Enzymes industrial uses, biotechnology methods processes optimum conditions temperature pH AQA Edexcel OCR Gateway science 21st century gcse chemistry revision notes

2. Enzymes and Biotechnology (see also rates notes at end of 2.)

Aspects of the vitamin, food and drugs GCSE chemistry are on the "Extra Organic Chemistry" page.

Living cells use chemical reactions to produce new materials. Living things produce catalysts called enzymes which allow chemical reactions to occur quite quickly at ordinary temperatures and pressures. Enzyme proteins are powerful 'biochemical catalysts' and are widely used in the food industry and are being used more and more to manufacture many other chemicals. These biological catalysts promote most of the reactions in living tissue. The names of enzymes end in ...ase e.g. amylase, protease, invertase, isomerase etc.

Enzymes are in all cells and keep all living things working and are always extracted from a living organism.
- Enzymes are always complex protein molecules and are sensitive to pH (mustn't be too acid or too alkaline) and temperature (mustn't be to low - too slow, or too high which causes denaturing of the enzyme).
- Enzymes are true catalysts because they are not consumed in the process and keep working as long as the substrate reactant is present e.g. as long sugar is left in an aqueous solution mixed with yeast.
- Enzymes have been used by humans since the beginning of recorded history e.g. use in fermentation to make wine.
- Enzymes are becoming increasingly important in the “biotechnology” industry.
- Enzyme reactions happen inside living cells. However, dead cells that have been broken open to release their enzymes are used to let the process happen in test tubes or for large scale industrial production situations.
- Enzymes have ability to reduce the activation energy needed (See rates of reaction notes)
- The reaction profile diagrams illustrate the point.
- By lowering the activation energy, more molecules have enough kinetic energy to react - bonds are more easily broken - so greater probability of fruitful collision forming the products of the reaction.
Cells contain protein molecules that act as biological or biochemical catalysts, they are known as ENZYMES.
- Enzymes catalyse everything in living cells from respiration, metabolism=digestion, protein synthesis and photosynthesis.
Specific enzymes catalyse specific chemical reactions, they only function with a specific substrate molecule or molecules - the reactants for a specific biochemical reaction.
At low concentrations of either enzyme or substrate the reaction mathematics is usually quite simple.
- Increase in concentration of either will give a proportionate increase in the rate of reaction.
- Extra ADVANCED notes on the kinetics of enzyme reactions, deals with the complications, but not for GCSE level.
The chemical reactions brought about by living cells are quite fast in conditions that are warm rather than hot.
This is because the cells use these enzyme catalysts. The 'key and lock' mechanism is explained later on.
Although they are fantastic catalysts, they can perform efficiently under quite narrow conditions in terms of temperature and pH of the reaction medium.
Enzymes are protein molecules which are usually damaged by temperatures above about 45º C. Although not damaged by lower temperatures, the reactions may be too slow to be of any use. (see rates notes at the end of this section).
- All enzymes have an optimum temperature range.
Different enzymes work best at different pH values.
- All enzymes have an optimum pH range.
The enzymes in yeast cells (living organism's) convert sugar into ethanol ('alcohol') and carbon dioxide in the brewing and drinks industry.
- Wine is made from fermented grapes and beer and whisky from fermented grain and hops.
- A similar process is used to convert sugar cane into ethanol and distilled to use as biofuel.
- The process does NOT need oxygen and occurs best under anaerobic conditions in a fermenter.
- e.g. glucose ==> ethanol and carbon dioxide in water and the absence of air.
- C₆H₁₂O_6(aq) ==> 2C₂H₅OH_(aq) + CO_2(g)
- This process occurs efficiently between 25 to 55^oC and is called fermentation and is used to produce the alcohol in beer and wine. The carbon dioxide dissolved in the final alcoholic drink produces the fizz!
- Note on raising agents in cooking: It is this reaction producing bubbles of carbon dioxide which make dough mixtures rise in the kitchen or food industry when yeast is used in baking bread or cake making etc.
  - An alternative to yeast is to use sodium hydrogencarbonate ('sodium bicarbonate' or 'baking soda') in baking. The rising action is also due to carbon dioxide gas formed from its reaction with an acid (e.g. tartaric acid), and nothing to do with enzymes:
    - self-raising baking powder = carbonate base + a solid organic acid, giving
    - sodium hydrogencarbonate + acid ==> sodium salt of acid + water + carbon dioxide
- A simple laboratory test for carbon dioxide is that it forms a milky precipitate with limewater.
