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Sourcing, storing and handling enzymes Introduction Enzymes are found in living organisms. Their function is to speed up chemical reactions which would otherwise be too slow to maintain life. Enzymes catalyse a specific reaction on a specific chemical or group of related chemicals, called their ‘substrate(s)’. Enzymes are proteins whose shape and structure are sensitive to the environment they are in. Therefore, enzymes are prone to degrade if exposed to conditions very different from those in which they normally occur. Nevertheless, many enzymes are stable enough to be extracted from their natural sources and kept as a dry powder or a solution. They can be used in artificial reaction vessels such as test-tubes, alginate balls or agar plates. However, some care needs to be taken to ensure they remain stable during the activity. Most enzyme preparations keep better if stored cold, though some newer preparations are less sensitive. Enzymes designed for use in washing powders have relatively long shelf lives. Main points to ensure success with enzyme activities • Check the enzyme’s properties to choose suitable conditions for the reaction • Store enzymes or natural sources of enzymes in the refrigerator • Prepare extracts freshly or make sure the purchased enzyme is within its use-by date • Prepare diluted enzymes and substrates freshly before use • Use any instructions provided, and Recipe Sheet 37, to prepare dilutions • Always trial activities to make sure the enzyme is active and will produce results in a reasonable time. If necessary adjust concentrations of enzyme solution and/or substrate. • Diluted enzymes tend to be less stable than other preparations. Keep diluted solutions in the fridge or on ice if they are to be used over a period of one or two days. If in doubt, re-check the enzyme activity shortly before use. Sources of enzymes It is usually cheaper and may be safer to use natural sources for enzymes if they are readily available. For example: • Saliva is generally an excellent source of amylase; commercial samples can be unpredictable and if good hygiene precautions are followed (Laboratory Handbook 14.4.3), pupils can use their own saliva • A number of enzymes can be extracted quite easily from fruits, vegetables and liver. Commercial suppliers derive enzymes from a variety of sources. These can be plant, animal or microbial, and should be stated. Preparations are usually impure, and include other bits of the source organism or their growing medium (which is partly why some of these fail to produce clear ‘solutions’). Although such enzymes may have the same name (eg ‘amylase’) the properties may differ markedly GL116 JS 05/13 Page 1 of 6 © CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk depending on the source organism. Always check very carefully before purchase that the enzyme is suitable for the intended use. The activity of naturally-sourced enzymes may vary but they are often more reliable than commercially-sourced extracts. In addition, suppliers may change their own suppliers, so different batches may have different properties. They may have suffered poor conditions in transit, affecting their activity. The short shelf life and the high cost of many commercial enzyme preparations also make their use quite expensive. Some appear to lose activity quite suddenly, so it is important to obtain fresh stocks for use in examinations and assessed practical work. Nevertheless, commercial preparations may be convenient to use, and many good, stable products are available. Enzymes in the slice of apple cause browning Liquid, tablet and powder forms of some commercial enzymes The table overleaf lists a number of enzymes commonly used in schools and suggests some natural sources and brief details of their actions and properties. GL116 JS 05/13 Page 2 of 6 © CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk Some enzymes used in school and some of their sources Enzyme Where it occurs naturally Amylase Saliva, gut. Natural sources for school use Saliva Bromelain Pineapple Pineapple Chymosin Diastase Lactase / beta galactosidase Alternative name for rennin Alternative name for amylase Gut, ‘lactic Yoghurt acid’ bacteria. Catalase Ubiquitous to cells of all living things. Catechol oxidase Some fruits Dopa oxidase (as for catechol oxidase) Catalyses one reaction in the synthesis of melanin, the pigment that makes skin and hair darker Invertase / sucraseisomaltase Lipase Gut, some bacteria. Breaks down sucrose to glucose and fructose Papain Pepsin Pectinase Phosphatases Phosphorylase s Proteases general Rennin Typical properties / uses / some notes Breaks down starch to glucose and maltose. Two main forms (alpha and beta) act differently Breaks