ACTIVITY BRIEF Enzyme technology The science at work Enzymes Enzymes have become big business. They are used in many industrial processes to catalyse biological reactions. Enzymes are exploited in a variety of manufacturing processes such as food processing and for the synthesis of medicines such as antibiotics like artificial penicillin. They are also used to clean up factory effluents and pollution in water and soil. Many processes can be made faster and cheaper by using the right enzyme and conditions. Optimum conditions are maintained during factory production by use of bioreactors. These are vessels which are designed to provide the ideal environment for reactions involving enzymes or living organisms. Your brief Most people in the UK produce an enzyme called lactase in their small intestine to break down lactose, the sugar found in milk. However, many people do not produce enough lactase and therefore have problems digesting the milk. They may get diarrhoea, wind and stomach cramps if they consume milk or milk products. This is caused by the action of bacteria in the gut, fermenting the lactose. This problem has been addressed by a number of companies who produce lactose free milk. Such companies have to address the problem of establishing the optimum conditions for their milk treatment process. Task 1 Investigating the use of a bioreactor Your task is to make a bioreactor containing an immobilised enzyme and use it to produce lactose reduced milk. You will assess the effect that temperature has on the enzyme driven reaction within the bioreactor. Use Study sheet: Investigating a bioreactor Task 2 Using Bioreactors You will make a detailed report of the findings of your investigation and relate them to the use of enzyme technology and bioreactors in industry. Use Study sheet: Using bioreactors Enzyme technology: page 1 of 10
STUDY SHEET Investigating a bioreactor Introduction Simple bioreactors can be constructed in the laboratory, using basic apparatus. These can be used to study the feasibility of processes and to establish the most favourable reaction conditions. The most promising processes can then be trialled using more sophisticated equipment. Eventually pilot studies yield the information that scientists and engineers need to be able to take the expensive step of designing and building chemical manufacturing plant on an industrial scale. In this task, you design, carry out and write up an experimental test of a bioreactor. In the next task, you will be asked to write a report to discuss your results in relation to industrial practice. The experiment Lactose reduced milk can be made by using a bioreactor to treat skimmed milk with immobilised lactase enzyme. Using the information provided and other sources, design and carry out a practical investigation of the effect of temperature on the activity of the enzyme lactase, when immobilised and used in a bioreactor. Summary You will need to: 1 Write a clear plan of action of your own to include: the method/procedure to be followed, with measurements of appropriate precision, repeated when appropriate a list of equipment and reagents, with quantities and concentrations timing and laboratory/other requirements detailed risk assessments consistent with COSHH guidelines, using secondary sources an explanation of the use of a range of techniques and equipment. 2 Obtain data: working with appropriate accuracy using appropriate precision making a detailed set of relevant observations collecting sufficient data to complete simple statistics. 3 Display and analyse the data accurately: using a range of methods illustrating trends clearly. 4 Draw simple conclusions: basing them solely on your data and observations relating them to your tables, charts and/or calculations. Enzyme technology: page 2 of 10
Doing it 1 Agree a time scale with your teacher. You must work to a deadline, but establish how much class time you will have. 2 Carry out research and decide on a suitable method. Use Fact sheet: Bioreactor techniques and other suitable resources to help you. 3 Write a provisional plan in outline and have it approved by your teacher. Use the Summary to check what you need to include. This first draft need not be in detail, just establish that you have chosen a workable safe method. 4 After you have consulted your teacher, modify or develop your plan. It would be a good idea to plan a short pilot study to test your chosen methods and establish the quantities and concentrations of the materials that you need. Include: requests for materials and resources risk assessments planned times for supervised laboratory work. Health and safety: Do not undertake any practical work until you have had your plans agreed by your teacher. Before carrying out any practical work a risk assessment must be carried out. Wear eye protection. Some people are allergic to lactase solution; avoid getting it on your skin or rubbing eyes after handling. Wash your hands before and after your practical work. 5 Carry out your investigation. You may need to repeat some things or modify your approach as you go along. Continue to submit revised plans as necessary and do not undertake any work without prior approval. Keep a record of all the work that you do as evidence of your planning, including any rough notes and references to sources that you use (paper, electronic or human). 