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School biology revision notes: All about the digestive system, its function and role of enzymes

Use the page sub-index, take time to study the content or [Use the website search box] re-edit 20/03/2023

DIGESTIVE SYSTEM and ENZYMES - the structure, function and optimum conditions of these amazing molecular biological catalysts, and enzyme reaction investigations

See also Enzymes and Biotechnology (GCSE chemistry notes)

IGCSE AQA GCSE Biology Edexcel GCSE Biology OCR GCSE Gateway Science Biology OCR GCSE 21st Century Science Biology Doc Brown's school biology revision notes: GCSE biology, IGCSE  biology, O level biology,  ~US grades 8, 9 and 10 school science courses or equivalent for ~14-16 year old students of biology.   Some of these biology revision notes on the digestive system and enzymes are suitable for UK KS3 Science-Biology (~US grades 6-8).  Some of these biology revision notes on the digestive system and enzymes are suitable for UK KS3 Science-Biology (~US grades 6-8). This page helps answer questions such as ...

 What is the structure of the digestive system (the alimentary canal) and what do the different parts do?  Why are gut bacteria important? What do gut bacteria do? What is the structure of enzymes? What is the microbiome?  What is the 'key and lock' mechanism?  What is the function of enzymes? Why are enzymes so important?   Why are enzymes most effective in a narrow pH or temperature range?

Sub-index for enzymes page

What are enzymes? and why are they so important in living systems

How do enzymes work? - the 'key and lock' mechanism theory - well supported by scientific evidence

Enzymes and digestion - big molecules to small molecules!

A summary of the human body's enzyme production sites and the digestive system

Appendix 1. Summary of the digestive system and the role of gut bacteria and fungi

Enzymes and synthesis of carbohydrates, proteins and lipids - small molecules to big molecules!

Factors that affect an enzyme's performance

1. What is the effect of changing concentration of the substrate for an enzyme catalysed reaction?

2. Effect of pH - What is the optimum pH of an enzyme catalysed reaction?

3. Effect of temperature - What is the optimum temperature for an enzyme catalysed reaction?

Methods of measuring enzyme activity

Method 1. A method of measuring the rate of product formation from an enzyme reaction - the decomposition of hydrogen peroxide by the enzyme catalase - collecting oxygen gas - investigating the effect of changing pH, temperature and concentration

Method 2. A colour change method of measuring the reduction of substrate concentration in an enzyme reaction - the starch to maltose reaction - effect of pH, temperature and concentration changes

Using a colorimeter to follow the speed of a reaction that can be monitored with a colour change

See also Surfaces for the exchange of substances in animal organisms (includes small intestine)

Doc Brown's Very Unoriginal Roman Bread Recipe! - but an excellent example of domestic enzyme technology!

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Introduction

What are enzymes? and why are they so important in living systems?

Enzymes are complex protein molecules made of chains of linked amino acids that catalyse most chemical reactions that go on in cells.

Each of these biological catalysts has a unique structure, particularly the shape of the active site.

Because of their structure, each enzyme catalyses a specific reaction.

Most enzyme catalysed reactions involve:

Breaking large molecules down into smaller ones.

Building small molecules into large molecules.

Changing one molecule into another.

(see later in the details of 'key and lock' mechanism theory).

Reminder - catalysts are substances that increase the speed of reactions by lowering the activation energy needed, BUT, they have not chemically changed overall or been used up after the reaction has taken place.

Most biochemical reactions have and require, a specific enzyme to catalyse it.

This is referred to as the specificity of an enzyme.

Enzymes are true biological catalysts, speeding up reactions without being used up in the chemical processes they facilitate.

Every protein an organism requires, including enzymes, is coded for by a different gene in the DNA.

This specific gene not only determines the sequence of the amino acids in an enzyme protein, but also its unique shape, and it is the shape that largely determines what an enzyme can do.

The enzyme shape is created by the folding and coiling of chains of amino acids joined together in the protein molecule.

The 3D shape of the enzyme determines the unique 3D shape of the active site.

Only one type of molecule can fit into the active site - if denatured, it becomes inactive.

The rate of chemical reactions are increased by increase in temperature, but higher temperatures may harm the structure and function of complex biological molecules like the enzyme protein molecules.

Therefore the catalytic power of enzymes speeds up the thousands of different chemical reactions which enable most organisms to live specifically at relatively low temperatures - in fact, to keep them alive at any temperature!

Extremophiles e.g. like bacteria growing near hot volcanic undersea hydrothermal vents still have enzymes but their activity is much less than in organisms at the typical lower temperatures of the Earth's surface.

Without enzymes there would be no life - no photosynthesis in plants, no protein synthesis and respiration in plants and animals, so without these processes there would be no life!

e.g. mitochondria contain all the enzymes needed for the chemical reactions involved in respiration - the source of energy to power cells,

and plant cells have all the enzymes needed for photosynthesis - chlorophyll (not an enzyme) is just one molecule in the many required for the process.

Moreover, all the chemical processes of life must well controlled to keep things in balance so any organism can function properly.

This involves enzymes and hormones for example e.g. the right levels of sugar in the blood (hormone control), what the organism can do with the sugars (enzyme controlled).

Hormones may control the appropriate concentrations of substrates and products as well as temperature - all important variables that need controlling.

BUT, if the rates of so many chemical reactions are not controlled in harmony with each other, then cells may be damaged beyond repair hence endangering the whole organism,

AND many of the reactions involved are facilitated by enzymes.

All of the descriptions of enzyme action on this page are greatly simplified, and most biochemical reactions are multi-stage process i.e.

initial substrate reactant 1 == enzyme 1 ==> product 1  == enzyme 2 ==> product 2 etc. == last enzyme ==> final product

and please remember this when studying my 'pretty' simplified diagrams!

See also Enzymes and Biotechnology (gcse chemistry notes)


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How do enzymes work? - the 'key and lock' mechanism theory

A substrate molecule is a reactant which is to be changed into the product by way of the specific enzyme.

The substrate molecule (or molecules) must fit neatly into the active site on an enzyme and weakly bond to it.

The enzyme, or more specifically, the active site, is referred to as the 'lock', and in an analogy with door locks, the substrate molecules are referred to as the 'key or keys'.

The action by which enzymes function is called the 'key and lock' mechanism. This is illustrated below.

The following diagrams illustrate two examples of the 'key and lock' mechanism - how an enzyme works.

The active site is where the chemical change from substrate to product takes place and its shape is very important.

The mechanism is discussed in more detail in the next section.

Many biochemistry reactions either involve synthesis of a larger molecule by joining smaller ones together or breaking down and splitting a larger molecule into smaller ones.

It is sometimes quoted as a hypothesis, but there is a vast amount of evidence to show this mechanism is correct.

Each enzyme is shaped precisely to accept the substrate molecules, otherwise the reaction will NOT take place. This is why a particular enzyme can only catalyse a specific reaction. The substrate must fit into the active site!

The complete molecular structure of some enzymes has been determined by X-ray crystallography.

From a computer database you generate the structure of the enzyme and with advanced computer graphics you can 'virtually' examine the 3D active site.

You can then bring in a substrate molecule (real or theoretical) to see how it fits (or not) into the unique structure of the active site.

It is now possible to design drugs to block enzyme reactions to treat a particular medical condition.

You can then synthesise the drug and thoroughly test to see if it works AND has no harmful side effects.

If the enzyme is not the right shape e.g. the protein structure-active site is damaged, the substrate molecule cannot 'key in' or 'dock in' so the enzyme cannot function and the reaction does not take place.

This protein structure damage is referred to as a denaturing of the enzyme.

Enzyme damage (denaturing) can be caused by too high a temperature or the medium may be too acid (too low a pH) or too alkaline (too high a pH) - see later section on factors affecting the rate of enzyme reactions.


