Download Sourcing, Storing And Handling Enzymes

Sourcing, storing and handling enzymes
Introduction
Enzymes are found in living organisms. Their function is to speed up chemical reactions which would
otherwise be too slow to maintain life. Enzymes catalyse a specific reaction on a specific chemical or
group of related chemicals, called their ‘substrate(s)’.
Enzymes are proteins whose shape and structure are sensitive to the environment they are in.
Therefore, enzymes are prone to degrade if exposed to conditions very different from those in which
they normally occur. Nevertheless, many enzymes are stable enough to be extracted from their natural
sources and kept as a dry powder or a solution. They can be used in artificial reaction vessels such
as test-tubes, alginate balls or agar plates. However, some care needs to be taken to ensure they
remain stable during the activity.
Most enzyme preparations keep better if stored cold, though some newer preparations are less
sensitive. Enzymes designed for use in washing powders have relatively long shelf lives.
Main points to ensure success with enzyme activities
•
Check the enzyme’s properties to choose suitable conditions for the reaction
•
Store enzymes or natural sources of enzymes in the refrigerator
•
Prepare extracts freshly or make sure the purchased enzyme is within its use-by date
•
Prepare diluted enzymes and substrates freshly before use
•
Use any instructions provided, and Recipe Sheet 37, to prepare dilutions
•
Always trial activities to make sure the enzyme is active and will produce results in a reasonable
time. If necessary adjust concentrations of enzyme solution and/or substrate.
•
Diluted enzymes tend to be less stable than other preparations. Keep diluted solutions in the
fridge or on ice if they are to be used over a period of one or two days. If in doubt, re-check the
enzyme activity shortly before use.
Sources of enzymes
It is usually cheaper and may be safer to use natural sources for enzymes if they are readily available.
For example:
•
Saliva is generally an excellent source of amylase; commercial samples can be unpredictable and
if good hygiene precautions are followed (Laboratory Handbook 14.4.3), pupils can use their own
saliva
•
A number of enzymes can be extracted quite easily from fruits, vegetables and liver.
Commercial suppliers derive enzymes from a variety of sources. These can be plant, animal or
microbial, and should be stated. Preparations are usually impure, and include other bits of the source
organism or their growing medium (which is partly why some of these fail to produce clear ‘solutions’).
Although such enzymes may have the same name (eg ‘amylase’) the properties may differ markedly
GL116 JS 05/13
Page 1 of 6
© CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ
Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk
depending on the source organism. Always check very carefully before purchase that the enzyme is
suitable for the intended use.
The activity of naturally-sourced enzymes may vary but they are often more reliable than
commercially-sourced extracts. In addition, suppliers may change their own suppliers, so different
batches may have different properties. They may have suffered poor conditions in transit, affecting
their activity. The short shelf life and the high cost of many commercial enzyme preparations also
make their use quite expensive. Some appear to lose activity quite suddenly, so it is important to
obtain fresh stocks for use in examinations and assessed practical work. Nevertheless, commercial
preparations may be convenient to use, and many good, stable products are available.
Enzymes in the slice of apple cause browning
Liquid, tablet and powder forms of some
commercial enzymes
The table overleaf lists a number of enzymes commonly used in schools and suggests some natural
sources and brief details of their actions and properties.
GL116 JS 05/13
Page 2 of 6
© CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ
Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk
Some enzymes used in school and some of their sources
Enzyme
Where it
occurs
naturally
Amylase
Saliva, gut.
Natural
sources
for school
use
Saliva
Bromelain
Pineapple
Pineapple
Chymosin
Diastase
Lactase / beta
galactosidase
Alternative name for rennin
Alternative name for amylase
Gut, ‘lactic
Yoghurt
acid’ bacteria.
Catalase
Ubiquitous to
cells of all
living things.
Catechol
oxidase
Some fruits
Dopa oxidase
(as for
catechol
oxidase)
Catalyses one reaction in
the synthesis of melanin,
the pigment that makes
skin and hair darker
Invertase /
sucraseisomaltase
Lipase
Gut, some
bacteria.
Breaks down sucrose to
glucose and fructose
Papain
Pepsin
Pectinase
Phosphatases
Phosphorylase
s
Proteases
general
Rennin
Typical properties /
uses / some notes
Breaks down starch to
glucose and maltose. Two
main forms (alpha and
beta) act differently
Breaks down proteins
Used in biological
washing powders.
