Enzymes are used in biotech for many different reasons. Examples? Proteases and lipases in biological washing powder Dig

2. How are Enzymes used in biotechnology? • Enzymes are used in biotech for many different reasons. Examples? • Proteases and lipases in biological washing powder • Digestive enzymes in cattle feed • Leather industry to prepare skins • Biofuel production • Fruit juice extraction using pectinase • Cheesemaking

3. Why are enzymes so useful? • They are very specific • They lower the temperature at which reactions take place

4. Manufacturing enzymes • To produce the enzymes: • Bacteria provided with carbon source – often waste product of agriculture such as left over maize – and nitrogen source – such as urea or ammonium salts. • Usually, batch culture. (why?) • The bacteria almost always aerobic so contents of the fermenter are well aerated. • Enzymes secreted in to medium or remain within the cells. • After fermentation, the culture is heated to kill the cells. Cells broken open if necessary and the enzymes will dissolve in the culture medium. • The medium is then concentrated and filtered, leaving the cell fragments behind and collecting the enzyme in solution. • Enzymes purified and packaged.

5. One of the useful things about enzymes is that they are unchanged by a reaction, so can be used again Eg – lactase is used to produce lactose free milk Add lactase If we are going to re-use the enzyme how do we get the enzyme back? Lactose in the milk is broken down = lactose free milk

6. Immobilised enzymes Enzymes that have been fixed to a surface and held separate from the reaction mix But they still work…..

7. Immobilised enzymes Enzymes that have been fixed to a surface and held separate from the reaction mix But they still work….. Milk poured through tube Alginate beads containing lactase What variable will effect the rate of break down of the lactose in the milk? Lactose free milk

8. Advantages and disadvantages of enzyme immobilization

9. How do you immobilize enzymes?

10. Methods – with support • Adsorption (carrier bound) – enzymes mixed with immobilising support (adsorbing material, porous carbon, glass beads, clays and resins ) and bind it due to hydrophobic interactions and ionic bonds. • Covalent bonding (cross linked) – enzymes bondedto the support medium by covalent bonds, these link the enzymes together and to an insoluble material (e.g. clay particles) using cross linking agent like gluteraldehyde or sepharose.

11. Methods – without support • Entrapment (inclusion) – enzymes trapped e.g in gel bead or network of cellulose fibres. • Enzymes not bound to anything so active site not affected. • Reaction rates can be reduced since substrate needs to get through trapping barrier so active site less easily accessible. • Membrane separation (microcapsule/encapsulation) – enzymes physically separated form the substrate by partially permeable membrane. • E.g. enzyme solution held at one side of membrane whilst substrate passes along other side. • Substrate molecules small enough to pass through membrane so that reaction can take place, and products small enough to pass back.

12. Explain how can immobilization affect enzymes activity • May alter tertiary structure, so altering shape of active site. • If altered significantly enzyme may not be able to catalyse reaction. • Bonding may slow or stop substrate entering active site. Also – leakage can be a problem

13. Compare and contrast methods