6 It is good practice to plot results in a graph or calculate averages as you go along, so you can see if results are reliable. If you have inconsistent or anomalous results, you should repeat measurements and parts of procedures to check for possible causes of errors and to get more accurate results. Make notes of any relevant observations. 7 Complete the analysis of your data and write a report of your investigation to meet the criteria shown in the Summary. Include: introduction method results conclusions. Do not include a detailed discussion of your findings. You will do this in your next task. Enzyme technology: page 3 of 10
FACT SHEET Bioreactor techniques Lactose reduced milk can be made by using a bioreactor to treat skimmed milk with immobilised lactase enzyme. The following techniques may be modified for investigating this process. Technique 1: Immobilising an enzyme Enzymes may be immobilised using calcium alginate beads. 1 Mix 10 cm 3 of 2% sodium alginate solution and 0.5 cm 3 of lactase solution in a small beaker (use a syringe or pipette to measure out the solutions). Mix well trying to avoid creating too many air bubbles. 2 Place 100 cm 3 1.5% w/v calcium chloride solution into a clean beaker. 3 Draw up the sodium alginate and lactase mixture into a disposable pipette or syringe and add it drop by drop to the calcium chloride. Hold the tip of the pipette or syringe about 5 cm above the surface, adding the mixture slowly as a steady stream of drops. Make sure the pipette or syringe doesn t touch the calcium chloride. Alginate beads will form in the solution. Try to make as many as possible, then leave them for about 5 minutes to harden. 4 Sieve off the alginate beads from the calcium chloride solution with the tea strainer. 5 Rinse the beads with distilled water. 6 If necessary, beads may be stored under water in a refrigerator overnight. Technique 2: Construction of a simple bioreactor 1 Take an empty syringe barrel or a separating/dropping funnel and put a piece of gauze in the bottom. A small piece of net curtain or glass wool [handle with forceps] may also be used. If using a syringe, attach a piece of plastic tubing to the nozzle and close it with a screw clip. Fix your apparatus on a clamp stand. 2 Add the beads to fill the syringe or funnel and gently tap them down. 3 If you wish, you may insulate the body of the bioreactor. Technique 3: Processing with a bioreactor 1 Use a water bath to heat samples of the substrate solution to be processed. Leave long enough to reach the desired temperature (varies). Enzymes have different optimum operating temperatures. Immobilised enzymes are more thermostable than those in solutions. 2 For continuous flow processing the tap or screw clip is left open. Substrate solution at a given temperature is poured or pumped into the body of the bioreactor so that it flows through the alginate beads and is collected using the tube at the bottom. The flow rate may be adjusted by partially opening or closing the screw clip or tap. Enzyme technology: page 4 of 10
3 For batch processing, close the tap and add enough distilled water to the bioreactor to cover the alginate beads. Run this off into a measuring cylinder and record the volume. This indicates the volume of substrate that may be processed in one batch in the bioreactor. Add enough substrate solution to the column/body of the bioreactor to cover the alginate beads. Leave for a measured period of time, then open the screw clip or tap fully and collect the product. Technique 4: Pilot studies with bioreactors Factors which affect enzyme activity may be varied to establish the optimum operating conditions for scaling up. One factor at a time should be varied (others should be kept constant or controlled) and a suitable method used to establish the rate of reaction, e.g. by measuring the rate at which a product appears or at which substrate is used up. 1 The bioreactor is set up and a given set of conditions is applied. 2 Measurements are made to establish the rate of reaction. 3 The column is washed through with a few cm 3 of distilled water. 4 One factor is altered and a second set of measurements are taken. 5 The process is repeated to establish any pattern. Sufficient results are taken to establish the reliability of the results and the optimum conditions for the processing of the substrate. Technique 5: Using a dipstick test for glucose concentration Diastix gives a colour change which varies with the glucose concentration of a solution. 