Leaving Certificate Biology Higher Level Chapter 9: Enzymes Leaving Certificate Biology Higher Level

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

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

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

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

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

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

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 body’s cells produce heat shock proteins which protect the folded shape of important enzymes]

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

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

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 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

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

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:

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

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.

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.

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).

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.