down proteins Used in biological washing powders. Some are inhibited by citrate Used as a meat tenderiser Breaks down lactose to glucose and galactose Bacteria with this enzyme are used to make milk into yoghurt. A very prolific and highly active enzyme. Catalyses the breakdown of hydrogen peroxide, a byproduct of the cell’s process of detoxification of free radicals Responsible for browning Gut, liver, blood vessels Papaya Inhibited by copper(II) Inhibited by phydroxybenzoic acid (competitive), phenylthiocarbamide (non-competitive) Used in the manufacture of softcentred chocolates. Used in biological washing powders Used as a meat tenderiser Breaks down fats to free fatty acids and glycerol Papaya Breaks down proteins, cleaving at basic amino acids Stomach Breaks down proteins; Optimal pH = 2 cleaves after the N-terminal of tyrosine, tryptophan and phenylalanine Some fruits Jam-making Breaks down the jelly-like Used to help extract ingredient pectin juice from fruit Cells of all Mung bean Remove the phosphate living things seedlings group from their substrate Cells of all Adding phosphate groups living things to their substrates See pepsin, rennin, trypsin. Biological washing powders contain proteases Stomach Trypsin Duodenum Urease Some beans; some soil, and pathogenic bacteria. GL116 JS 05/13 Page 3 of 6 Animal and vegetable sources eg, potato, liver, blood. Banana, apple, other fruit What it does Cooking ingredient Jack bean, soya bean Breaks down proteins Breaks down proteins, cleaving after the Cterminal of lysine and arginine Breaks down urea to carbon dioxide and urea Used to coagulate milk Optimal pH = 8.5 A number of proteins in beans have urease activity © CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk Preparing enzymes for use Using diluted enzyme preparations Diluted enzymes lose their activities relatively quickly. It is best to prepare dilutions freshly. For activities taking place over one or two days, keep the diluted solutions in the fridge or on ice to preserve their activity. Even so, it is worth checking their activity before beginning the practical and, for critical work (eg assessment), during the day. Follow any instructions provided with the enzyme or activity protocol and also see also Recipe Sheet 37 which includes important safety points and guidance. Allergic responses are rare, particularly with the small quantities used in schools but take sensible precautions as described on the Recipe Sheet. • Always trial an activity to ensure the enzyme is working effectively under the conditions which will be provided in the lesson. Preparing enzyme solutions Many enzymes work best within a certain pH range. If this is close to neutral (pH 7), tap water can often be used. Distilled water is often acidic, and may be suitable for enzymes with pH optima below 7. For enzymes which have an optimum which is acidic or alkaline or for testing how enzyme activity changes with pH, the table below gives simple solutions that will provide a stable enough pH during the duration of many practical activities. In tests at CLEAPSS, these solutions maintained their pH during incubation of amylase for 10 minutes. pHs of water and some common solutions Solution Approximate pH 0.1 M hydrochloric acid 1 0.01 M hydrochloric acid 2 (distilled water) Can be pH 4 (tap water) Can be pH 7 0.1 M sodium carbonate 11-12 0.1 M sodium hydroxide (IRRITANT) 13 Occasionally a practical requires a very stable pH which uses a buffer to resist pH change. However, buffers must be used with caution because the chemicals involved can act as either a co-factor or an inhibitor to the enzyme, completely altering how it works and producing unexpected and incorrect results. If a buffer is required, the type of buffer should be specified. Recipes for some commonlyused buffers are given on Recipe Sheet 18. Tablets, designed to be made up to certain pHs, should be used with caution unless their chemical composition is known, and known to not affect living systems. Enzyme ‘concentration’ Enzyme concentrations are usually given as %, sometimes followed by m/v or v/v (mass per volume or volume per volume; i.e the mass or volume needed to make up 100 ml of solution). It is not appropriate to try to work out molar concentrations of enzymes. For school use, simply make up the enzyme as a percentage concentration, trial the activity and decide the appropriate concentration to use to obtain results in a reasonable time. Why isn’t our enzyme ‘solution’ clear? Not all enzyme preparations make up clear ‘solutions’; sometimes the enzyme is insoluble in water, or insoluble products are also present in the preparation. In some cases, filtering might remove the activity (which may be in the non-soluble fraction) and you need to use the unclear