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The 'key and lock' mechanism for enzyme action

A good example of using a scientific model and well supported by scientific evidence

(Stage 1) is the 'docking in' of the substrate molecules into the active site, they are held there just sufficiently strongly to allow the chemical transformation to take place.

The active site is considered the 'lock'.

(Stage 2) happens on the active site where the substrates are catalytically changed to products which are then released from the enzyme.

The substrate reactant molecule is considered the 'key'.

 

e.g. the key and lock mechanism for synthesising a larger molecule from smaller molecules.

Sequence key e.g. for a larger molecule being made from two smaller molecules, perhaps a stage in protein synthesis

E = free enzyme (the 'lock')

S = free substrate reactant molecules (the 'keys'), the three then combine together - 'lock' together

ES = enzyme-substrate complex ==> EP = enzyme-product complex

The chemical change at the active site, this complex then breaks down to give the free enzyme and product.

E = free enzyme, P = free product

The diagram simulates two amino acids joined together to make a dipeptide, or you can just think of one of the substrate molecules being a longer partially made protein molecule and another amino acid is added to the end of the chain.

 

Key and lock mechanism for producing smaller molecules from larger ones

Sequence key e.g. for a larger molecule being broken down into two smaller molecules, perhaps in digestion where large carbohydrate molecules are broken down into small sugar molecules like glucose.

E = free enzyme (the 'lock')

S = free substrate reactant molecule (the 'key'), the two then combine together - 'lock' together

ES = enzyme-substrate complex ===> EP = enzyme-products complex

The chemical change at the active site, this complex then breaks down to give the free enzyme and products.

E = free enzyme, P = free products

 Apart from water molecules, the diagram actually matches the hydrolysis of sucrose to glucose and fructose by the enzyme invertase.

sucrose  +  water  ===> glucose + fructose

C12H22O11  +  H2O  ===> C6H12O6  +  C6H12O6 

The anaerobic fermentation reaction

glucose (sugar) == enzyme zymase ==> ethanol + carbon dioxide

C6H12O6(aq) ===> 2C2H5OH(aq) + 2CO2(g) 

is another example of an enzyme breaking down a larger molecule into four smaller ones, there are only two shown in the diagram!

Please bear in mind that these reactions are more complicated than the simple scheme above.

They often involve multiple stages and several enzymes.

 

See also Enzymes and Biotechnology (gcse chemistry notes)


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More examples of enzyme controlled reactions: explaining how we digest food

See also organ systems e.g. an overview of the human digestive system

Enzymes and digestion is all about big molecules to small molecules!

When we take in food it contains many large molecules, like carbohydrates, fats and proteins, all of which must be broken down by digestive enzymes to produce the useful smaller molecules to supply cells with necessary nutrients.

Enzymes control the chemical processes of digestion, but there are important physical processes too.

In the mouth, the action of your teeth cut and grind up your food into a pulp of smaller pieces and moistened with saliva containing enzymes.

This allows the food to move more easily through the digestive system.

It also increases the surface for the enzymes to react with the food particles.

Muscles in the stomach wall compact the food as well as churning and mixing everything up.

Other muscles in the digestive system squeeze the food along by the process of peristalsis.

The contraction and relaxation of the tissue lining muscles in the wall of the digestive system produce a wave-like movement that pushes the balls of food along the digestive system.

gcse biology peristalsis stomach oesophagus gullet small intestines pushing food balls along wave like movement

Even the tiniest bits of food cannot pass through permeable membranes into the blood.

Therefore the food must be broken down at the molecular level ...

AND so to digestive enzymes!

Enzymes are produced at certain points in the digestive system to break the food down into small soluble molecules that can be absorbed into the bloodstream - the process of chemical digestion.

Digestive enzymes break down e.g.

(i) carbohydrates like starch into sugars by carbohydrase enzymes like amylase,

(ii) fats are broken down into glycerol and long chain fatty acids by lipase enzymes

(iii) proteins are broken down into amino acids by protease enzymes.

Apart from fatty acids, sugars, amino acids and glycerol are all soluble in water and readily pass through the walls of the digestive system and so easily absorbed into the bloodstream for the body to use.

The smaller molecules can now pass through cell membranes for the cells to use.

The small digested molecules can then used for a variety purposes, all involving enzyme catalysed reactions e.g.

Muscle tissue is built from protein synthesised from amino acids in the ribosomes (examples of growth).

Fatty tissue is made from newly synthesised lipid molecules, these are used in building cell membranes (examples of growth). Lipid molecules are made from fatty acids and glycerol. Fat molecules are used as an chemical energy store and in synthesising hormone molecules.

Glycogen, made from glucose, can be used as a chemical energy store in the body, needed for ATP production in respiration.

On hydrolysis (enzyme catalysed), glycogen breaks down to reform the smaller molecule glucose - the main 'fuel' for respiration.

In plants, the carbohydrate starch is used as an energy store. When a plant needs energy, the starch is broken down by enzymes and converted to small sugar molecules

The sugars are then used to provide energy for the cells from respiration.

The simple sugars can also be converted into cellulose, the infrastructure of the plant.

 

Fats and fatty acids are not soluble in water, but they are essential nutrients.

The body uses bile to neutralise stomach acid and aid the emulsification of fat.

Bile is produced in the liver and stored in the gall bladder prior to release into the small intestine.

The stomach acid, hydrochloric acid (HCl) makes the pH too low, too acidic, for most enzymes to operate efficiently in the small intestine.

However, bile is alkaline, and neutralises the stomach acid and makes the ambient pH over 7, so the digestion medium is made alkaline.

The enzymes in the small intestine work best under alkaline conditions.

The bile helps emulsify the fats by reducing them to tiny droplets which are readily suspended and dispersed in the digestion fluids.

The emulsification into tiny fat drops greatly increases the surface for the lipase enzymes to act on, and so increases the rate of enzyme reaction - increases the speed of digestion.

For more on the theory see Effect on rate of reaction on changing the surface area

 

Some examples of digestion chemical reactions - ALL catalysed by specific enzymes

Three examples of 'big' molecules to 'little' molecules

1. Carbohydrases

Carbohydrates are compounds containing the elements carbon, hydrogen and oxygen e.g. C6H12O6.

They range in size from small simple sugar molecules like glucose, fructose, sucrose etc. to the huge complex carbohydrate polymer molecules of glycogen, starch and cellulose.

In many respects, analogous to synthetic polymers like poly(ethene) or nylon, simple sugars can be considered the monomer molecules and the complex carbohydrates the 'natural' polymer molecules

Carbohydrases break down carbohydrates into simple sugars.

Carbohydrase enzymes are made in salivary glands, pancreas and small intestine.

The carbohydrase enzyme amylase, breaks down starch into small sugar molecules - an important digestion reaction.

Enzyme reaction word equation: starch  +  water  === amylase enzyme ==> maltose, glucose (dextrose) etc.

2(C6H10O5)n  +  nH2O  ====>  nC12H22O11

or  (C6H10O5)n  +  nH2O  ====>  nC6H12O6  

(n is a very large number!)

To effect this conversion, the amylase enzyme  is produced in salivary glands, small intestine and the pancreas, they work best close to a neutral pH with an optimum around pH 6 to pH 7.

It is important in a living organism, complex carbohydrates like starch can be broken down to provide small molecules like glucose - used up in respiration chemistry to power the life of cells - energy source and facilitate the synthesis of other molecules.

 

2. lipases

Lipids, like many organic molecules, only contain the elements carbon, hydrogen and oxygen.

Lipase enzymes break down lipids like natural fats and oils (triglyceride esters) into glycerol and long chain fatty acids. Lipids are NOT polymers because they are not very long chain molecules.

Lipase enzymes are made in the pancreas.

