Science and Plants for Schools – Student Project Starter
Enzymes and their activity . . . in fruits and vegetables
This is a project starter, suitable for Advanced Higher biology investigations or
A-level extended projects. Don’t forget to credit this resource in your
bibliography by including the title, the website, the web address and the date
you accessed it.
Have you cut open a freshly picked apple (or newly dug potato) and seen how
quickly it goes brown? Then compare this with an apple or potato stored for
several weeks. Which do you think goes brown more quickly?
The metabolic changes that occur in ripening fruit and vegetables are a
reflection of changes in the activity of enzymes within their tissues. An
observed change in one physical character of the fruit, such as softness or
colour, may be the result of changes in activity of one or more of a bewildering
variety of enzymes. An enzyme may, for example, be responsible for
degrading a particular component of the plant cell wall, or degradation of a
particular pigment leading to a change in colour. Some of these naturally
occurring enzymes, such as pectinase and cellulase, are used in the food
processing industry - for example, in fruit juice manufacture.
In the process of ripening, followed by senescence or spoilage, the activity of
a range of different enzymes may change and indeed may be different in
different fruits or vegetables. This offers possibilities of a wide range of
enzyme-based investigations, examining some of the following:





the changes in the activity of an enzyme during ripening and / or storage
of a particular fruit or vegetable
the activity of the same enzyme in different species
the properties of the same enzyme extracted from different species or
varieties (comparing optimum pH, temperature, etc.)
the loss of an enzyme substrate (such as pectin or starch) during the
ripening process
the appearance of an enzyme product (such as glucose or galactose)
during the ripening process
Practical protocols and other suggestions to explore
Each of the enzymes listed below is likely to show variations in activity during
ripening and storage of fruits and vegetables. These give relatively simple
methods that can be used to assay their activity.
An investigation from Science & Plants for Schools, www.saps.org.uk/students
Updated June 2012
1. Amylase
2. Cellulase
3. Pectinase
4. Polygalacturonase
5. Polyphenoloxidase (Catechol oxidase)
6. Protease
Other enzymes you could explore
1. Catalase
2. Peroxidase
3. Phosphatase
Cellulase assays
Cellulase enzymes show activity during the ripening of some fruits, where
their effects on cell walls results in softening of the fruit. In cases of
programmed cell death, such as the formation of aerenchyma (large air
spaces in the cortex of plants in flooded soils), and in the abscission zones of
leaves and fruits, cellulases are once again very active, breaking down the
cellulose walls of the dead cells.
Viscosity reduction method
The technique is based on the action of cellulase enzymes which shorten the
lengths of cellulose molecules in a viscous solution of wallpaper paste and
cause it to become less viscous (runnier).

Make up a 2% (w/v) wallpaper paste solution, sufficient to provide 25
cm3 for each sample to be tested.

Place 25 cm3 of the paste in a boiling tube and add 2 to 5 cm3 of fruit
extract. Mix thoroughly.

Then pour the mixture into the barrel of a syringe, held in a retort stand,
pointing downwards into a small beaker. Note the time taken for all the
mixture to drain through the syringe nozzle into the beaker.

Incubate the mixture in a water-bath at 30°C, checking the change in
viscosity about every 30 minutes.
The more active the enzyme, the greater the reduction in viscosity, and so the
shorter the drainage times.
An investigation from Science & Plants for Schools, www.saps.org.uk/students
Updated June 2012
Pectinase assay
Pectinases are actually a mixture of enzymes, which, along with others such
as cellulase, are widely used in the fruit juice industry where they are widely
used to help extract, clarify and modify fruit juices. (See the foot of this page
for more information)
Pectins are large polysaccharide molecules, made up (mainly) of chains of
several hundred galacturonic acid residues. Enzymes in this pectinase group
include polygalacturonases, pectin methyl esterase and pectin lyases. These
pectinase enzymes act in different ways on the pectins, which are found in the
primary cell walls and in the middle lamella. Pectins are well known also for
their ability to form gels.
Pectinases are produced during the natural ripening process of some fruits,
where together with cellulases, they help to soften their cell walls. These
enzymes are also secreted by plant pathogens such as the fungus Monilinia
fructigena and the soft-rot bacterium Erwinia carotovora, as part of their
strategy for penetrating the plant host cell walls. In fact, the products of such
enzyme assaults (oligosaccharins) act as a signal which induces uninfected
cells to defend themselves.
The principle of this assay depends upon measuring the amount of watery
juice released from tinned apple puree (which is very rich in pectin) as a result
of pectinase action.
Tinned or bottled apple puree (sauce) can be purchased for this assay.