Some are inhibited by
citrate
Used as a meat
tenderiser
Breaks down lactose to
glucose and galactose
Bacteria with this
enzyme are used to
make milk into
yoghurt.
A very prolific and
highly active enzyme.
Catalyses the breakdown of
hydrogen peroxide, a byproduct of the cell’s
process of detoxification of
free radicals
Responsible for browning
Gut, liver,
blood vessels
Papaya
Inhibited by copper(II)
Inhibited by phydroxybenzoic acid
(competitive),
phenylthiocarbamide
(non-competitive)
Used in the
manufacture of softcentred chocolates.
Used in biological
washing powders
Used as a meat
tenderiser
Breaks down fats to free
fatty acids and glycerol
Papaya
Breaks down proteins,
cleaving at basic amino
acids
Stomach
Breaks down proteins;
Optimal pH = 2
cleaves after the N-terminal
of tyrosine, tryptophan and
phenylalanine
Some fruits
Jam-making Breaks down the jelly-like
Used to help extract
ingredient
pectin
juice from fruit
Cells of all
Mung bean
Remove the phosphate
living things
seedlings
group from their substrate
Cells of all
Adding phosphate groups
living things
to their substrates
See pepsin, rennin, trypsin. Biological washing powders contain proteases
Stomach
Trypsin
Duodenum
Urease
Some beans;
some soil, and
pathogenic
bacteria.
GL116 JS 05/13
Page 3 of 6
Animal and
vegetable
sources eg,
potato, liver,
blood.
Banana,
apple, other
fruit
What it does
Cooking
ingredient
Jack bean,
soya bean
Breaks down proteins
Breaks down proteins,
cleaving after the Cterminal of lysine and
arginine
Breaks down urea to
carbon dioxide and urea
Used to coagulate
milk
Optimal pH = 8.5
A number of proteins
in beans have urease
activity
© CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ
Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk
Preparing enzymes for use
Using diluted enzyme preparations
Diluted enzymes lose their activities relatively quickly. It is best to prepare dilutions freshly. For
activities taking place over one or two days, keep the diluted solutions in the fridge or on ice to
preserve their activity. Even so, it is worth checking their activity before beginning the practical and,
for critical work (eg assessment), during the day.
Follow any instructions provided with the enzyme or activity protocol and also see also Recipe Sheet
37 which includes important safety points and guidance. Allergic responses are rare, particularly with
the small quantities used in schools but take sensible precautions as described on the Recipe Sheet.
•
Always trial an activity to ensure the enzyme is working effectively under the conditions
which will be provided in the lesson.
Preparing enzyme solutions
Many enzymes work best within a certain pH range. If this is close to neutral (pH 7), tap water can
often be used. Distilled water is often acidic, and may be suitable for enzymes with pH optima below 7.
For enzymes which have an optimum which is acidic or alkaline or for testing how enzyme activity
changes with pH, the table below gives simple solutions that will provide a stable enough pH during the
duration of many practical activities. In tests at CLEAPSS, these solutions maintained their pH during
incubation of amylase for 10 minutes.
pHs of water and some common solutions
Solution
Approximate pH
0.1 M hydrochloric acid
1
0.01 M hydrochloric acid
2
(distilled water)
Can be pH 4
(tap water)
Can be pH 7
0.1 M sodium carbonate
11-12
0.1 M sodium hydroxide (IRRITANT)
13
Occasionally a practical requires a very stable pH which uses a buffer to resist pH change. However,
buffers must be used with caution because the chemicals involved can act as either a co-factor or an
inhibitor to the enzyme, completely altering how it works and producing unexpected and incorrect
results. If a buffer is required, the type of buffer should be specified. Recipes for some commonlyused buffers are given on Recipe Sheet 18. Tablets, designed to be made up to certain pHs, should
be used with caution unless their chemical composition is known, and known to not affect living
systems.
Enzyme ‘concentration’
Enzyme concentrations are usually given as %, sometimes followed by m/v or v/v (mass per volume or
volume per volume; i.e the mass or volume needed to make up 100 ml of solution). It is not
appropriate to try to work out molar concentrations of enzymes. For school use, simply make up the
enzyme as a percentage concentration, trial the activity and decide the appropriate concentration to
use to obtain results in a reasonable time.
Why isn’t our enzyme ‘solution’ clear?