- However other enzymes in living material can also catalyse oxidation with the oxygen in air. When alcoholic drinks turn sour it is due to the alcohol being oxidised to the weak organic acid ethanoic acid, commonly know as 'vinegar'!
Enzymes are involved in the following processes in the home
- Bread dough raising using yeast (see above), the rising action is due to the formation of carbon dioxide.
- Biological detergents may contain protein-digesting protease enzymes and fat-digesting enzymes lipase enzymes.
In industry and agriculture, enzymes are used to bring about reactions at normal temperatures and pressures that would otherwise require more expensive and more energy demanding equipment e.g.
- In the dairy industry yoghurt and cheese are formed by the action of bacteria (and their enzymes) on milk.
- Proteases break down proteins and are used to 'pre-digest' the protein in some baby foods.
- Carbohydrases are used to convert starch syrup into sugar syrup.
- Invertase is used to make the sugar for the soft centres of chocolates, but is quite expensive.
- Isomerase* is used to convert glucose syrup into fructose syrup, which is much sweeter and therefore can be used in smaller quantities e.g. in slimming foods.
  - (* The name comes from the word 'isomers' which means molecules of the same molecular formula but different molecular structures. Glucose and fructose both have the molecular formula C₆H₁₂O₆).
- Pectinase breaks down insoluble pectin polysaccharides and so is used in clarify fruit juices.
- Amylases break down carbohydrates and Lipases break down fats.
- Enzymes are used in genetic engineering and penicillin production.
- The dairy industry uses enzymes made by microorganisms (bacteria) to produce yoghurt and cheese from milk.
  - The bacteria enzymes convert the sugar in milk (lactose) into lactic acid.
- Enzymes in biological detergents - the enzyme lipase (together with protease) is used in biological detergents to break down (digest) the substances in stains into smaller water soluble molecules
- The antibiotic penicillin is made by the action of enzymes in the penicillium mould which is mixed with a particular sugar solution and other ingredients in a fermentation tank and as the mould grows the penicillin is produced. The penicillin is then extracted from the penicillium mould and ends up as a solution in a little bottle ready for injection into a patient.
Successful industrial processes often depend on the immobilization of the enzyme:
- Traditional use of enzymes have all been “batch” processes, which is not very efficient on a commercial scale. Modern biotechnology industries have developed techniques to isolate and then immobilise enzymes, thereby allowing continuous processes to be developed. The enzyme is isolated, usually from a culture of bacteria, then 'immobilised' or trapped in an unreactive material, so that they remain stable.
- Methods of immobilisation:
  - The enzymes can be trapped in a silica gel lattice or a collagen matrix (a mesh that traps the enzymes) or cellulose fibres (again enzyme trapped in a molecular mesh). In principle all that is needed is a stable inert surface (the larger the better for a more efficient rate of reaction) on which the enzyme is attached and stable.
  - Encapsulating in beads of alginate or polymer microspheres can also be used immobilize enzymes but these beads need to be selectively permeable allowing substrate molecules in and reactant products out.
    - In all cases the use of a matrix, mesh or small particles means the solvent containing reactant molecules can readily flow by the enzyme giving a good rate of reaction - its effectively giving a good 'surface area' of contact between reactants and enzymes.
- Advantages of immobilisation:
  - Stabilising the enzyme keeps it functioning for a longer period because it can be easily recovered for further use.
  - To immobilise the enzyme allows a continuous process, this means a continuous input of raw materials and output of product, so can run 24 hours a day for many weeks or months efficiently.
  - A batch process means loading the reactor vessel with reactants, extract the products, clean out the reactor/fermenter, re-load with reactants etc. etc. i.e. less efficient, costs time and so is less economic!
  - There is less contamination of the product with the enzyme because of ease of separation

Rates of Reaction - Kinetics of Enzymes - Optimum Conditions (full rates of reaction notes)

Note extra notes on the kinetics of enzyme reactions, for advanced level students only

Concentration: If either the substrate reactant e.g. sugar, or the yeast cell (enzyme) concentration is increased, the rate of reaction increases in a simple proportional way.

The graph on the left shows what happens as you gradually increase the substrate molecule concentration for a fixed constant concentration of enzyme. Initially the rate of reaction steadily increases, in fact it rises proportionately with increase in substrate concentration.

Kinetic particle theory: The greater the concentration of substrate or enzyme, the greater the probability of a fruitful collision leading to the formation of products. See Rates of Chemical Reactions Notes

What happens if the enzyme gets overloaded with substrate molecules?