1 Remove one strip from the container and replace the cap. 2 Dip the test area of the strip into the test solution and remove immediately. 3 Tap the edge of the strip against the container or a clean, dry surface to remove excess liquid. 4 Compare the test area to the Colour Chart exactly one minute after wetting. 5 Record the amount of glucose in mmol dm -3. 6 By comparing each Diastix in a time series it is possible to estimate the glucose concentration for intermediate colours. 7 If a change in concentration is being found, the glucose concentration of the original solution should be established. Health and safety using lactase Before carrying out any practical work a risk assessment must be carried out. Wear eye protection. Some people are allergic to lactase solution: avoid getting it on your skin or rubbing eyes after handling. Enzyme technology: page 5 of 10
STUDY SHEET Using bioreactors Introduction You should carry out the work in Study sheet: Investigating a bioreactor before starting this activity. You need to have carried out your experiment using a bioreactor to investigate the effect of temperature on the activity of lactase. You should have written up the first half of your report and formed conclusions based on your data. You now need to carry out some research on the use of bioreactors in industry and write a detailed discussion. Discusssion To conclude your report you will need to write a detailed discussion. Start by making notes on the following, keeping a record of any sources that you use: 1 List and make short notes on the uses of enzyme technology in medicine and industry, including named examples. 2 What reaction is used to reduce the lactose content of milk? 3 What affect did temperature have on the rate of this reaction? Explain why temperature had this effect. 4 What other factors might affect the rate of this reaction and how were they kept constant during this experiment? 5 For the industrial process of removing lactose from milk (i) Why would a company want to immobilise lactase and not just add it directly to milk? (ii) What are any disadvantages of immobilising an enzyme? 6 How does your bioreactor relate to: (i) batch-system (ii) continuous-system? Which of these is better for producing large amounts of lactose-free milk? Explain your answer. 7 How does your bioreactor compare to those used in industry? Now write the final part of your report under the sub-heading Discussion. Use information from your findings and your notes to meet the marking criteria which include: interpreting the results in detail using secondary sources to support your findings drawing conclusions relating your results to the use of bioreactors and enzyme immobilisation, specifying named examples in either medicine or industry discussing the significance of your findings in terms of how enzymes work and the advantage of enzyme technology to industry Enzyme technology: page 6 of 10
Resources Resources on the production of lactose-free milk http://www.lactolite.co.uk/lactofree/ http://www.nutraingredients.com/news/ng.asp?id=36761-valio-extends-lactose Resources on enzymes http://www.bbc.co.uk/schools/gcsebitesize/chemistry/chemicalreactions/5enzymesrev5.shtml http://biotech.about.com/od/casestudies/tp/dairyenzymes.htm http://urc.cc/bio_r.htm Companies that produce enzymes for use in industry Advanced Enzymes: http://www.enzymeindia.com/ Baxel: http://www.microtack.com/index.html Enzyme technology: page 7 of 10
Teacher notes This activity links to OCR A2 Unit 15 Applications of biotechnology. The specification includes: evidence of your investigation into the use of biotechnology to solve agricultural, medical and industrial problems This evidence needs to include: AO3: a practical investigation into enzyme technology, to include construction of a simple bioreactor and the effect of temperature on enzyme activity for the highest marks: 1 produce a clear plan of action of your own, including detailed risk assessments consistent with COSHH guidelines, using secondary sources 2 carry out measurements from the constructed bioreactor, using an immobilised enzyme system, on factors affecting your bioreactor 3 explain the use of a range of techniques and equipment and will have repeated measurements when appropriate 4 work with an appropriate degree of accuracy 5 make and record a detailed set of relevant observations with limited help, using the appropriate precision in your measurements 6 display the scientific data accurately in a range of ways, and process them in a manner chosen to best illustrate the trends in data 7 collect sufficient data to complete simple statistics on the results 8 interpret the results in detail using secondary sources to support your findings 9 draw conclusions relating your results to the use of bioreactors