Not all enzyme preparations make up clear ‘solutions’; sometimes the enzyme is
insoluble in water, or insoluble products are also present in the preparation. In
some cases, filtering might remove the activity (which may be in the non-soluble
fraction) and you need to use the unclear mixture. If a clear mixture is needed eg,
GL116 JS 05/13
Page 4 of 6
© CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ
Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk
for measuring a product in a colorimeter, it may be better to filter the mixture after incubation.
In the photo, the urease, despite thorough mixing, remains as a cloudy suspension.
Enzyme Glossary
Term
Explanation
Enzyme
Usually a relatively large protein molecule that catalyses a specific
reaction on a particular substrate or group of substrates. The enzyme
does not get used up in the reaction, though, as with other natural
substances, over time its activity may decline.
Substrate
A chemical whose reaction can be catalysed by the enzyme.
Catalyse, catalysis
To speed up a chemical reaction without taking part in it. Thus a
catalyst is a substance that speeds up a reaction. Enzymes are
catalysts. When the reaction is complete and the products have moved
away, the enzyme is ready to catalyse the reactions of further
substrate(s).
Specificity
Each enzyme will bind to a specific substrate or group of substrates
and carry out a specific reaction or type of reaction with it/them.
Active site
The part of the enzyme where the substrate and other chemicals taking
part in the reaction bind to the enzyme and where they react.
Bind, binding
The substrate or substrates attach to the enzyme via chemical bonds.
These bonds are just strong enough to hold the substrate(s) in the
correct position to react. The product(s) do not bind so strongly and
are able to leave the active site.
Enzyme-substrate complex
The enzyme and substrate combined during the reaction.
Lock-and-key hypothesis
The hypothesis that the substrate fits into the active site like a key
fitting into its lock.
Induced-fit hypothesis
The hypothesis that when the substrate attaches to the active site it
causes changes to improve the way they are combined and to make
the reaction work better.
Inhibitor
A substance that interferes with the enzyme’s activity. Inhibitors may or
may not act competitively (see Competitive inhibitor and Noncompetitive inhibitor). Inhibitors might act reversibly or irreversibly,
depending on whether they inflict permanent damage to the enzyme or
merely distort it while they are present.
Competitive inhibitor
An inhibitor that competes with the substrate for its place in the active
site of the enzyme. Competitive inhibitors tend to be similar to the
substrate in some way. The relative concentrations of the substrate
and the competitive inhibitor affects enzyme activity.
Non-competitive inhibitor
An inhibitor that affects the enzyme’s ability to catalyse the reaction
without competing with its substrate. Non-competitive inhibitors could
deform the enzyme, either preventing the substrate from binding to the
active site or from reacting once it is bound. Heavy metals are common
non-competitive inhibitors for many enzymes. Only the concentration of
the non-competitive inhibitor affects enzyme activity.
Cofactor or coenzyme
Usually small molecules or substances that are needed to enable the
enzyme to work. Some vitamins and metals ions are cofactors.
GL116 JS 05/13
Page 5 of 6
© CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ
Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk
Continued
Enzyme Glossary continued
Term
Explanation
Optimum / optimal
conditions
The most favourable conditions for enzyme activity. Eg, the pH,
temperature and concentration of other reagents needed to enable it
to perform at its fastest rate.
Denature
When a protein loses its shape it is denatured. If the protein is an
enzyme, this affects its activity.
In vitro
In artificial reaction vessels (eg, test tubes, agar plates or alginate
balls): refers to activities carried out under these conditions.
In vivo
In a living organism: refers to research carried out on a whole living
organism.
Stability
An enzyme is usually only fully active when it is intact and in good
condition. The conditions needed to maintain the enzyme’s activity or
‘stability’ may differ from the optimal conditions.
Turnover rate
A measure of the number of substrate molecules that can be ‘turned
over’ (react) per active site per unit time.
Activity
The turnover rate per unit mass of the purified enzyme (eg, units per
mg). Due to their large size, instead of referring to the molarity of an
enzyme preparation it is common to refer to its activity. Both
commercial and natural enzyme preparations are impure. In industry
and research, the activity of an enzyme preparation is calculated under
standard conditions. For school work, absolute activity is not relevant. It
is more useful to work out a concentration of the enzyme preparation
that produces meaningful results in a reasonable time, and to express
this as a percentage by mass (eg, 1% m/v).
GL116 JS 05/13
Page 6 of 6
© CLEAPSS®, The Gardiner Building, Brunel Science Park, Kingston Lane, Uxbridge UB8 3PQ
Tel: 01895 251496; Fax: 01895 814372; E-mail: [email protected]; Web site: www.cleapss.org.uk