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NAME:_______________________ OPTION GROUP:__________________
ENZYMES 2
IMMOBILISED ENZYMES:
Medical and Industrial
Uses
WORKBOOK
2
Instructions
Regular revision throughout the year is essential. It’s vital you keep a track of what you understand and what you don’t understand. This booklet is designed
to help you do this. Use the following key to note how well you understand the work after your revision. Put the letter R, A or G in the table. If you place an
R or an A then you should make a note of what you are struggling with and the end of this book under the relevant section and seek help with this.
Key R = Red. I am not confident about my knowledge and understanding A = Amber. I am fairly confident about my knowledge and understanding G = green. I am very confident about my knowledge and understanding
STUDY CHECKLIST AND ASSESSMENT OBJECTIVES
3
Assessment Objective Description AO1 Demonstrate knowledge and understanding of scientific ideas, processes, techniques and procedures.
AO2 Apply knowledge and understanding of scientific ideas, processes, techniques and procedures:
• In a theoretical context
• In a practical context
• When handling qualitative data
• When handling quantitative data
AO3 Analyse, interpret and evaluate scientific information, ideas and evidence, including in relation to issues, to:
• Make judgments and reach conclusions
• Develop and refine practical design and procedures
Medical and Industrial Applications of Enzymes
1. Can you define what an immobilised enzyme is?
2. Can you describe several ways in which enzymes are immobilised?
3. Can you distinguish between a qualitative and a quantitative biosensor?
4. Can you describe the functions of the various components of a quantitative (digital) biosensor?
5. Can you list the advantages of a digital biosensor?
6. Can you describe a medical use of a biosensor?
7. Can you describe the industrial production of lactose free milk using immobilised enzymes?
8. Can you explain the effects of bead size and flow rate on how much product is formed during the industrial use of immobilised enzymes.
9. Can you list the advantages of using immobilised enzymes in industrial processes?
10. Can you explain the effect of increasing temperature on immobilised enzymes and compare this to non-immobilised (free) enzymes.
4
Below is a list of some key words and phrases you will need to learn and understand in this immobilised
enzyme topic
1. Biosensor
2. Immobilised enzyme.
3. Qualitative
4. Quantitative
5. Alginate beads
6. Inert matrix
7. Transducer
8. Glucose oxidase
9. Peroxidase
10. Selectively permeable membrane
11. Gluconic acid
12. Hydrogen peroxide
WORD BANK
5
1.0.1 to 1.0.3 Making immobilised enzymes
1.0 WHAT ARE IMMOBILISED ENZYMES?
1.0.1 Definition. An immobilised enzyme is an enzyme that is attached or trapped in or
on an inert matrix
1.0.2 Concept immobilised enzymes can be made by absorption, covalent binding, entrapment and cross linking to the inert matrix.
Adsorption method.
Formed by weak bonds or
interactions between the
enzyme (blue circle) and the
inert matrix.
Entrapment.
Enzymes are not chemically
bonded but trapped in a gel
matrix.
Covalent bonding.
The enzyme is covalently
bonded to a matrix often
made of cellulose or collagen
Cross-linking.
A molecule (small blue
circle) binds to the
enzyme forming cross
links.
6
1.0.3 Prac Concept Enzymes can be immobilized by entrapment in alginate beads.
The procedure is summarised below:
Representation of enzymes (red
circle) immobilised in alginate
gel (blue circle).
7
2.0 MEDICAL USES OF IMMOBILISED ENZYMES
Colour chart
2.0.1 to 2.0.2 Qualitative biosensors
2.0.1 Concept Immobilised enzymes are used in medicine to measure the
concentration of a substance in the blood or the urine. In the examples that
follow I will be discussing the measurement of glucose levels; however, many
other substances can be detected using immobilised enzymes. The
measurement of concentration levels using immobilised enzymes is achieved
by a device called a biosensor. There are two main types of biosensor one is
a colour change, or qualitative, biosensor and the other is a digital, or
qualitative, biosensor.
2.0.2 Prac Concept Clinistix are qualitative biosensors that measure the
concentration of glucose in the blood by producing a colour change. Two
enzymes, along with dye molecules, are immobilised onto an absorbent
matrix pad that is attached to a plastic strip. The two enzymes are glucose
oxidase and peroxidase.
Pad to which the glucose oxidase and
peroxidase enzymes are immobilised.
