduated cylinder showed the most
  enzyme action.

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Experiment to investigate effect of temperature
on enzyme action

• Three graduated cylinders with celery (catalase
  enzyme), pH buffer 7 and 1 drop washing-up liquid
  set up in three separate water baths of 0C,
  25C, 80C.
• Hydrogen peroxide added to all three.
• Volumes in graduated cylinders noted at 0 min, 1
  min and 2 min.
• Rate of enzyme action calculated by 2 min volume
  minus 1 min volume.
• Result 25C graduated cylinder showed the most
  enzyme action.

19
Experiment to investigate effect of heat
denaturation on enzyme action

• Two graduated cylinders with celery (catalase
  enzyme), pH buffer 7 and 1 drop washing-up liquid
  set up in three separate water baths of 25C and
  100C and allowed to reach temperature.
• Hydrogen peroxide added to both.
• Volumes in graduated cylinders noted at 0 min, 1
  min and 2 min.
• Rate of enzyme action calculated by 2 min volume
  minus 1 min volume.
• Result 25C graduated cylinder showed enzyme
  action and 100C graduated cylinder showed no
  enzyme action (denaturation).

20
Experiment to immobilise an enzyme and examine
its application

• Yeast cells are immobilised using sodium
  alginate.
• Yeast suspension is mixed with sodium alginate
  solution and dropped into calcium chloride to
  solidify.
• Beads of alginate beads are washed three times
  and placed in a dropping funnel.
• A sucrose solution is placed into funnel and
  immobilised yeast allowed to act on sucrose.
• Product is released by opening tap.
• Product is tested for reducing sugar (glucose and
  fructose) using clinistix or glucose test strips.
• Result Immobilised yeast converted the sucrose
  to glucose fructose without contaminating the
  product.