Enzyme reaction word equation: lipid == lipase enzymes ==> glycerol + long chain fatty acids

To effect this conversion, the lipase enzymes are produced in the pancreas and the small intestine.

(Fats and oils are a sub-group of a class of molecules called lipids)

The sort of molecular change that takes place - details you do not need to know for GCSE level biology.

See a 'decay' investigation using milk and lipase  gcse biology revision notes

Its part of the web page Carbon cycle, nitrogen cycle, water cycle, decomposition - decay investigation 

 

3. proteases

All amino acid molecules contain carbon, hydrogen, oxygen and nitrogen and are the 'monomer' molecules for making the natural 'polymers' we call protein.

Proteases break down proteins into amino acids - they can work at a very low optimum pH of 2 - caused by the presence of the strong stomach acid (hydrochloric acid) which is important because it kills most bacteria in the stomach.

Note that your stomach produces a thick mucous to coat the lining wall of the stomach to protect the tissue being irritated or harmed by the hydrochloric acid.

Protease enzymes are made in the stomach, pancreas and small intestine.

Enzyme reaction word equation:

one protein molecule === protease ===> several different amino acids

To effect this conversion, the protease enzymes are produced in the stomach (here the protease enzyme is called pepsin), the pancreas and the small intestine.

It is important that in a living organism, proteins from meat, fish and plant foods can be broken down into amino acids, which are required to make the specific proteins required by that organism.


Summary of the human body's enzyme production sites and digestive system

The digestive system is essentially a long tube running from the mouth to the anus.

It consists of a succession of organs working together to digest and absorb food molecules, water and mineral ions.

Each organ is adapted to perform a particular different function.

The digestion is completed in the small intestine and the soluble food passes through the intestine wall into the blood - this is the process is called absorption and enables the blood to carry the absorbed nutrients to all the cells of the body.

The enzymes our digestive system uses to break down food are produced by specialised cells in glands and the gut lining.

A variety of different enzymes are required to catalyse the breakdown to produce molecules that can be absorbed into the bloodstream.

The organs of the digestive system their adaptations and what they do - emphasis on enzymes

For other details see Appendix 1. Summary of the digestive system

Salivary glands in mouth - these moisten food and produce saliva containing the enzyme amylase which catalyses the breakdown of carbohydrates like starch. The chewing action of the teeth mashes up the food and increases the surface area the enzymes can act on - chemistry - rates of reaction factor.

The oesophagus (Gullet) - connects the mouth with the stomach - has muscular walls that move food along by peristalsis.- wave-like movement of the tissue - a sort of squeeze and push effect when the muscle linings contract and then relax.

The stomach - mixes and mashes up food using strong muscular walls. It produces the protease enzyme, pepsin that breaks down proteins. It also produces hydrochloric acid to kill harmful microbes like bacteria  AND create the right pH ~2 for enzymes like protease to work function properly. Note the double function.

The liver - produces alkaline bile that neutralises stomach acid to modify the pH for other enzymes to act, bile emulsifies fats-lipids to help in their breakdown by enzymes and also stores carbohydrates as glycogen. The emulsification increases the surface area of the fat drops to increase the rate of the enzymic breakdown - rates of reaction factor - more chance of fruitful enzyme-substrate collision producing fatty acids and glycerol which diffuse into the lymphatic system.

The small bag of the gall bladder is where bile is stored before it is released into the small intestine.

Pancreas - glandular tissue that produces and releases enzymes into the small intestine - protease (breaks down proteins into amino acids), amylase (breaks down carbohydrates like starches) and lipase enzymes (breaks down lipid fats).

The small intestine (duodenum and ileum) - produces protease, amylase and lipase enzymes and is the site where the small digested food molecules are absorbed into the bloodstream. Note that large insoluble molecules cannot be absorbed into the body.

Specialised cells in the  large intestine absorb excess water and makes solid waste left over from digested food.

The anus and rectum is where most indigestible food ends up with the rest of the body's waste and stored as faeces, which we eject through our anus with the help of strong muscles!

See APPENDIX 1 For more on the digestion system

See also Surfaces for the exchange of substances in animal organisms (includes small intestine)


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More examples of enzyme controlled reactions

Enzymes and the synthesis of carbohydrates, proteins and lipids

Three examples of 'small' molecules to 'big' molecules!

The previous section on digestion was all about enzymes breaking large molecules down to small molecules to aid digestion and absorption of nutrients.

Here we consider the opposite biochemistry - building up essential large molecules from small substrate molecules.

The molecules so formed are examples of naturally occurring polymers.

Some examples of 'big molecule' synthesis reactions - ALL catalysed by specific enzymes

(Most are the opposite of the digestive chemistry described above)

1. Protein synthesis

Several enzyme catalysts join amino acids together to form proteins - its a complicated process!

Proteins are an example of a natural polymer.

Enzyme reaction word equation: proteins == enzymes ==> protein

For more details see DNA structure and Protein Synthesis  (gcse biology revision notes)

 

2. Lipid synthesis

Again, it takes multiple enzymes to synthesise lipid fats from glycerol and long chain fatty acids.

Enzyme reaction word equation: glycerol + 3 fatty acids === enzymes ===> lipid oil or fat

The molecular details of lipid oils and fats are not required for GCSE biology, but you should recognise aspects of their structure from your GCSE chemistry course on the equation diagrams below.

e.g. the functional groups: carboxylic acid -COOH, unsaturated' alkene group >C=C< and alcohol C-OH.

Lipids are NOT polymers

 

3. Complex carbohydrate synthesis

Complex carbohydrates are synthesised by combing together lots of small sugar molecules such as glucose into long polymer molecules such as glycogen (in animals) and starch and cellulose in plants.

The enzyme glycogen synthase (the name says it all), can join lots of simple glucose molecules into long chain glycogen molecules - a natural polymer.

Enzyme reaction word equation: glucose == glycogen synthase ==> glycogen

Glycogen is used by animals, like ourselves, as a chemical energy store.

Other enzymes can rapidly convert glycogen back to glucose to fuel respiration - to provide energy for cells to perform all their functions.


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Factors that affect an enzyme's performance

Enzymes perform best in their 'optimum' ambient conditions

BUT, first consider a basic view of enzyme activity and collision theory.

1. Suppose you mix the reactant molecules without the appropriate enzyme - the reaction is very slow.

2. The appropriate enzyme is added and the reaction speeds up - the activation is lowered by the enzyme and the rate of fruitful collisions increases producing more product.

3. From 2. to 3. the reaction proceeds at a steady rate, enzymes molecules fully active and ample substrate molecules, plenty of fruitful collisions.

4. Eventually the enzyme is running out of substrate molecules, less fruitful collisions possible, less product per unit time, so the rate of reaction steadily decreases to zero when all the substrate is used up.

The reaction profile of a catalysed reaction compared to an uncatalysed reaction.

The enzyme helps break reactant molecule bonds more easily than without a catalyst, so facilitating a faster reaction without increasing concentration or temperature - the collision rate doesn't increase, but there is more chance a fruitful collision producing the product molecules.

These arguments apply irrespective of whether the enzyme is functioning in its optimum conditions.

1. What is the effect of changing concentration of the substrate for an enzyme catalysed reaction?

When investigating the effect of concentration on enzyme activity, three factors must be kept constant, (i) either the concentration of substrate or enzyme, (ii) the temperature and (iii) the pH of the solution (see experimental methods).

(c) doc b

Concentration: If the substrate reactant e.g. sugar, concentration is increased, the rate of reaction increases in a simple proportional way as long as the enzyme concentration is constant.

Kinetic particle theory: The greater the concentration of substrate (or enzyme, see below) The greater the probability of a fruitful collision leading to the formation of products. For more details see Effect on rate of changing reactant concentration

The graph on the above-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, usually, it rises proportionately with increase in substrate concentration, but ....