To make an extract of the fruit or vegetable, blend 2 cm3 of water for
every 1 g of fruit. Prepare at least 25 cm3 of extract.

Collect and label two boiling tubes and place 25 cm3 of apple sauce in
each of them.

Add 25 cm3 of extract to one of the tubes and 25 cm3 of water to the
other, to act as a control.

Use a glass rod to mix thoroughly the contents of both tubes and then
leave them in a boiling tube rack in a water bath at 35°C for at least 30
minutes.

Take two similar sized funnels (funnel size about 100 cm3 is suitable)
and support them over two similar small (25 cm3) measuring cylinders.

After the incubation period, pour the contents of the two boiling tubes
into the two funnels, and allow the juices to drain into the measuring
cylinders. Allow at least five minutes for the draining to finish and note
the volumes of juice obtained.
An investigation from Science & Plants for Schools, www.saps.org.uk/students
Updated June 2012
The differences in the volume of juice between the two tubes gives a measure
of the pectinase activity in the extract.
If this experiment is being carried out on a large scale, the initial drainage
from the funnels could be collected in test tubes, and then the volume of juice
collected is measured later.
Note: when using commercially prepared enzymes in your investigations . . .
Many industrial enzyme products are mixtures of different enzymes. Thus a
'pectinase' preparation might contain a range of pectinases and cellulases.
Other preparations might contain only a single type of enzyme, especially if
the enzymes are produced by genetically-modified strains or are highly
purified.
When planning your own investigations, it is therefore very important to
study the data sheet supplied with each enzyme. This will indicate whether
the product is a mixture or contains just one type of enzyme.
The data sheet also provides a rough guide to how the enzyme might
behave. In practice, however, enzyme activity is affected by many things (e.g.
pH, temperature, the presence of inhibitors or cofactors) which will affect the
results you obtain. In addition, specimen activity graphs are often prepared
using simple substances under ideal conditions rather than the complex
substrates and sub-optimal conditions that may be encountered in an
industrial or school context.
Polygalacturonase assay
This enzyme is famous for being involved in the development of the GMO
tomatoes (more information from the link at the foot of this page). The cells of
these tomatoes have been genetically modified to contain a reversed copy of
the gene for this enzyme. This produces anti-sense mRNA which combines
with the normal mRNA for polygalacturonase, effectively supressing its
synthesis. As a result the GM tomatoes have only 1% of usual
polygalacturonase activity, and soften much more slowly than unmodified
tomatoes - thus extending their shelf-life.
The normal role of polygalacturonase is to hydrolyse pectins during fruit
ripening, which leads to softening of the fruit. The plant hormone ethene has
been shown to promote the translation of polygalacturonase mRNA, raising
the levels of the enzyme in ripening fruit.
Some plant pathogenic fungi, such as Phytophthora infestans (potato blight),
secrete polygalacturonase during their attack of host plant cell walls.
Another interesting fact is that the regulation of the production of the enzyme
is partly due to ethene (ethylene), which induces the translation of
polygalacturonase mRNA, rather than regulating the transcription step.
An investigation from Science & Plants for Schools, www.saps.org.uk/students
Updated June 2012
The principle of this assay is to mix an extract of fruit (or vegetable) with a
viscous solution of polygalacturonic acid, and then note the decrease in
viscosity due to its breakdown by the enzyme.

Make the extract of the fruit (or vegetable) in a 40 mM sodium acetate
solution (1), buffered to pH 5.0 with hydrochloric acid. Use a volume of
buffer equivalent to the mass of the fruit or vegetable sample (e.g. 10 g
fruit + 10 cm3 buffer). Grind the sample in a mortar and pestle, or if
larger quantities are required, do this in a blender.

