1. Biotechnology – Biotechnological techniques 1.Use of micro-organisms 2.Industrial production of enzymes 3.Tissue cultures

2. Industrial Production of Enzymes  Use of micro-organsims  Production of enzymes  Product recovery

3. Use of micro-organisms Enzymes have many applications within industry e.g. food and drink industries and textile industries  Naturally occurring enzyme production Cellulases  Source is bacteria  Used in laundry industry for fabric softening and as a detergent Pectinase  Source is fungi  Used in wine and fruit juice industries in pressing and clarification of juices

4. Use of micro-organisms cont… Amylases  Sources – fungi or bacteria  Used in paper making, as sugar syrups (sweeteners) and to degrade stains on cloths See table 5.2 for further examples of enzymes produced by microbes

5. Use of micro-organisms cont…  Manipulation of micro-organisms to produce enzymes Chymosin (AKA rennin)  Chymosin is used in cheese manufacture to clot the milk creating solid curds (and liquid whey which is drained)  Lactic acid also helps clot the milk  Traditionally chymosin comes from the stomach lining of calves

6. Use of micro-organisms cont…  Fungi have been used to produce forms of protein-clotting enzymes as a substitute for chymosin  The best solution is the cloning of chymosin gene in E. coli Gene for the enzyme is removed from the source Placed in plasmid and inserted in E. coli Microbe is cultured in a fermenter Enzyme removed and purified

7. Use of micro-organisms cont…  Cloned chymosin is Of consistent quality Acceptable to vegetarians and people with religious objections to eating calf derived products Produced on a large scale

8. Production of enzymes  Enzymes are produced in industrial quantities by growing the micro-organism in fermenters  Remember to consider the following when using fermenters Sterility (pure culture) Nutrients Oxygen pH Temperature Anti-foaming agents Time

9. Products Microbial products can be classified as either primary or secondary metabolites.  Primary metabolites Produced by metabolic activity that is essential to cell survival Produced during the exponential growth phase Examples  Ethanol (bakers yeast)  Citric acid (Aspergillus)  Some enzymes

10. Products cont…  Secondary metabolites By-products of metabolism not essential to immediate cell survival Produced during the stationary phase Examples Antibiotics (e.g. Penicillin by Penicillium chrysogenum)

11. Product Recovery The majority of enzymes are produced in an extracellular form by the microbe. Enzymes are separated from the microbes, they can subsequently be concentrated and purified

12. Product Recovery ( Table 5.1, p.101)  Separation - the removal of enzymes from cells, proteins, other metabolites and growth media Flocculation  Addition of a chemical that causes the enzyme to precipitate Filtration Centrifugation  Spinning solution at high speeds which separates products according to mass

13. Product Recovery cont…  Concentration Ultrafiltration  Filtration through a semi-permeable membrane that only allows very small molecules through Vacuum Evaporation  Pressure of the culture is lowered until the water boils at room temperature  Water evaporates concentrating the product

14. Product Recovery cont…  Purification Chromatography  The analyte (substance to be purified) is mixed with a solvent  The analyte is separated according to its properties and the style of chromatography used

15. Past Paper Questions  2005 Q1  2003 Q1  2006 Q3  2005 Q3