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 active sites for the 'key and lock' mechanism are all in use and the maximum rate of reaction depends on the rate of diffusion of substrate in, and diffusion of product out from the active sites.

This shows up as the later horizontal portion on the graph - meaning the speed of the reaction has become constant because there are no more active sites to bring into operation.

What happens if you change the enzyme concentration?

At constant substrate concentration, the rate of reaction (usually) increases linearly with increase in enzyme concentration. The greater the concentration of enzyme, the greater the probability of a fruitful collision leading to the formation of products. Hence the initial linear part of the graph.

However, if the enzyme concentration is very high and the substrate concentration low, the rate tails off and becomes constant. This is because there are lots of 'active sites' available, in excess of those needed, and adding more of the enzyme makes no difference.

 

See a 'decay' investigation using milk and lipase  gcse biology revision notes

Its part of the web page Carbon cycle, nitrogen cycle, water cycle, decomposition - decay investigation 


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2. Effect of pH - What is the optimum pH of an enzyme catalysed reaction?

When investigating the effect of pH on enzyme activity, three factors must be kept constant, (i) the temperature and the concentrations of both the (ii) substrate and (iii) enzyme must all be kept constant (see experimental methods).

General description of graphs: For any enzyme, initially, as you increase the pH, the rate of reaction increases.

Then as the pH increases, the reaction rate reaches a maximum at the optimum pH and then decreases with further increase in pH.

The optimum pH varies quite a bit from one enzyme to another.

(c) doc bpH 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 (very low pH) or too alkaline (very high pH), the structure of the protein is changed and it is 'denatured' affecting the shape of the 'active site' which becomes less effective. The acid or alkali may chemically react with the enzyme at or near the active site affecting the shape of the active site or the ability of the substrate molecule to 'dock in' to the active site - a denaturing effect due to interfering with the bonds holding the enzyme together in its unique 3D shape.

The two graphs above illustrate how the rate of reaction varies with pH for many enzymes.

The enzyme catalase breaks down harmful hydrogen peroxide into water and oxygen, with an optimum pH range around pH 7 (top left graph).

Another enzyme may have an optimum pH range of 3.5 to 4.5 (lower-right graph).

Note that an enzyme is active over a pH range of perhaps several pH units, but beyond this range it is relatively ineffective.

In the optimum pH range, the enzyme catalysis is at its most efficient. In the denaturing process the 'active site' (see 'key and lock' mechanism details above) may be damaged by highly acid or alkaline conditions, and changed in such a way that the enzyme cannot perform its catalytic function on the substrate molecules - they don't fit in the active site.

If the enzyme does not have the correct 'lock' structure in the protein (the 'active site'), 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. Our own body fluids e.g. in blood or cells have a pH of ~pH 7.2 to 7.4, so its no coincidence that many of our enzymes have an optimum operating pH ~7, but it does depend on where you are in your body!

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

Examples of optimum pH values for enzyme activity

(c) doc b   activity of selected enzymes versus pH 

Increase in acidity or alkalinity creating a pH well away from the optimum, can affect the protein structure of the enzyme and so affecting the active site, and, the substrate molecule can no longer readily lock into place into the active site and cannot be transformed into the product molecules.

The first diagram is typical of many enzymes operating in near neutral solutions (~pH 7)

The other two diagrams shows the wide range of pH that different enzymes can operate in

e.g pepsin breaks down proteins in the very acid conditions of the stomach.

Blood has a pH of ~7.4 and carbonic anhydrase (optimum pH ~7) is found in red blood cells. This enzyme enables the efficient conversion of carbon dioxide and water into the carbonic acid and the hydrogen carbonate ion ('bicarbonate ion') and operates in near neutral conditions.

Trypsin is a protease enzyme from the pancreas that breaks down proteins (peptides) in the alkaline conditions (~pH 8.5) of the smaller intestine, so its optimum rate of reaction is around that value.


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3. Effect of temperature - What is the optimum temperature for an enzyme catalysed reaction?

When investigating the effect of temperature on enzyme activity, three factors must be kept constant, (i) the pH and the concentrations of both the (ii) substrate and (iii) enzyme must all be kept constant (see experimental methods).

General description of graphs: For any enzyme, initially, as you increase the temperature, the rate of reaction increases.

Then as the temperature increases, the reaction rate reaches a maximum at the optimum temperature and then decreases with further increase in temperature.

The optimum temperature varies from one enzyme to another, but for many warm blooded animals like us, it is often close to 37oC - but beware of enzymes in extremophile organisms in hot springs, volcanic vents and 10 km down at the bottom of an ocean,

(c) doc b

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.

A too high a temperature can affect the shape of the active site or the ability of the substrate molecule to 'dock in' to the active site - some kind of change is promoted by a high temperature - a denaturing effect due to interfering with the bonds holding the enzyme together in its unique 3D shape - the active site is damaged and the substrate molecule can't fit in.

Explaining the left graph of enzyme reaction rate versus temperature

(a) 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 to increase the chance of the product forming from the higher KE 'fruitful collisions.

In other words more molecules have sufficient energy to overcome the activation energy to break bonds and change from reactants to products.

There is also an increase in the rate of collision of the substrate and enzyme molecules.

For more details see Effect on rate of changing the temperature of reactants

(b) At higher temperatures the rate goes through a maximum and then slows down!

However as the temperature rises further, the increasing thermal vibration of the enzyme molecule causes its 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). It means the substrate molecule cannot properly dock into the active site on the enzyme.

This may be due to the failure of weak intermolecular bonding forces or actual ionic or covalent bonds being broken, but the 3D molecular structure  of the enzyme is changed so that the substrate molecule cannot 'dock into' the active site on the enzyme to be changed into products.

This damage to the enzyme's active site at higher temperatures - the denaturing of the enzyme, is NOT reversible. The enzyme will not go back to its normal shape even if the reaction mixture is cooled down.

The above diagram shows the effect of high temperatures on an enzyme molecule - the crucial and effective 3D shape is destroyed when bonds in the protein molecule are disrupted.

The optimum temperature for the fastest rate of reaction is often around 30-40oC (note our body temperature is about 37oC, no coincidence!). Eventually at high temperatures the enzyme completely ceases to function.

Note: NOT every enzyme reaction has an optimum of ~30-40oC!, some organisms exist and survive at very low temperatures and some at very high temperatures - extremophiles.

See Adaptations, lots of examples explained including extremophiles

By combining the points made in (a) and (b) we can now completely explain the shape of the graph.

The actual graph that you obtain from experiments is effectively the result of adding two trends together,

(a) The increase in rate due to increase in temperature - 'normal chemical behaviour,

and (b), the decrease in rate as denaturing of the enzyme increases with increase in temperature.

The resulting graph then has two minimums at the lower and higher temperatures, and one maximum - the hump in the graph is the point of maximum speed of the reaction, 'highlighting' the optimum most effective temperature range.

(c) doc b 

So, the first graph diagram is typical for temperature controlled enzyme activity.

The 2nd temperature graph shows what happens to the speed of the enzyme catalysed process of photosynthesis as the temperature is increased.

As the temperature increase the rate of catalysis increases (normal effect on the speed of reaction as the average kinetic energy of the molecules increases), but at high temperatures the protein structure of the enzyme is destroyed, so the active site on the enzyme is damaged.

More extreme condition: Some enzymes in bacteria found in:

(i) hot springs have an optimum temperature of over 80oC - at this temperature most our enzymes would be denatures,

(ii) bacteria living in cold deep ocean water have optimum temperatures as low as 0oC - at this temperature, most of our enzymes would only function very slowly.

See a 'decay' investigation using milk and lipase  gcse biology revision notes

Its part of the web page Carbon cycle, nitrogen cycle, water cycle, decomposition - decay investigation

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METHODS OF MEASURING ENZYME ACTIVITY

This section is about ideas and possible experimental procedures for investigating the factors that control the rates of enzyme catalysed chemical reactions.