To obtain a clear enzyme extract from the mashed up fruit, then either
filter it (could be speeded up on a Buchner funnel) or centrifuge to
produce a clear supernatant containing the enzymes (faster and
simpler).

Then add an equal volume of this enzyme extract / supernatant to the
polygalacturonic acid solution (2). Set up a control by adding an equal
volume of sodium acetate to the same volume of another
polygalacturonic acid sample.

Incubate these mixtures in a water bath at 40°C. Measure the viscosity
at the start and after incubation for about one hour (see next paragraph).

Measure the initial vicosity of these mixtures by drawing up 1 cm3 into a
glass pipette, and then time how long it takes the mixture to drain out
under gravity, to the 0.9 cm3 mark. Repeat the measurement three
times and calculate a mean time. Re-measure the viscosity after
incubation (at 40°C, for 1 hour).
Because of variations in the diameter etc. of pipettes, it would be best to use
the same pipette for all readings, carefully rinsing it out between them. Make
sure the pipette is held vertically, possibly with a retort stand and clamp.
Preliminary experiments with a ripe tomato have shown a 50% decrease in
viscosity after incubation for 1 hour.
(1) Sodium acetate buffer - 40 mM solution made up, then add 1 M HCl
dropwise, whilst pH is monitored with a pH meter.
(2) Make up a 3.2 % (w/v) of polygalacturonic acid (sodium salt), by mixing
the powder with warm distilled water, stirring it thoroughly with a glass rod and
then placing the mixture in a boiling water bath for 10 minutes - stirring
occasionally. Then filter the warm solution or centrifuge it to provide a clear,
viscous solution of polygalacturonic acid for these experiments.
Polyphenoloxidase (catechol oxidases) assay
Browning of the cut surface of some fruits and vegetables is due the presence
of a group of enzymes called polyphenoloxidases. These enzymes are
An investigation from Science & Plants for Schools, www.saps.org.uk/students
Updated June 2012
released by the broken cells and they catalyse the reaction between
colourless molecules called polyphenols and molecular oxygen. This reaction
creates coloured compounds and these new compunds can spontaenously
cross react with one another to form black-brown complexes called melanins.
One example of a substrate for these enzymes is catechol, hence the
alternative name ‘catechol oxidases’ for these enzymes. Catechol is oxidised
initially to the orange compound benzoquinone which is then converted to
melanins.The conversion to melanin is spontaneous but slow.
Polyphenoloxidase
(slowly)
catechol + oxygen --------> benzoquinone + water ------> melanins
Food processing and cooking often involve procedures which are intended to
inhibit the action of polyphenoloxidases. Why do you think a cook immediately
places freshly peeled potatoes into a pan of water? Or why do people
squeeze a few drops of lemon juice on to a freshly cut avocado? Mushrooms
contain high levels of polyphenoloxidases, so how do you think pre-sliced
packaged ones can be prevented from going brown?
The assay
In this technique, the change from a colourless solution of catechol to
coloured benzoquinone is followed with a colorimeter. A fruit extract is added
to a solution of catechol and the rate of formation of coloured benzoquinone is
measured. The faster the rate of increase in absorbance of the reaction
mixture, the greater the polyphenoloxidase activity of the fruit extract.

Make an extract of fruit or vegetable, by grinding in a mortar or blending
with an equivalent mass of water.

Strain the extract through muslin, then centrifuge the filtrate to remove
the remaining solids. (Alternatively, the filtrate could be filtered using a
Buchner funnel, or just allowed to stand - so the solids form a sediment
at the bottom of the vessel. Then draw the liquid off into another vessel.)
Note that if the plant tissues contain much polyphenoloxidase activity,
then the extract itself will become quite darkly coloured — but this need not be
a problem as only a very small volume is used in the reaction mixture. The
enzyme activity of the extract lasts for at least a couple of days, provided it is
stored in a refrigerator.