However, if the concentration of enzyme is low but the substrate concentration becomes very high, the rate of reaction rises to a maximum and then stays constant.

This is because the maximum number of catalyst sites for the 'key and lock' mechanism are all in use and the rate of reaction depends on the rate of diffusion of substrate in and diffusion product out of the active sites.

What is the optimum pH of an enzyme?

pH effect: The structure of the protein enzyme can depends on how acid or alkaline the reaction medium is, that is, it is pH dependent. If it is too acid or too alkaline, the structure of the protein is changed and it is 'denatured' and becomes less effective. In the optimum pH range, the enzyme catalysis is at its most efficient. In the denaturing process the 'active site' may be damaged or changed so the enzyme cannot perform its catalytic function on the substrate molecules.

If the enzyme does not have the correct 'lock' structure, it cannot function efficiently by accepting the 'key' substrate molecule. Most enzymes have an optimum pH of between 4 and 9, and quite frequently near the neutral point of pH 7.

However, the enzyme pepsin has a peak at pH 2 and can operate in the very acid (hydrochloric) conditions of the stomach to help breakdown proteins for digestion in the small intestine.

Enzyme	Optimum pH	Function
Amylase (pancreas) enzyme	pH 6.7 - 7.0	This would be the graph for an enzyme with an optimum pH of 7.0 A pancreatic enzyme that catalyzes the breakdown and hydrolysis of starch into soluble sugars that can readily be digested and metabolised for energy generation.
Amylase (malt) enzyme	pH 4.6 - 5.2	Catalyzes the breakdown and hydrolysis of starch into soluble sugars in malt carbohydrate extracts.
Catalase enzyme	pH~7.0	Catalyses the breakdown of potentially harmful hydrogen peroxide to water and oxygen. Important in respiration metabolism chemistry. 2H₂O_2(aq) ==> 2H₂O_(l) + O_2(g)
Invertase enzyme	pH~4.5	Catalyses the breakdown/hydrolysis of sucrose into fructose + glucose, the resulting mixture is 'inverted sugar syrup'. C₁₂H₂₂O₁₁ + H₂O ==> C₆H₁₂O₆ + C₆H₁₂O₆
Lipase (pancreas) enzyme	pH~8.0	Lipases catalyse the breakdown dietary fats, oils, triglycerides etc. into digestible molecules in the human digestion system.
Lipase (stomach) enzyme	pH 4.0 - 5.0	As above, but note the significantly different optimum pH in the acid stomach juices, to optimum pH in the alkaline fluids of the pancreas.
Maltase enzyme	pH 6.1 - 6.8	Breaks down malt sugars.
Pepsin enzyme	pH 1.5 - 2.0	Catalyses the breakdown/hydrolysis of proteins into smaller peptide fragments.
Trypsin enzyme	pH 7.8 - 8.7	Catalyses the breakdown/hydrolysis of proteins into amino acids. Note again, the significantly different optimum pH to similarly functioning pepsin.
Urease enzyme	pH~7.0	Catalyzes the hydrolysis breakdown of urea into ammonia and carbon dioxide. (NH₂)₂CO_(aq) + H₂O_(l) ==> 2NH_3(aq) + CO_2(aq)
Zymase enzyme	pH~6	catalyses the fermentation of sugars into ethanol C₆H₁₂O_6(aq) ==> 2C₂H₅OH_(aq) + 2CO_2(g) I've seen on the internet quoted values from 4 to 8 for the optimum pH for the maximum, most economic, speed of reaction for fermentation. The pH in yeast cells is apparently 6 which is the mid-value of those quoted. There are also different strains of yeast. See also chemistry of ethanol
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Note in the data/information table above, that there are several instances, amongst many, where two different enzymes perform the same function, BUT at very different optimum pHs. This results in versatility ie where same chemistry is needed in different organs of the body - which incidentally, functions very nicely with its ~3000 different enzymes. Almost every chemical reaction in living organisms requires a specific catalyst or enzyme.

What is the optimum temperature of an enzyme?

Temperature: The structure of the protein enzyme can depend the temperature. If the enzyme does not have the correct 'lock' structure, it cannot function efficiently. The shape of the graph is due to two factors.

(1) The initial rise in rate of reaction is what you normally expect for any chemical reaction. The increase in temperature increases the average kinetic energy (KE) of the molecules. This gives more particles to have the necessary activation to break bonds and increase the chance of the product forming from the higher KE 'fruitful collisions.

