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2. Enzyme technology is concerned with the application of enzymes as tools of industry, agriculture and medicine Enzymes are biological catalysts that fulfil their role by binding specific substrates at their active sites This specificity is one property of enzymes that makes them useful for industrial applications The value of using enzymes over inorganic catalysts in the technological field is their efficiency, selectivity and specificity Enzymes are able to operate at room temperature, atmospheric pressure and within normal pH ranges (around 7) – all of which create energy savings for industry Enzymes possess specifically shaped active sites for reacting with one specific substrate thereby generating pure products free from unwanted by-products Enzymes are biodegradable and, unlike many inorganic catalysts, cause less damage to the environment Enzyme Technology

3. The micro-organisms (such as yeast) are really used as a source of enzymes during the manufacture of these products of biotechnology Many industrial processes now make use of pure sources of enzymes, i.e. the enzymes have been ISOLATED from the micro-organisms before use Micro-organisms have been used for thousands of years for making products such as wine, beer, vinegar, soy sauce, bread and cheese Products of Enzyme Technology

4. Microbial enzymes are ISOLATED from a variety of sources and these include bacteria, fungi and yeast cells Micro-organisms produce enzymes that function inside their cells (intracellular enzymes) and they may also produce enzymes that are secreted and function outside the cells (extracellular enzymes) Electron micrograph of bacteria (Bacillus)

5. The large scale production of enzymes involves culturing micro-organisms in chambers called FERMENTERS or BIOREACTORS Micro-organisms are suitable for use in the large scale production of enzymes in fermenters because: They have rapid growth rates and are able to produce larger numbers of enzyme molecules per body mass than many other organisms Micro-organisms can be genetically engineered to improve the strain and enhance yields Micro-organisms are found in a wide variety of different habitats such that their enzymes are able to function across a range of temperatures and pH Micro-organisms have simple growth requirements and these can be precisely controlled within the fermenter Micro-organisms can utilise waste products such as agricultural waste as substrates Large Scale Production of Enzymes

6. MODIFICATION – possible application of genetic engineering to improve the microbial strain LABORATORY SCALE PILOT – to determine the optimum conditions for growth of the Micro-organism PILOT PLANT – small scale fermenter to clarify optimum operating conditions SCREENING – choosing an appropriate micro-organism for the desired enzyme INDUSTRIAL SCALE FERMENTATION The Biotechnological Process of Enzyme Production

7. Pectin is an insoluble substance found in the cell walls of plants In the drinks industry, juice extracted from fruits appears cloudy due to the presence of pectin PRODUCTION OF PECTINASE Pectinase is an enzyme that is used in the industry to break down the pectin The effect of pectinase is to clarify the fruit juice and to make it flow more freely Pectinase is obtained from the fungus Aspergillus niger Aspergillus niger produces pectinase as an extracellular enzyme Commercial Enzyme Production - An Example

8. PRODUCTION OF PECTINASE Aspergillus niger is grown in a fermenter with a source of nitrogen, with sucrose as the carbon source and the substrate pectin to stimulate pectinase production by the fungus Filtration or centrifugation to obtain a cell-free system containing pectinase in solution Evaporate to concentrate the enzyme Precipitate the pectinase out of the solution and filter the solid Dry and purify the crude pectinase Pure, powdered pectinase

9. Enzymes are used in industrial processes and as analytical reagents in medicine Immobilisation of enzymes is an important technique used in industry as it enables economical operation of a process and protection of enzymes during their use Because of their sensitivity and specificity, enzymes are used as analytical reagents in systems such as the detection of glucose in human blood and urine Thermostability and an ability to withstand extremes of pH are essential properties for enzymes used in many industrial processes Enzymes in Biotechnology

10. Thermophilic bacteria inhabit hot springs

11. Thermophilic bacteria inhabit volcanic vents

12. The costs associated with the use of enzymes for industrial purposes can also be reduced by immobilising the enzymes Enzymes for industrial processes are more valuable when they are able to act in an insolubilised state rather than in solution Enzymes are immobilised by binding them to, or trapping them in a solid support Various methods for immobilising enzymes are available Immobilised Enzymes

13. Enzymes are held on to a solid support (matrix) by weak forces such as hydrogen bonding Enzymes are trapped within the structure of a solid polymer (usually in the form of beads) – the enzyme is trapped rather than bound Methods for Immobilising Enzymes