and enzyme immobilisation, specifying named examples in either medicine or industry 10 discuss the significance of your findings in terms of how enzymes work and the advantage of enzyme technology to industry. This activity gives students an opportunity to meet OCR assessment objective. Students design an investigation and make a simple bioreactor containing an immobilised enzyme (lactase) and investigate the effect of increased temperature on the enzyme. It does not cover introductory aspects of Unit 15.2.4: Enzyme Technology. There is already a wealth of information available. For example, there are many good practicals that allow students to find out how enzymes work by investigating rates of reaction and limiting factors (see other resources). Information about the lock and key and induced fit hypothesises can be found in most A- level biology text books. You may wish to cover some of these aspects before using the investigation for assessment. Enzyme technology: page 8 of 10
Organisation Students will need to begin their investigation by reading through the activity and planning the experiment. In particular they will need to carry out risk assessments and plan which temperatures they wish to investigate. Students should preferably carry out the investigation by themselves. However, quite a lot of Diastix may be required per experiment. The exact number of Diastix required will depend upon the number of temperatures tested and the number of repeats. Diastix cost around 4 to 5 for 50. The same column should be used for collecting each complete set of results. This will eliminate discrepancies in results due to variations in the concentration of enzyme in different columns (dependent on accurate measurement and the number/size of beads put into each column). As long as students carry out sufficient individual work, class results could be pooled for the final analysis, providing results using separate columns are not combined for calculations. Students who have difficulty designing their investigation may be given extra help (taken into account for marking). If necessary, ideas from the following protocol could be provided, up to giving the whole method to follow. Students will still need to carry out risk assessments and decide on the temperatures to investigate. Using a bioreactor to reduce the lactose content of milk The following procedure may be used to investigate lactose reduction of skimmed milk using immobilised lactase enzyme. Health and safety Wear eye protection. Before carrying out any practical work a risk assessment must be done. Some people are allergic to lactase solution; avoid getting on the skin or rubbing eyes after handling. Equipment and materials 0.5 cm 3 lactase enzyme solution (available from Philip Harris or NCBE) [Irritant] 10 cm 3 of 2% sodium alginate solution 100 cm 3 of 1.5% calcium chloride solution skimmed milk (not UHT, 25 cm 3 for each test) Diastix (one for each test) small beakers (25 or 50 cm 3 ) stirring rod 150 cm 3 beakers nylon gauze or net curtain (1-3 cm 2 ) If glass wool is used, forceps are needed for handling it tea strainer spatula 10 cm 3 graduated syringe 25 cm 3 pipette Enzyme technology: page 9 of 10
2 cm 3 pipette or syringe test tubes boiling tubes ice (if requested) insulating material (if requested) water baths at several temperatures thermometers stop watch Bioreactor: 10 cm 3 graduated syringe tubing clip about 10 cm of 4 mm diameter aquarium airline tubing to fit syringe screw clip retort stand, boss and clamp or use separating funnel instead of syringe for the bioreactor Resources A number of enzyme experiments are available from the NCBE: http://www.ncbe.reading.ac.uk/ncbe/protocols/pracbiotech.html NCBE also supply lactase (as lactozym) at about 14 per 100 cm 3. Details and background information on the commercial use of lactose reduction in dairy products can be found at: http://www.ncbe.reading.ac.uk/ncbe/materials/enzymes/lactozym.html Investigating the effect of competitive and non-competitive inhibitors on the enzyme ß-galactosidase from SAPs may be useful to investigate how enzymes work: http://www-saps.plantsci.cam.ac.uk/worksheets/scotland/inhibit.htm Many practicals on enzymes may be found in: King, T., Reiss, M. & Roberts M. (2001). Practical Advanced Biology Nelson Thornes Ltd. ISBN 0-17-448308-2. Technical notes Make sure distilled or deionised water is used when making up solutions, since calcium salts in tap water will cause the sodium alginate to set. Sodium alginate doesn t dissolve easily. The best way is to mix the sodium alginate and distilled water in a beaker, then place the beaker on a magnetic stirrer/heater on a low heat (just to warm slightly) and leave for about 1-2 hours. The solution can go mouldy after about 1 week, so a fairly fresh solution is required. Lactase needs to be kept refrigerated. Enzyme technology: page 10 of 10