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Enzymes: 8. Method 2. Investigating the enzyme catalysed hydrolysis of starch to maltose with amylase - the effects of changing temperature, pH and concentration, apparatus and chemicals required, experimental procedure described

Doc Brown's Biology exam study revision notes

There are various sections to work through, after 1 they can be read and studied in any order.

needs sub-index

Sub-index of biology notes on enzymes and digestion


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!

For this reaction there are three experimental situations are fully described

(a) Changing concentration of substrate molecule or enzyme

(b) Changing the temperature of the reaction mixture - looking for the optimum

(c) Changing the pH of the reaction mixture - looking for the optimum

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 the enzyme 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


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

(starch to maltose using the enzyme 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


(c) The effect of changing pH for an enzyme reaction - method 2

(starch to maltose using the enzyme 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


Summary of learning objectives and key words or phrases

Know about the experimental methods for investigating the enzyme catalysed hydrolysis of starch to maltose with amylase by amylase and how to investigate the effects of changing temperature, changing pH and changing the concentration.

Know what apparatus is needed and the main points of the experimental procedure for the enzyme catalysed hydrolysis of starch to maltose with amylase, including the apparatus and chemicals required.


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