14. Enzymes are covalently bonded to a matrix such as cellulose or collagen Another more expensive method involves enzymes which are both covalently bonded to, and cross-linked within, a matrix Cross-linking and covalent bonding may cause some enzymes to lose their catalytic activity especially if the active site is involved in forming the linkages

15. Compared with free enzymes in solution, immobilised enzymes have a number of advantages for use in industrial processes The stability of many enzymes is increased when they are in an immobilised state; they are less susceptible to changes in environmental conditions such as temperature and pH fluctuations Immobilised enzymes can be recovered and re-used, reducing overall costs The products of the reaction are not contaminated with enzyme eliminating the need to undertake costly separation of the enzyme from the product Immobilising enzymes allows for continuous production of a substance with greater automation Advantages of Immobilising Enzymes

16. Enzyme Immobilisation and Thermostable Enzymes in The Production of High Fructose Syrup This industrial process involves the conversion of cheap corn starch into a high fructose syrup for use as a sweetener in confectionary and drinks Starch Paste Starch paste is incubated with the thermostable enzyme alpha amylase at 90 o C for a couple of hours Dextrins (short chains of glucose molecules) Alpha amylase catalyses the hydrolysis of the starch into short glucose chains called dextrins The temperature is raised to 140 o C to denature the amylase and then lowered to around 55 o C before adding the fungal enzyme amyloglucosidase Glucose Amyloglucosidase catalyses the hydrolysis of dextrins into glucose molecules

17. Fructose syrup emerges from the end of the column free from contamination with enzyme The final stage involves the conversion of glucose syrup into the much sweeter fructose syrup using the enzyme glucose isomerase Glucose isomerase is immobilised in rigid granules and packed into a column Glucose syrup is poured into the top of the column and is hydrolysed as it contacts the immobilised enzyme

18. The sensitivity and specificity of enzymes makes them useful tools in medicine for the detection and measurement of chemicals in fluids such as blood and urine Because of their specificity, enzymes will bind to only one substrate – they can therefore be used for the identification of a specific substance in a biological sample Because of their sensitivity, enzymes are able to detect the presence of specific molecules even when they are present at very low concentrations The enzyme glucose oxidase is used in an immobilised form for the detection of glucose in biological fluids Enzymes as Analytical Agents

19. This method relies upon the specificity of the enzyme glucose oxidase, allowing glucose to be detected in the presence of other sugars N.B. Benedict's test is not specific for glucose as it gives a positive reaction with ALL reducing sugars At the tip of the clinistix is a cellulose fibre pad on to which glucose oxidase, peroxidase and a chromagen dye are immobilised When the clinistix is dipped into a urine sample (containing glucose), the glucose oxidase catalyses the conversion of glucose to hydrogen peroxide: This test uses a plastic strip (clinistix) for the detection of glucose in the urine of diabetics Glucose + O 2 gluconic acid + hydrogen peroxide (H 2 O 2 ) In the presence of the enzyme peroxidase, the chromagen dye is oxidised by the hydrogen peroxide to produce a colour change on the fibre pad DH 2 (chromagen dye) + H 2 O 2 2H 2 O + D The amount of coloured compound (D) produced is a direct measure of the amount of glucose in the sample Glucose Measurement using 'Clinistix'

20. The colour of the pad on the clinistix is compared with a colour chart to determine the amount of glucose present in the sample Increasing amounts of glucose No glucose Glucose Measurement using 'Clinistix'

21. Biosensors are electronic monitoring devices that make use of an enzyme’s specificity and the technique of enzyme immobilisationBiosensors

22. Biosensors are electronic monitoring devices that make use of an enzyme’s specificity and the technique of enzyme immobilisation Transducer Amplifier Read-out Immobilised enzymes bind with specific molecules even when they are present in very low concentrations The enzyme reaction brings about a change that is converted into an electrical signal by a transducer The electrical signal is amplified and gives a read-out on a small display screenBiosensors

23. A biosensor has been developed for detecting glucose in the blood of diabetics Glucose oxidase oxidises any glucose present in the blood to release electrons – these are detected by the transducer and converted into an electrical current Transducer Amplifier The current generated is proportional to the amount of glucose present in the sample and this is displayed as a digital read-out Glucose molecules in the blood Glucose oxidaseBiosensors