I do not give detailed recipes for the experiments - just lots of ideas, there is much 'stuff' to think about!

See also  'decay' investigation using milk and lipase  gcse biology revision notes

Its part of the web page Carbon cycle, nitrogen cycle, water cycle, decomposition - decay investigation


How might the factors controlling enzyme activity be investigated in the laboratory?

Introduction

In principle you can measure the rate of an enzyme reaction in two ways:

Method 1. Measuring the rate of formation of a product (product/time)

e.g. how much product is formed in a give time.

In method 1. the decomposition of hydrogen peroxide by catalase is followed by measuring how much oxygen is formed.

Method 2. Measuring the rate at which the substrate is used up

e.g. the time taken for a given amount of substrate to react and be used up.

In method 2. the hydrolysis of starch to maltose by amylase is followed by measuring the time a given amount of starch is used up.

 

The experiments described below can be adapted to look at the four main factors (variables) which can affect and control the speed of an enzyme catalysed reaction.

Theoretically there are four main variables you can investigate for any enzyme reaction.

All of the following can all affect the speed of an enzyme catalysed reaction.

The concentration of the substrate.

The concentration of the enzyme

The temperature of the reaction medium.

The pH (how acidic/alkaline) of the medium

This means three of the variables involved in an experimental investigation must be kept constant except for the variable you are investigating e.g.

(a) Varying the concentration of the substrate or enzyme at a constant temperature and constant pH.

Room temperature is convenient, but reaction might be too slow - trial and error!

You must keep either the substrate concentration, or the enzyme concentration constant, and monitor the rate effect of changing the concentration of the other.

The laboratory temperature can vary even in a single lesson so using a thermostated water bath is advisable - if available. A temperature of 25oC to 35oC is likely to be suitable, but must be kept constant.

The pH should be automatically fairly constant and no buffer should be needed unless any acidic or alkaline substances are formed or removed as a consequence of the enzyme catalysed reaction.

If in doubt use a buffer of pH close to the optimum pH of the enzyme.

A buffer is a special solution of chemicals that resists pH changes if small amounts of acid or alkali are added to, or removed from, a solution.

If in doubt, use a buffer solution that matches the optimum pH of the enzyme.

Your body automatically does this by various chemical means, to keep most of your cell and body fluids around ~pH 7.3, safe!

(b) Varying the temperature of the reaction mixture keeping the concentrations of both substrate and enzyme constant and at a constant pH.

If in doubt about the constancy of the pH, use a buffer matching the enzyme's optimum pH.

Again, using a thermostated water bath is advisable and the best way to vary the temperature e.g. conducting experimental runs at 20oC, 25oC, 30oC, 35oC, 40oC etc.

(c) Varying the pH of the reaction medium using different buffer solutions. Ideally using a range of at least five buffers ranging from pH 2 to pH 11. The temperature and concentrations of substrate and enzyme must ALL be kept constant.

Again, using a thermostated water bath is advisable to keep the temperature constant e.g. somewhere in the range 25oC to 35oC.

 

To get good results takes a lot of hard careful work and sometimes 'trial and error', but the experiments can be divided out amongst a class ad the pooled data analysed by all the class.

To get good data in one lesson the work may be split between groups of 2-3 students.

See also GCSE chemistry notes: How we measure the speed or rate of a chemical reaction


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Method 1. A method of measuring the rate of product formation from an enzyme reaction

The decomposition of hydrogen peroxide by the enzyme catalase

In this experiment you are measuring the rate at which oxygen is formed from the enzyme catalase decomposing hydrogen peroxide. Here, the product oxygen gas, provides the means of following the rate of the reaction.

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

2H2O2(aq)  ====>  2H2O(l)  +  O2(g)

The basic procedures for method 1.


(a) Investigating the effect of changing the concentration for an enzyme reaction

(hydrogen peroxide to oxygen using catalase)

(vary either the hydrogen peroxide or the enzyme catalase).

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

2H2O2(aq)  ====>  2H2O(l)  +  O2(g)

Mashed up potato made into a fine slurry diluted with acts as a source of the enzyme catalase. It needs to be well shaken before use so that each portion measured out has the same amount of catalase in it.

A slurry is a pulverized solid mixed in a liquid.

You need a series of hydrogen peroxide solutions of known different concentrations and a fixed concentration of the potato mix to investigate the effect of changing the hydrogen peroxide concentration.

You may have to do some 'trial and error' experiments to find out which amounts give 'reasonable' results.

You can also keep the hydrogen peroxide concentration constant and do the investigation with a set of different concentrations of the potato-catalase mixture.

The water bath is set to a constant temperature e.g. 25oC. The apparatus is setup as illustrated above.

The optimum conditions for human catalase are pH 7 (so no need for buffer) and 37oC

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - but record and monitor the room temperature.

You can use a beaker of heated water but its difficult to keep it at a constant temperature.

The 'stock' solutions of potato-catalase or hydrogen peroxide should be initially in separate boiling tubes and placed in the water bath so that everything starts at the right temperature. Or, if no water bath available, they can be just put together in test tube racks on the laboratory bench, but you should monitor and record the room temperature.

Depending on what accuracy you require, you can measure out a fixed amount of the potato slurry and a varied amount of the same hydrogen peroxide solution into the boiling tube using a pipette, or 10 cm3 measuring cylinder or plastic syringe. You should keep the total volume of the reaction mixture the same.

There shouldn't be a need for a buffer, but the mixture should have a constant pH of ~7.

If in doubt build a fixed volume of a pH 7 buffer into your method.

You should make up the reaction mixture of hydrogen peroxide and potato slurry as quickly as possible and shake well.

Your reaction mixture to vary the hydrogen peroxide concentration may be as follows

w cm3 of buffer (if used)

x cm3 of potato slurry - kept constant

y cm3 of hydrogen peroxide solution - variable

z cm3 of water - variable

w + x + y + z = total constant volume and volume y + z must also be kept constant.

You can vary y and z to give different concentrations if different stock solutions of

By varying volumes y and z you can produce a range of hydrogen peroxide concentrations if a variety of stock solutions are not available..

The boiling tube and mixture is quickly connected to the delivery tube rubber bung and placed in the water bath and the stop watch started. Make sure the boiling tube is fully immersed in water so it and the contents are at the right temperature.

Start the stopwatch. You can now measure how much oxygen is formed in a set time e.g. 1 minute, and repeat the experiment several times with the same volumes of reactants at the same temperature.

This will allow a more accurate mean value of the rate of reaction to be used in the final analysis.

(Or set of volume readings for one run over a longer time, and plot graph of volume versus time and measure the initial gradient, but more work for repeats - see graph on the right)

However you get the results, the rate is calculated as follows:

From the initial gradient of the graph, the rate of enzyme reaction is expressed as:

rate = volume of O2 formed ÷ time taken (cm3/s)

(c) doc bYou then draw a graph of the mean values of the rate of reaction (in cm3/s) at each temperature versus concentration.

You should find that the rate increases with increase in either hydrogen peroxide or enzyme concentration, if you have been very accurate you may get a nice linear graph like the one on the right or else!

You then repeat the whole exercise with different concentrations of the enzyme using the kind of x + y + z 'recipe' described above using a fixed concentration of hydrogen peroxide.


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Gas syringe system

It is possible to get more accurate results using a gas syringe system, as long as the flask can be set up in a water bath (omitted from the diagram below!) or the laboratory temperature stays constant.

The investigation is conducted in the same way as already described above.

Factors affecting the rates of Reaction - theory and methods of measuring the speed of a reaction (c) Doc Brown

You can get more accurate data of the volume of oxygen formed over time and from the graphs work out the initial rate of reaction from the initial gradient (see right-hand side of above diagram).