To prepare a colorimeter tube, add 2 cm3 standard pH 7 buffer and 2
cm3 of 0.1% catechol. Note the time and then add 0.1 cm3 enzyme
extract, quickly mixing the contents of the tube. Place it into a
colorimeter (previously zeroed using a tube with 4 cm3 water and 0.1
cm3 of the extract). Take readings of the absorbance at regular intervals
(e.g. every 10 seconds).
An investigation from Science & Plants for Schools, www.saps.org.uk/students
Updated June 2012

Plot a graph of the change in absorbance against time. An increase in
absorbance is due to the formation of benzoquinone, the product of the
reaction. The initial slope of the graph gives a measure of the
polyphenoloxidase actvity of the fruit or vegetable extract.
Ideas for investigations into the activity of these enzymes in plant tissues

the effects of pH, or ionic concentration, or temperature

the effects of plant pathogen infection

the effects of enzyme inhibitors, such as metal ions

the effects of anti-oxidant chemicals
Alternative techniques for monitoring the progress of the reaction include a
‘low-tech’ method, such as following the reaction by allowing the formation of
melanins overnight, or a ‘high-tech’ method, by following the uptake of
dissolved oxygen from a reaction mixture using an oxygen electrode.
For more information, you will find suggestions for experiments with potato
polyphenoloxidases at http://food.oregonstate.edu/ref/plant/weaver/. Typing
the search term polyphenoloxidase on WWW search engines will provide
plenty of hits.
Protease assay
In certain fruits, such as pineapples and mangoes, the flesh contains proteindigesting enzymes (proteases). These may play a part in helping to soften the
fruit tissues as the fruit ripens, making it even more attractive to animals that
might disperse the seeds. So perhaps the activities of these proteases
enzymes will increase during the ripening process.
Applications of plant proteases, such as ‘bromelain’ from the stems and fruits
of pineapples, include uses in the pharmaceutical industry as a blood anticoagulant, and in the prevention of proteinaceous hazes in chill-proof beers!
The assay
Protease enzymes are added to a milky colloidal suspension of egg albumen.
As the protease enzymes digest the suspended particles of proteins, the
mixture becomes more transparent. The absorbance changes in the reaction
mixture are followed with a colorimeter. The protease content, for example in
extracts of fruits, can be assayed by measuring the rate at which the solution
of egg albumen and extract becomes clearer.
Preparation of the egg albumen colloidal suspension (enzyme substrate)

Separate the white of a single egg into a 250 cm3 beaker and add 150
cm3 tap water, stirring the mixture thoroughly. The mixture becomes
An investigation from Science & Plants for Schools, www.saps.org.uk/students
Updated June 2012
quite cloudy as a result of the denaturation of some of the egg albumen
by the water.

Place the beaker on a tripod and gauze and heat with a bunsen until the
mixture boils, stirring it regularly.

Allow the mixture to cool, then decant it through two or three layers of
muslin into another beaker. This creates a homogenous milky colloidal
solution.
Carrying out the reaction

Select a test-tube that fits into the colorimeter. Add 2 cm3 of an
appropriate buffer (e.g. pH7), followed by 2 cm3 of the albumen
substrate solution and 1 cm3 of fruit extract.

Mix the contents of the colorimeter tube, and place it in the colorimeter
(previously zeroed using a tube with 4 cm3 buffer and 1 cm3 of fruit
extract). Read the absorbance and note the time.

Place the reaction mixture(s) in a water bath at 30°C to promote the
activity of any protease enzymes present.

Take further absorbance readings at regular time intervals (say every 5
or 10 minutes), until no further change (decrease) in absorbance is
detected.

Plot a graph of the change (decline) in absorbance against time.
Measuring the time taken for a 50% reduction in absorbance value gives
an indication of the protease activity of the original fruit extract.
Ideas for investigations with this system (with fruits and vegetables)

Follow the changes in protease enzymes during ripening

Compare the effects of pH on proteases from different fruits

Compare the effects of temperature on proteases from different fruits

Investigate the presence of protease-inhibitors in the seeds of legumes

Monitor the release of amino acids from the digested proteins, using
paper chromatography
Please note that following recent advice on the health risks of using the stain
Congo red the Gel Diffusion Method cellulase assay has been removed from
this document (June 2012). SAPS will search for alternative methods to
undertake this activity. Students are advised against following the previous
versions of this document or using Congo red.
An investigation from Science & Plants for Schools, www.saps.org.uk/students
Updated June 2012