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.
Ribbon diagram of the structure of amylase

Chapter 9: Enzymes notes page

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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
The lock and key model of enzyme action

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 and the enzyme returns to its original shape
The induced fit model of enzyme action

Factors affecting enzyme function

pH and temperature are two important environmental conditions that affect the rate at which enzymes act. All enzymes have an optimum pH and optimum temperature at which their activity is a maximum.

Optimum Activity of an Enzyme

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

pH

  • pH refers to the level of acidity or basicity of a liquid/solution.
  • Enzymes are affected by pH because acids and bases can affect the shape of an enzyme.
  • Most enzymes have a pH at which they work best, called the optimum pH.
  • The optimum pH for most enzymes present in animals is pH 7.4.
  • Some animal enzymes have an optimum pH of 2 (e.g. pepsin in the stomach).
Effect of pH on enzyme activity

Temperature

  • Environmental temperatures affect enzymes because very warm or hot temperatures can cause the enzyme to change shape.
  • Cooler temperatures tend not to change the shape of enzymes – they just slow their activity.
  • All enzymes have an optimum temperature – at which they work best.
  • Most human enzymes work best at a temperature of 37 ºC.
  • Plant enzymes work best between the temperatures of 10 – 30 ºC, depending on their natural habitat.
Effect of temperature on enzyme activity

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 carried out in bioreactors

  • Bioprocessing is the use of micro-organisms, or their components, such as enzymes to make useful products.
  • 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.

Examples of bioprocessing:
– 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 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.
Structure of a bioreactor

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: when the enzyme is attached to water-insoluble substances such as cellulose or agarose
  2. Cross-linking method: when enzymes are attached together covalently using glutaraldehyde
  3. Entrapment method: when the enzyme is trapped in a gel such as alginate
Yeast cells trapped in alginate

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):

  • 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 and microorganisms are allowed to proceed through some or all of the stages of the microorganism growth curve (lag, log, stationary, decline phases)
  • Product is collected at end of the reaction/process
  • Bioreactor is then cleaned out for the next batch

Continuous-flow Culture

  • Substrate is continually put into the bioreactor
  • Reaction and microorganisms are maintained in the log phase of growth
  • Product is continually collected

Mandatory Experiment: to investigate effect of pH on enzyme action.

Equipment:

  • Lab coat
  • Safety goggles
  • Graduated cylinders
  • Celery
  • Knife
  • pH buffers 4, 7 and 13
  • Washing up liquid
  • Stopwatch
  • Droppers
  • Waterbath

Method:

  • 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 cylinders at the same time.
  • Volumes in graduated cylinders noted at 0 min, 1 min, 2 min and 3 min, if needed.
  • Rate of enzyme action calculated by a simple subtraction of the difference in volume during one of the selected minutes.
Investigating the effect of pH on the rate of enzyme action

Result:

  • pH 7 graduated cylinder showed the most enzyme action (greatest amount of bubbles/foam produced).

Conclusion:

  • pH 7 is the optimum pH for catalase.

Mandatory Experiment: to investigate effect of temperature on enzyme action.

Equipment:

  • Lab coat
  • Safety goggles
  • Graduated cylinders
  • Celery
  • Knife
  • pH buffer 7
  • Washing up liquid
  • Stopwatch
  • Droppers
  • Waterbaths (0 ˚C, 25 ˚C and 80 ˚C)

Method:

  • 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 (sitting in ice), 25˚C, and 80˚C.
  • Hydrogen peroxide added to all three cylinders at the same time.
  • Volumes in graduated cylinders noted at 0 min, 1 min, 2 min and 3 min, if needed.
  • Rate of enzyme action calculated by a simple subtraction of the difference in volume during one of the selected minutes.
Investigating the effect of temperature on the rate of enzyme action

Result:

  • 25 ˚C graduated cylinder showed the most enzyme action (greatest amount of bubbles/foam produced).

Conclusion:

  • 25 ˚C is the optimum temperature for catalase.

Mandatory Experiment: to investigate effect of heat denaturation on enzyme action.

Equipment:

  • Lab coat
  • Safety goggles
  • Graduated cylinders
  • Celery
  • Knife
  • pH buffer 7
  • Washing up liquid
  • Stopwatch
  • Droppers
  • Waterbaths (0 ˚C and 100 ˚C)

Method:

  • Two graduated cylinders with celery (catalase enzyme), pH buffer 7 and 1 drop washing-up liquid set up in two separate water baths of 25 ˚C and 100 ˚C
  • Contents of each graduated cylinder were allowed to reach required temperature (approximately 15 minutes).
  • Hydrogen peroxide added to both cylinders at the same time.
  • Volumes in graduated cylinders noted at 0 min, 1 min, 2 min and 3 min, if needed.
  • Rate of enzyme action calculated by a simple subtraction of the difference in volume during one of the selected minutes.
Investigating the effect of heat denaturation on the rate of enzyme action

Result:

  • 25 ˚C graduated cylinder showed enzyme action (by seeing formation of bubbles/foam) and the 100 ˚C graduated cylinder showed no enzyme action (no bubbles/foam produced).

Conclusion:

  • The enzyme in the 100 ˚C graduated cylinder had been denatured by the hot temperature.

Mandatory Experiment: to immobilise an enzyme and examine its application.

Equipment:

  • Lab coat
  • Safety goggles
  • Dried Yeast
  • Sodium alginate
  • Calcium chloride
  • Beakers
  • Dropping funnel
  • Syringe
  • Tea strainer
  • Sucrose
  • Clinistix strips or Benedict’s solution

Method:

  • Yeast cells and sodium alginate are dissolved in water separately and then mixed
  • The mixture is dropped slowly (drop by drop) using a syringe into a solution of calcium chloride (to create and solidify the beads)
  • Beads of Yeast-alginate are washed three times using water and a tea strainer and placed in a dropping funnel.
  • A sucrose solution is placed into funnel and the immobilised yeast is allowed to act on sucrose for a few minutes.
  • Product is released by opening tap of the dropping funnel.
  • Product is tested for reducing sugar (glucose and fructose) using clinistix or glucose test strips or Benedict’s solution (+heat).

Result:

  • The product was clear – no yeast was present in the product.
  • The product tested positive for reducing sugar.

Conclusion:

  • Immobilised yeast converted the sucrose to glucose + fructose without contaminating the product.
Preparing the immobilised yeast beads
Washing the immobilised yeast beads
Examining the application of immobilised yeast