Chapter 9: Enzymes

Chapter 9: Enzymes

  • Metabolism is the sum of all the chemical reactions in an organism

All energy in living organisms all came from the Sun.

  • Enzymes are folded, globular-shaped protein catalysts that speed up reactions without being used up.

Examples of catabolic enzymes:
– Pepsin – digests proteins into peptides
– Amylase – digests starch into maltose
– Lipase – digests fats into fatty acids and glycerol

Examples of anabolic enzymes:
– Potato phosphorylase – makes starch from glucose
– DNA polymerase – makes DNA from its building blocks (nucleotides)

Active site theory of enzyme action
All enzymes have an active site where the enzyme combines with its specific substrate.

Definitions:

  • Active site: area of the enzyme where substrate enters and is changed into product(s).
  • Specificity: refers to the enzyme’s ability to react with only one substrate.
  • Substrate: substance upon which the enzyme acts.
  • Product: substance that results from the action of an enzyme.

Active site theory involves two models of enzyme action:

  1. Lock and key model
  2. Induced fit model

Lock and Key Model

  1. Enzyme has a rigid shape.
  2. The substrate enters the active site of the enzyme and fits snugly, much like a key fits in a lock
  3. An enzyme-substrate complex is formed
  4. Substrate is changed into product(s)
  5. Product(s) exit the active site
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Induced Fit Model

  1. Substrate enters active site
  2. The enzyme changes its shape slightly to accept substrate
  3. An enzyme-substrate complex is formed
  4. Substrate is changed into product(s)
  5. Product(s) exit the active site upon which point the enzyme returns to its original shape
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Optimum Activity of an Enzyme

  • Optimum activity refers to the conditions under which an enzyme works best.

Generally, most human enzymes work best at 37 ºC and pH 7. An exceptions is pepsin, which acts on proteins in the stomach at a pH of 1 – 2.
Plant enzymes work best between the temperatures of 10 – 30 ºC depending on their natural habitat,

Heat Denaturation of Enzymes

  • Denaturation involves a permanent change in the shape of an enzyme so that it does not act on its substrate

Enzymes can become denatured at high temperatures. 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 body’s cells produce heat shock proteins which protect the folded shape of important enzymes].

Bioprocessing

  • Bioprocessing is the use of micro-organisms, 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

Bioprocessing is carried out in bioreactors

  • bioreactor is a vessel in which a product is formed by a cell or cell component, such as an enzyme.

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

Bioprocessing with Immobilised Enzymes

  • Immobilised enzymes are enzymes that are attached to or trapped in an inert insoluble material


Bioprocessing is carried out in bioreactors

  • 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

Three ways in which enzymes are immobilised:
1. Carrier-binding method
– Physical adsorption
– Ionic binding
– Covalent binding
2. Cross-linking method
3. Entrapment method

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


Advantages of Immobilised Enzymes
Immobilised enzymes have advantages over free enzyme (enzyme in solution):

  • Immobilised enzymes are 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


Bioprocessing is carried out using one of two general procedures:

  1. Batch culture
  2. Continuous-flow culture


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


Continuous-flow Culture

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

Mandatory 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 25˚C 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.

Mandatory 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 0˚C, 25˚C, 80˚C.
  • 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: 25˚C graduated cylinder showed the most enzyme action.

Mandatory 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 25˚C and 100˚C 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: 25˚C graduated cylinder showed enzyme action and 100˚C graduated cylinder showed no enzyme action (denaturation).

Mandatory 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.
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