Graph line A (steeper gradient) compared to graph line B may represent (i) an increase in concentration of either substrate or enzyme, or (ii) an increase in temperature or (iii) a solution pH nearer the optimum value for that particular enzyme.

See also GCSE chemistry notes: Effect on rate of changing reactant concentration in a solution


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(b) Investigating the effect of changing temperature for an enzyme reaction

 (hydrogen peroxide to oxygen using catalase)

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

2H2O2(aq)  ====>  2H2O(l)  +  O2(g)

Mashed up potato made into a fine slurry diluted with water acts as a source of the enzyme catalase. It needs to be well shaken before use so that each portion measured out has the same amount of catalase in it.

You need a hydrogen peroxide solution of known and constant concentration.

You may have to do some 'trial and error' experiments to find out which amounts give 'reasonable' results.

You should also use the same volume of the well shaken potato slurry.

The water bath is set to the start temperature e.g. 20oC. The apparatus setup is illustrated above.

You could start as low as 10oC perhaps by cooling the water with ice, not sure how well it would work?

The two 'stock' solutions should be initially in separate boiling tubes and placed in the water bath so that everything starts at the right temperature  - solutions and boiling tube.

Depending on what accuracy you require, you can measure out a fixed amount of the potato slurry and a fixed amount of the same hydrogen peroxide solution into the boiling tube using a pipette or a 10 cm3 measuring cylinder or plastic syringe. You should keep the total volume of reaction mixture constant.

(There shouldn't be a need for a buffer, the mixture should have a constant pH of ~7)

(If in doubt use a buffer to match the optimum pH of the enzyme catalase).

You make up the reaction mixtures as quickly as possible in a boiling tube and shake well.

The boiling tube and mixture is quickly connected to the delivery tube rubber bung and the stop watch started.  Make sure the boiling tube is fully immersed in water so it and the contents are at the right temperature.

Start the stopwatch. You can now measure how much oxygen is formed in a set time e.g. 1 minute, and repeat the experiment several times with the same volumes of reactants at the same temperature.

Repeats will allow a more accurate average value of the rate of reaction to be used in the final analysis.

  (or set of volume readings for one run, plot graph of volume versus time and measure the initial gradient, but more work for repeats - see the graph on the right)

From the initial gradient of the graph, the rate of enzyme reaction is expressed as:

rate = volume of O2 formed/time taken (cm3/s)

(c) doc bYou then repeat the whole exercise at 30oC, 40oC, 50oC etc. adjusting the thermostat temperature control.

You should find from 20oC to 40oC an increase in the rate of oxygen production, but an increasingly slower rate of reaction from 50oC to 70oC (see graph on bottom right).

You then draw a graph of the mean values of the rate of reaction (in cm3/s) at each temperature versus temperature.

See the end of method 1. (a) for a gas syringe method

See also GCSE chemistry notes: Effect on rate of changing the temperature of reactants


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(c) Investigating the effect of changing pH for an enzyme reaction

(hydrogen peroxide to oxygen using catalase)

Enzyme reaction equation: hydrogen peroxide === catalase ===>  water  +  oxygen (gas)

2H2O2(aq)  ====>  2H2O(l)  +  O2(g)

Mashed up potato made into a fine slurry diluted with water acts as a source of the enzyme catalase. It needs to be well shaken before use so that each portion measured out has the same amount of catalase in it.

You need a hydrogen peroxide solution of known and constant concentration AND stock solution of the potato slurry to provide the enzyme catalase.

You may have to do some 'trial and error' experiments to find out which amounts give 'reasonable' results.

You need a range of at least five stock solutions of buffers giving a variety of pH values e.g. ideally from pH 2 to pH 11.

A buffer solution keeps the pH constant in a reaction medium - it can neutralise small amounts of acid or alkali formed.

The water bath is set to a constant temperature e.g. 25oC-35oC.

The higher temperature is faster - do a trial run, if too slow raise the temperature, but don't go above 35oC).

The apparatus setup is illustrated above.

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - measure and monitor.

The 'stock' solutions of catalase, hydrogen peroxide and the buffer solutions should be initially in separate boiling tubes and placed in the water bath so that everything starts at the right temperature. Or, if no water bath available, they can be just together in test tube racks on the laboratory bench, but you should monitor and record the room temperature.

Depending on what accuracy you require, you can measure out a fixed amounts of the potato slurry, hydrogen peroxide solution and the buffer solutions into the boiling tube using a pipette or more accurately with a 10 cm3 measuring cylinder.

Whatever your 'recipe', keep the total volume of the three solutions constant for the final reaction mixture.

The three solutions are mixed in a boiling tube and well mixed, the total volume should be constant and you use the same concentrations of the hydrogen peroxide and potato-catalase. The pH of the buffer should be the only variable.

The boiling tube and mixture is quickly connected to the delivery tube rubber bung and the stop watch started.  Make sure the boiling tube is fully immersed in water so it and the contents are at the right temperature.

Start the stopwatch. You can now measure how much oxygen is formed in a set time e.g. 1 minute, and repeat the experiment several times with the same volumes of reactants at the same temperature.

This will allow a more accurate mean value of the rate of reaction to be used in the final analysis.

(or set of volume readings for one run, plot graph of volume versus time and measure the initial gradient, but more work doing repeats - see the graph on the right)

From the initial gradient of the graph, the rate of enzyme reaction is expressed as:

rate = volume of O2 formed/time taken (cm3/s)

(c) doc bYou then repeat the whole exercise with different pH buffer solutions.

You then draw a graph of the mean values of the rate of reaction (in cm3/s) versus the pH, and it 'may' look like the graph on the right.

See the end of method 1. (a) for a gas syringe method


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Method 2. A method of measuring the reduction of substrate concentration in an enzyme reaction

The starch to maltose reaction - method 2 - monitoring a colour change

In this case you are detecting when the substrate (starch) is used up when it is broken down to maltose by the enzyme amylase.

Enzyme reaction equation: starch  +  water  === amylase ==> maltose

2(C6H10O5)n  + nH2O  ====> nC12H22O11

Starch is a natural polymer where n is a very large number.

This reaction is called a 'hydrolysis' because a molecule reacts with water to give two or more products.

You can use 'baby rice' for this experiment!

The method for this particular reaction relies on detecting the presence of starch using iodine (illustrated above). You use a dilute solution of iodine dissolved in potassium iodide solution, which is orangey-brown in colour.

When the iodine solution is added to a starch solution a blue-black colour is seen.

This is a simple standard biological laboratory test for starch and you simply take a sample from the reaction mixture and test it for starch using the iodine solution in the wells of a spotting tile.

When you no longer see a blue-black colour, all the starch is used up and you note the time taken.

The rate of the enzyme reaction can be expressed as the reciprocal of the reaction time: 1 / time in s-1 or min-1

This involves using a continuous sampling system - all the experimental details further down.

 

Using a colorimeter to follow the speed of a reaction

This method is fine in a school/college laboratory where simple apparatus will be available for several groups in a class.

However it is possible to measure the intensity of the blue-black colour using an electronic instrument called a colorimeter.

A sample of the reaction mixture is diluted and put into a special tube and the colorimeter apparatus can measure the intensity of the blue-black colour.

The dilution must done with the same volumes of reaction mixture and water used to dilute it.

A special light filter allows you to measure just the intensity of the colour you are interested in.

You also take a blank reading without the starch present. From a calibration graph of known starch concentrations versus their colour intensity with iodine you can actually measure the concentration of the starch as it decreases with time.

This method does avoid the uncertainty of when the blue-black colour actually completely disappears. In the methods described below you are measuring the average rate and the rate is varying all the time. This colorimeter method avoids errors due to the varying speed of the reaction, which is always slowing down!

Light from a suitable source is passed through a light filter to select the most appropriate wavelength of light, some of which is then absorbed by the solution held in a special glass cuvet (a sort of 'test tube'). The amount of light absorbed is called, and measured as, the absorbance which is a function of the coloured solute concentration.


(a) The effect of changing concentration for an enzyme reaction - method 2.

(starch to maltose using amylase)

(vary either the starch or amylase concentration)

Enzyme reaction equation: starch  +  water  === amylase ==> maltose

2(C6H10O5)n  + nH2O  ====> nC12H22O11

The theory of the method is explained at the start of the method 2. section

The basic experimental procedure for method 2.

You can start with 1% starch solution and 1% amylase solutions are suitable concentrations for this investigation.

The presence of starch is detected with orangey-brown dilute iodine solution which gives a blue-black colour with starch.

When all the starch is broken down, the iodine solution colour is unchanged i.e. it remains an orangey-brown.

The water bath is set to a constant temperature e.g. 25oC-35oC.

The higher temperature is faster - do a trial run, if too slow raise the temperature, but don't go above 35oC).

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - but record and monitor the room temperature.

The boiling tubes of stock solutions of starch and amylase of varying concentrations should be put into the water bath, so everything involved is at the same start temperature.

A portion of dilute iodine solution is placed into all the wells of the white spotting tile with a pipette.

Portions of the starch and amylase are carefully measured out into a boiling tube and shaken well and immediately placed in the water bath and the stop watch started.  Make sure the boiling tube is fully immersed in water so it and the contents are at the right temperature.

You need

x cm3 of amylase solution (constant)

y cm3 starch solution

z cm3 water

x + y + z = constant volume.

y + z must be kept constant, but you can vary them to give different concentrations of the starch.

The pH should stay constant, but if you think you need one then you need a buffer solution that matches the optimum pH of catalase. The volume of the buffer would have to be kept constant.

Start the stopwatch. Once the experiment is underway, with a clean teat pipette you sample every 10 seconds and spot some of the reaction mixture into a well of the iodine solution. At first the iodine solution is turned blue-black showing the presence of starch.

Eventually the iodine solution is unchanged, no blue-black colour observed, then you note the total time elapsed.

Repeat several times so you can calculate a more accurate average reaction time for the final analysis.

You then repeat the whole exercise with different concentrations of starch, keeping the amylase concentration constant OR varying concentrations of amylase and keeping the starch concentration constant.

(c) doc bThe reciprocal of the time (1 ÷ time) for the starch to be used up gives you a relative measure of the rate of the enzyme reaction.

(The rate has arbitrary units of reciprocal seconds or minutes)

The rate of the enzyme reaction can be expressed as the reciprocal of the reaction time: 1 / time in s-1 or min-1

As the concentration of either starch or enzyme is increased, the reaction time should decrease - upper graph on the right.

(c) doc bYou then draw a graph of the mean values of the rate of reaction (1/time) at each temperature versus concentration - lower graph on the right. The graph should show an optimum value of the maximum rate of reaction for that particular mixture.

See also GCSE chemistry notes: Effect on rate of changing reactant concentration in a solution


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(b) The effect of changing temperature for an enzyme reaction - method 2.

(starch to maltose using amylase)

Enzyme reaction equation: starch  +  water  === amylase ==> maltose

2(C6H10O5)n  + nH2O  ====> nC12H22O11

The theory of the method is explained at the start of the method 2. section

1% starch solution and 1% amylase solutions are suitable concentrations for this investigation.

The presence of starch is detected with orangey-brown dilute iodine solution which gives a blue-black colour with starch.

When all the starch is broken down, the iodine solution colour is unchanged i.e. it remains an orangey-brown.

The thermostated water bath should be set to an initial temperature e.g. 20oC an the experiment repeated at the same temperature raising the temperature to higher values e.g. ideally the range from 25oC, 30oC, 35oC, 40oC, 45oC and 50oC.

The boiling tubes of stock solutions of starch and amylase should be put into the water bath, so everything involved is at the same start temperature.

A portion of dilute iodine solution is placed into all the wells of the white spotting tile.

 

The basic experimental procedure for method 2.

Portions of the starch and amylase are carefully measured out into a boiling tube which is immediately placed in the water bath and start the stop watch. Make sure the boiling tube is fully immersed in water so it and the contents are at the right temperature.

With a teat pipette you sample every 10 seconds and spot some of the reaction mixture into a well of the iodine solution. At first the iodine solution is turned blue-black showing the presence of starch.

Eventually the iodine solution is unchanged, no blue-black colour observed, then you note the total time elapsed.

Repeat several times so you can calculate a more accurate mean reaction time for the final analysis.

You then repeat the whole exercise at 30oC, 35oC, 40oC, 45oC and 50oC etc. adjusting the thermostat temperature control.

The reciprocal of the time (1/time) for the starch to be used up gives you a measure of the rate of the enzyme reaction.

The rate of the enzyme reaction can be expressed as the reciprocal of the reaction time: 1 / time in s-1 or min-1

e.g. if all the starch is gone in 90 seconds, the rate = 1/90 = 0.011 (arbitrary units of reciprocal seconds)

(c) doc bYou should find from 20oC to 40oC a decrease in total reaction time for the starch to be used up, but an increasingly longer reaction time from 50oC to 70oC.

You then draw a graph of the mean values of the rate of reaction (1/time) at each temperature versus temperature. The graph should show an optimum value of the maximum rate of reaction for that particular mixture.

See also GCSE chemistry notes: Effect on rate of changing the temperature of reactants


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(c) The effect of changing pH for an enzyme reaction

(starch to maltose using amylase)

Enzyme reaction equation: starch  +  water  === amylase ==> maltose

2(C6H10O5)n  + nH2O  ====> nC12H22O11

The theory of the method is explained at the start of the method 2. section

1% starch solution and 1% amylase solutions are suitable concentrations for this investigation.

You need a good range of buffer solutions, preferably at least five ranging from pH 2 to pH 11.

A buffer solution keeps the pH constant in a reaction medium - it can neutralise small amounts of acid or alkali formed.

The presence of starch is detected with orangey-brown dilute iodine solution which gives a blue-black colour with starch.

When all the starch is broken down, the iodine solution colour is unchanged i.e. it remains an orangey-brown.

The basic experimental procedure for method 2.

The thermostated water bath should be set to a suitable temperature e.g. 25oC to 35oC.

If no thermostated water bath is available you can get reasonable results if the laboratory temperature stays reasonably constant - but record and monitor the room temperature.

The boiling tubes of stock solutions of starch, amylase and various buffer solutions of differing pH should be put into the water bath, so everything involved is at the same start temperature.

You can use plastic syringes to measure out the volumes.

You should use different syringes for the buffer solutions, starch solution and the amylase solution.

You should keep the total volume of the final mixture constant, but varying the buffer pH.

A portion of dilute iodine solution is placed into all the wells of the white spotting tile.

Portions of the starch, amylase and buffer solution are carefully measured out into a boiling tube which is immediately placed in the water bath and start the stop watch.  Make sure the boiling tube is fully immersed in water so it and the contents are at the right temperature.

One the experiment is underway, with a teat pipette you sample every 10 seconds and spot some of the reaction mixture into a well of the iodine solution. At first the iodine solution is turned blue-black showing the presence of starch.

Eventually the iodine solution is unchanged, no blue-black colour observed, then you note the total time elapsed.

The rate of the enzyme reaction can be expressed as the reciprocal of the reaction time: 1 / time in s-1 or min-1

Repeat several times so you can calculate a more accurate average reaction time for the final analysis.

 You then repeat the whole exercise with buffers of differing pH.

The reciprocal of the time (1/time) for the starch to be used up gives you a measure of the rate of the enzyme reaction.