mixture. If a clear mixture is needed eg, GL116 JS 05/13 Page 4 of 6 © CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk for measuring a product in a colorimeter, it may be better to filter the mixture after incubation. In the photo, the urease, despite thorough mixing, remains as a cloudy suspension. Enzyme Glossary Term Explanation Enzyme Usually a relatively large protein molecule that catalyses a specific reaction on a particular substrate or group of substrates. The enzyme does not get used up in the reaction, though, as with other natural substances, over time its activity may decline. Substrate A chemical whose reaction can be catalysed by the enzyme. Catalyse, catalysis To speed up a chemical reaction without taking part in it. Thus a catalyst is a substance that speeds up a reaction. Enzymes are catalysts. When the reaction is complete and the products have moved away, the enzyme is ready to catalyse the reactions of further substrate(s). Specificity Each enzyme will bind to a specific substrate or group of substrates and carry out a specific reaction or type of reaction with it/them. Active site The part of the enzyme where the substrate and other chemicals taking part in the reaction bind to the enzyme and where they react. Bind, binding The substrate or substrates attach to the enzyme via chemical bonds. These bonds are just strong enough to hold the substrate(s) in the correct position to react. The product(s) do not bind so strongly and are able to leave the active site. Enzyme-substrate complex The enzyme and substrate combined during the reaction. Lock-and-key hypothesis The hypothesis that the substrate fits into the active site like a key fitting into its lock. Induced-fit hypothesis The hypothesis that when the substrate attaches to the active site it causes changes to improve the way they are combined and to make the reaction work better. Inhibitor A substance that interferes with the enzyme’s activity. Inhibitors may or may not act competitively (see Competitive inhibitor and Noncompetitive inhibitor). Inhibitors might act reversibly or irreversibly, depending on whether they inflict permanent damage to the enzyme or merely distort it while they are present. Competitive inhibitor An inhibitor that competes with the substrate for its place in the active site of the enzyme. Competitive inhibitors tend to be similar to the substrate in some way. The relative concentrations of the substrate and the competitive inhibitor affects enzyme activity. Non-competitive inhibitor An inhibitor that affects the enzyme’s ability to catalyse the reaction without competing with its substrate. Non-competitive inhibitors could deform the enzyme, either preventing the substrate from binding to the active site or from reacting once it is bound. Heavy metals are common non-competitive inhibitors for many enzymes. Only the concentration of the non-competitive inhibitor affects enzyme activity. Cofactor or coenzyme Usually small molecules or substances that are needed to enable the enzyme to work. Some vitamins and metals ions are cofactors. GL116 JS 05/13 Page 5 of 6 © CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk Continued Enzyme Glossary continued Term Explanation Optimum / optimal conditions The most favourable conditions for enzyme activity. Eg, the pH, temperature and concentration of other reagents needed to enable it to perform at its fastest rate. Denature When a protein loses its shape it is denatured. If the protein is an enzyme, this affects its activity. In vitro In artificial reaction vessels (eg, test tubes, agar plates or alginate balls): refers to activities carried out under these conditions. In vivo In a living organism: refers to research carried out on a whole living organism. Stability An enzyme is usually only fully active when it is intact and in good condition. The conditions needed to maintain the enzyme’s activity or ‘stability’ may differ from the optimal conditions. Turnover rate A measure of the number of substrate molecules that can be ‘turned over’ (react) per active site per unit time. Activity The turnover rate per unit mass of the purified enzyme (eg, units per mg). Due to their large size, instead of referring to the molarity of an enzyme preparation it is common to refer to its activity. Both commercial and natural enzyme preparations are impure. In industry and research, the activity of an enzyme preparation is calculated under standard conditions. For school work, absolute activity is not relevant. It is more useful to work out a concentration of the enzyme preparation that produces meaningful results in a reasonable time, and to express this as a percentage by mass (eg, 1% m/v). GL116 JS 05/13 Page 6 of 6 © CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk