metabolism
is the sum of all the chemical reactions in an organism
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
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
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
Induced Fit Model
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
optimum activity
refers to the conditions under which an enzyme works best
Optimum activity of an enzyme
Generally, most human enzymes work best at 37 ÂşC 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 ÂşC depending on their natural habitat
denaturation
involves a permanent change in the shape of an enzyme so that it does not act on its substrate
heat denaturation of enzymes
Enzymes can become denatured at high temperatures. For example human enzymes will begin to denature at around 40 ÂşC. During infections the temperature of the human body can reach 42 ÂşC [the body's cells produce heat shock proteins which protect the folded shape of important enzymes]
bioprocessing
is the use of micro-organisms, or their components, such as enzymes to make useful products. 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
Bioprocessing with enzymes
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
immobilised enzymes
are enzymes that are attached to or trapped in an inert insoluble material
Bioprocessing with immobilised enzymes
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
Carrier-binding method
Physical adsorption
Ionic binding
Covalent binding
Cross-linking method
Entrapment method
uses of immobilised enzymes
â—ľImmobilised lactase breaks down lactose in milk for lactose-intolerant people â—ľImmobilised rennin is used in the cheese-making process â—ľImmobilised glucose isomerase is used in sweet manufacture as fructose is sweeter than glucose
Advantages of immobilised enzymes
â—ľImmobilised enzymes are more stable â—ľEfficiency of the enzyme is unaffected â—ľEasy recovery of product and enzyme at end of reaction â—ľImmobilised 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
â—ľA fixed amount of substrate is placed in bioreactor â—ľReaction is allowed to proceed â—ľProduct is collected at end of reaction â—ľBioreactor is then cleaned out for the next batch
continuous-flow culture
â—ľSubstrate is continually put into the bioreactor â—ľReaction proceeds and conditions within are strictly controlled using sensors â—ľProduct is continually collected
Mandatory Experiment: to investigate effect of pH on enzyme action
â—ľThree graduated cylinders with celery (catalase enzyme), pH buffer (4, 7, 13) and 1 drop washing-up liquid set up in 25ËšC water bath. â—ľHydrogen peroxide added to all three. â—ľVolumes in graduated cylinders noted at 0 min, 1 min and 2 min. â—ľRate of enzyme action calculated by 2 min volume minus 1 min volume. â—ľResult: pH 7 graduated cylinder showed the most enzyme action.
Mandatory Experiment: to investigate effect of temperature on enzyme action.
â—ľThree graduated cylinders with celery (catalase enzyme), pH buffer 7 and 1 drop washing-up liquid set up in three separate water baths of 0ËšC, 25ËšC, 80ËšC. â—ľHydrogen peroxide added to all three. â—ľVolumes in graduated cylinders noted at 0 min, 1 min and 2 min. â—ľRate of enzyme action calculated by 2 min volume minus 1 min volume. â—ľResult: 25ËšC graduated cylinder showed the most enzyme action.
Mandatory Experiment: to investigate effect of heat denaturation on enzyme action.
â—ľTwo graduated cylinders with celery (catalase enzyme), pH buffer 7 and 1 drop washing-up liquid set up in three separate water baths of 25ËšC and 100ËšC and allowed to reach temperature. â—ľHydrogen peroxide added to both. â—ľVolumes in graduated cylinders noted at 0 min, 1 min and 2 min. â—ľRate of enzyme action calculated by 2 min volume minus 1 min volume. â—ľResult: 25ËšC graduated cylinder showed enzyme action and 100ËšC graduated cylinder showed no enzyme action (denaturation).
Mandatory Experiment: to immobilise an enzyme and examine its application.
â—ľYeast cells are immobilised using sodium alginate. â—ľYeast suspension is mixed with sodium alginate solution and dropped into calcium chloride to solidify. â—ľBeads of alginate beads are washed three times and placed in a dropping funnel. â—ľA sucrose solution is placed into funnel and immobilised yeast allowed to act on sucrose. â—ľProduct is released by opening tap. â—ľProduct is tested for reducing sugar (glucose and fructose) using clinistix or glucose test strips. â—ľResult: Immobilised yeast converted the sucrose to glucose + fructose without contaminating the product.