(The rate has arbitrary units of reciprocal seconds)

You then draw a graph of the mean values of the rate of reaction (1/time) at each temperature versus the pH of the buffer solution.

You should get a peak around pH 5, a bit like the graph on the right.


See a 'decay' investigation using milk and lipase  gcse biology revision notes

Its part of the web page Carbon cycle, nitrogen cycle, water cycle, decomposition - decay investigation 


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Doc Brown's Very Unoriginal Roman Bread Recipe! - but an excellent example of domestic enzyme technology!

This recipe makes about 2 kg of bread (just over 4 lbs).

1. Weight out 500 g strong wholemeal bread flour plus 500 g strong white flour and mix in large bowl.

(I use Doves Farm organic flour in next village, other brands are available!)

2. Have two sachets of dried yeast (local village Co-Op!) and a whisk handy.

3. Add one teaspoon of sugar to 450 to 500 (max) ml of warm water (~30oC) and whisk in the dried yeast - thoroughly mix evenly.

Mix 3 tablespoons of honey (or 1 of honey + 1 of golden syrup + 1 of treacle, a sweeter mix I personally enjoy) plus 1 teaspoon of salt in 450 to 500 (max) ml of warm water.

I then mix the two in a very large jug with a large desert spoon of olive oil.

4. Rapidly add the yeast/sugars/olive oil mixture to the flour, mix well and knead the dough for 15 minutes.

The kneading slightly oxygenates the yeast and 'energises it', even though its an anaerobic respiration reaction!

Research has shown that yeast is activated by the presence of a little oxygen - not everything is anaerobic chemistry in bread making!

5. More great chemistry. Silicone based non-stick baking pans, which I use without greasing - they have proved a most excellent buy.

6. Find warm place and allow bread to rise thanks to the production of carbon dioxide gas.

glucose (sugar) == enzyme zymase ==> ethanol + carbon dioxide

C6H12O6(aq) ====> 2C2H5OH(aq) + 2CO2(g) 

However, the ethanol (alcohol) a liquid at room temperature, will vapourise in the dough in the oven to give a little bit more of a rising action.

7. It should rise nicely over at least an hour in a warm location.

Then bake for 40-45 minutes in a pre-heated oven at 180oC (~356oF).

8. Time to sample the produce with its lovely earthy texture and taste - as the Roman's would have enjoyed?!

9. Add butter and eat! Even better, toast it.

The flavour of the honey really comes out of this crispy delicacy  and the butter becomes optional!


See also Enzymes and Biotechnology and ethanol from fermentation (gcse chemistry revision notes)


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APPENDIX 1 Summary of the digestion system

The parts and function of the parts of the alimentary canal and the role of gut bacteria

  • You need to know about the structure and functions of the  tissues and organs of the human digestive system, including adaptations to function and how the digestive system digests food using enzymes as biological catalysts.

  • Through the digestive organs food and liquids travel as they are swallowed, digested, absorbed, and leave the body as faeces.
  • You should also know the importance of bacteria in the human digestive system

  • These organs include the mouth, pharynx (throat), oesophagus, stomach, small intestine, large intestine, rectum, and anus.
  • The alimentary tract is part of the digestive system and is also called digestive tract and gastrointestinal tract.
  • Know that organs are made of different tissues acting together to perform some particular function.

    • Know that one organ may contain several tissues.

    • Know that the stomach is an organ that contains:

      • muscular tissue, to churn the contents and break up the food into smaller chunks to aid digestion,

      • glandular tissue, to produce digestive juices containing enzymes to break food down at the molecular level,

      • epithelial tissue, to cover the outside and the inside of the stomach.

  • Know that organ systems are groups of organs that work together to perform a particular function.

    • Know that the digestive system is one example of a system in which humans and other mammals exchange substances with the environment.

      • The digestion process requires a variety of enzymes to breakdown food into soluble products we can absorb from the digestive system, and these enzymes are produced by specialised cells in the glands and gut system.

      • Large insoluble molecules like proteins, starch like carbohydrates and oils/fats cannot pass through the membranes of the cell walls of the gut system.

      • However, smaller soluble molecules like amino acids, sugars and fatty acids can pass through the walls of the digestive system.

      • Some examples of the enzymes responsible for the breakdown of large insoluble molecules into small soluble absorbable molecules are ...

        • protease enzymes like pepsin convert proteins to amino acids,

        • carbohydrase enzymes like amylase convert carbohydrates like starch to sugars such as glucose,

        • lipase enzymes convert oils/fats to fatty acids and glycerol,

        • and these smaller molecules can now pass from the small intestine into the blood capillaries.

There are 8 sections to the alimentary canal.

Know that the digestive system includes, and their functions:

(1) In the mouth, salivary glands produce the enzyme amylase which can break down carbohydrates like starch. The saliva also moistens the food and, together with the chewing action (mastication) of the mouth muscles, balls of food are formed that are easily swallowed. The chewing action of the teeth mashes the food and increases the surface area the enzymes can act on.

(2) The oesophagus (gullet) is a tube that connects the mouth to the stomach and its lined with muscles that help move the balls of food along (this action is an example of peristalsis).

(3) In the stomach the food is churned and broken up into smaller chunks by the muscles of the stomach wall. The protease enzyme pepsin is secreted which can break down proteins to amino acids. At the same time hydrochloric acid is produced, killing most of the bacteria present and creates the right acidic pH conditions (~pH 2) for the protease enzyme, which works best in these acid conditions to break down proteins into amino acids.

(4) The liver produces alkaline bile, which neutralises excess stomach acid (most enzymes can't work in very acid conditions), and bile helps to emulsify oils/fats. The emulsification is essential for the efficient faster digestion of oils/fats, the oils/fats are more dispersed giving greater surface area (greater surface area - think of the oil/fat as broken down into smaller droplets/particles).

(5) The gall bladder stores bile before its released into the small intestine to help with digestion.

(6) The pancreas gland tissue produces digestive juices containing the enzymes (i) protease pepsin (breaks down proteins), (ii) amylase (breaks down starches) and (iii) lipase (breaks down oils/fats), which are released into the small intestine.

(7) The small intestine is where digestion process continues with the release of the enzymes from the pancreas.

Here the absorption of soluble food into the blood stream occurs from the digestive system e.g. smaller molecules like amino acids, sugars and fatty acids - the products of enzyme breakdown of larger food molecules (x-reference with the enzymes above, which are released into the small intestine).

These smaller molecules can then be absorbed by diffusion through the gut wall into the bloodstream and transported to wherever they are needed in the body i.e the cells and tissues of the organ systems.

Note that large insoluble molecules cannot be absorbed into the body.

For more details on the structure and function of the small intestine see The small intestine and villi

(8) In the large intestine excess water is absorbed from the undigested food, producing faeces which are initially stored in the rectum before release through the anus!

(9) The role of gut bacteria and fungi (known as the microbiome)

The microbiome, or gut flora, are the microorganisms, including bacteria, archaea, fungi, and viruses that live in the digestive tracts of animals. The gut is the main location of the microbiome in human beings.

Bacteria are unicellular organisms and there are over 100 million bacterial cells in our alimentary canal.

Most of these are in the lower end of the small intestine and in the large intestine.

These bacteria are naturally found in your body and are not usually harmful to you.

These gut bacteria ...

(i) produce enzymes that help digest food,

(ii) synthesise important vitamins such as vitamin K,

(iii) produce important useful hormones,

(iv) reduce the possibility of harmful bacteria growing in your intestine and causing illness.

You should be able to recognise the organs of the digestive system on a diagram and know their function where described.

  • eg salivary glands, stomach, gall bladder, liver, large intestine, pancreas, small intestine, rectum

See also Surfaces for the exchange of substances in animal organisms (includes small intestine)


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