See Rates of Chemical Reactions Notes

(2) However as the temperature rises further, the increasing thermal vibration of the enzyme molecule causes its protein structure to break down (denature) and so the 'lock' is damaged so the enzyme is less efficient in interacting with the substrate molecule (see key-lock below).

This may be due to the failure of weak intermolecular forces or actual ionic/covalent bonds, but the 3D molecular structure of the enzyme is changed so that the substrate molecule cannot 'dock in' to be changed into products.
The optimum temperature for the fastest rate of reaction is often around 30-40^oC (note our body temperature is about 37^oC, no coincidence!). Eventually at high temperatures the enzyme completely ceases to function.

(3) The graph that you see is effectively the result of adding two graphs together, (1) the increase in rate due to increase in temperature and (2) the decrease in rate as denaturing of the enzyme increases with increase in temperature. The resulting graph than has two minimums and one maximum, the hump in the graph being the optimum temperature range.

Explaining enzyme biochemical catalysis - the 'key and lock' mechanism

Diagram showing how an enzyme converts substrates into products (c) doc b

KEY in sequence: E = free enzyme, S = free substrate reactant molecule, E-S = enzyme-reactant complex, E-P = enzyme-products complex, E = free enzyme, P = free product
The enzyme is a complex protein molecule, but there is a particular 'active site' where the reactant molecule 'docks in' by random collision. The enzyme is sometimes referred to as the 'lock' and the initial reactant substrate molecule as the 'key', hence this is called the 'key and lock mechanism'. This is also explains why enzymes are very specific - you need the right molecular key for a particular molecular lock.
(1) Once the 'reactant-enzyme complex' is formed the enzyme function changes the reactant molecule into the new product molecule or molecules (2).
(3) The 'enzyme-new molecule complex' breaks down to free the new product molecule and the enzyme reactive site can now be re-used by another reactant molecule.
- Note (a). Compared to the un-catalysed reaction, the enzyme provides a 'chemical change route' with a much lower activation energy, and so this greatly increases the rate of reaction as more molecules have enough kinetic energy to react at the same temperature.
- Note (b). The products are shown as two molecules, because there are quite often two products for each step of the breakdown of a bigger molecule into smaller molecules e.g. protein to 'smaller protein' + amino acid, or starch to 'smaller starch' plus a glucose molecule etc. But there can be just one product molecule e.g. when isomerase changes glucose into fructose. There can also be two substrate reactant molecules being combined to form a bigger molecule or a long natural polymer molecule like starch being broken down to small sugar molecules. In other words there are lots of possibilities!
- Note (c). Many drugs work by blocking the sites normally used by enzymes. The molecular key (the drug) goes onto the reactive enzyme site, but stays there, so inhibiting enzyme activity which promotes harmful chemical-organism effects in the body. The harmful effect might be the production of toxic chemicals from a bacteria or the reproduction of a harmful organism etc.
- Note (d). "Rates of Reaction Notes" fully explains all the factors affecting the rate of any chemical reaction, including explaining experimental methods and reaction profile diagrams and activation energy.
- Note (e). Different reactions need different enzymes, and also if enzymes, which bring about the same chemical change, are quite likely to have different optimum rate pH's or temperatures. this phenomena is known as the specificity of enzymes is related to the unique structure of each enzyme and its 'reactivity' limited to interaction with particular substrate molecules.

See also ENZYMES - structure, function, optimum conditions, experiments gcse biology notes

Extra advanced A level chemistry notes on Enzyme structure on the stereochemistry page

and extra notes on the kinetics of enzyme reactions, for advanced A level students only

Where next?

Index of selected pages describing industrial processes:

Limestone, lime - uses, thermal decomposition of carbonates, hydroxides and nitrates
Enzymes and Biotechnology
Contact Process, the importance of sulfuric acid
How can metals be made more useful? (alloys of Al, Fe, steel etc.)
Instrumental Methods of Chemical Analysis
Chemical & Pharmaceutical Industry Economics & Sustainability and Life Cycle Assessment
Products of the Chemical & Pharmaceutical Industries & impact on us
The Principles & Practice of Chemical Production - Synthesising Molecules
Ammonia synthesis/uses/fertilisers
Oil Products
Extraction of Metals
Halogens - sodium chloride Electrolysis
Transition Metals
Extra Electrochemistry - electrolysis and cells

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