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SAPS Student Project - enzymes and browning

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					Science and Plants for Schools – Student Project Starter

Enzymes and their activity . . . in fruits and vegetables
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.

 1. Amylase

 2. Cellulase

 3. Pectinase

 4. Polygalacturonase


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 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. Two possible
protocols are described here, a gel diffusion assay method and a viscosity reduction
assay method.

Gel diffusion method

A fruit extract is placed in a well in some agar containing carboxymethylcellulose
(CMC), and, as cellulase molecules diffuse outwards from the well, they destroy
cellulose molecules. The more concentrated the enzymes in the well, the larger the
zone of cellulose destruction.

     Prepare an agar gel containing 1.7% agar and 0.5% CMC
      (carboxymethylcellulose). Pour this gel into petri dishes and allow it to set.

     After the gel has set, use a narrow cork-borer to punch small cylinders in the
      gel. Then, using a mounted needle, remove each of these cylinders to create a
      series of similar sized wells in the agar. Four or more wells can be put in a
      single dish, provided they are spaced apart.

     Place similar volumes of extracts of fruits in the each of the wells. In one well,
      place some distilled water, as a control. Incubate the dishes for at least 24 hours
      at 30 °C.

     After the incubation period is finished, use tap water to rinse out the contents of
      the wells, and then flood the dishes with Congo red solution for 15 minutes.
      Then rinse the dishes with 1 M NaCl solution for at least 10-15 minutes.

Wells containing cellulase should have a clear zone around them, and the diameter of
the zone gives a measure of the cellulase activity in that well.

Viscosity reduction method



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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.

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.



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     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.

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


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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.

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.


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(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 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


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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).

     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 protein-digesting
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 anti-coagulant, 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


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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 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




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