Also present is the dye molecules
In the presence of glucose oxidase, the glucose is oxidised to gluconic acid and
hydrogen peroxide. The colourless dye molecule is then converted to a
coloured molecule by the reaction with hydrogen peroxide – this reaction is
catalysed by the peroxidase enzyme. The colour change is compared to a
standard colour chart to determine the glucose concentration. The colour
change is directly proportional to the glucose concentration. A numerical
concentration value is not generated.
8
2.0.3 Concept A digital biosensor can measure the concentration of a biological
molecule and express the concentration as a numerical value.
2.0.4 Concept A digital biosensor has the following components:
semipermeable membrane, immobilised enzymes, transducer, amplifier and
digital display.
2.0.5 Concept The semi-permeable membrane prevents large substances like
proteins from entering the biosensor but still allows the substrate to enter.
2.0.6 Concept The immobilised enzyme will convert the substrate into a
products.
Blood proteins
Blood cells
Substrate molecule
Blood sample
A digital biosensor used
to measure blood glucose
concentration
Diagram of a digital biosensor showing the main components.
Semipermeable
membrane
2.0.3 to 2.0.5 Quantitative/digital biosensors
Immobilised
enzyme Transducer Amplifier Digital
display
3.25
Product molecule
9
2.0.7 Concept The transducer is the component that generate electrical
impulses. To do this the transducer must absorb the product from the
enzyme catalysed reaction and then depending on the concentration of this
product will generate electrical signals that are directly proportional to the
original molecule concentration in the blood.
2.0.8 Concept A digital biosensor has the following advantages:
1. They are specific
The immobilised enzymes make the biosensor specific to just one molecule.
The molecule to be measured will have a complementary shape to the
enzymes active site. No other molecule will affect the activity of this enzyme
as it cannot bind to the active site to form an enzyme substrate complex.
2. They are sensitive
A biosensor has the ability to detect very low concentrations of a molecule.
3. They are rapid
The biosensor can generate results within minutes.
4. A small sample is required
Biosensors only need a small sample of, for example, blood/urine to
determine the concentration of a molecule.
5. They produce quantitative results
Quantitative results are those with numerical values.
10
3.0 INDUSTRIAL USES OF IMMOBILISED ENZYMES
3.0.1 Concept Immobilise enzymes can be used in industrial processes to
produce a particular product. Immobilised enzymes are particularly suited to
an industrial process called a continuous production process. An example of
a produce produced by a continuous production process would be lactose
free milk. Lactose free milk is produced for people who suffer from a
condition called lactose intolerance. This condition occurs due to the
inability of someone to produce the enzyme lactase. Lactase is an enzyme
found in the digestive system and hydrolyses lactose to glucose and
galactose. So, in lactose intolerant sufferers they cannot digest lactose and
suffer from abdominal pain and diarrhoea due to an accumulation of carbon
dioxide produce by the fermentation of lactose by the gut bacteria. Lactose
is the sugar found in milk, so lactose free milk is produced using immobilised
lactase enzyme for the consumption of lactose intolerant sufferers.
Diagram to show a lab scale set up to produce lactose
free milk. A column is packed with lactase
immobilised beads and milk is introduced at the top.
The lactose is hydrolysed to glucose and galactose so
lactose free milk is produced.
11
3.0.2 Concept Flow rate can affect the yield of product formed.
Explanation of flow rate on the yield of product
The flow rate is the speed at which the milk flows through the column of immobilised
enzyme. The faster the flow rate the quicker the milk will flow through the column. An
increase flow rate will reduce the amount of product being formed because the substrate
will be in contact with the immobilised enzyme for less time so there will be less enzyme
substrate complexes formed and hence less products formed. A too higher flow ate can
lead to a product being contaminated with substrate, so in the case of producing lactose
free milk there would be lactose contaminating the product.
A reduction in flow rate will have the opposite affect and will increase the product yield
(with no contamination with substrate) due to the substrate being in contact with the
enzyme for a longer period of time so forming more enzyme substrate complexes.
3.0.3 Concept The diameter of the beads used to immobilise the enzymes can
affect the yield of the product.
Explanation of bead size on the yield of product.
The diameter of the bead can affect the total surface area and the flow rate. A smaller
bead diameter can pack closer together than a larger bead size. Due to the closer packing
of the smaller bead size there is less space between the beads and this adds to the
resistance the fluid will experience as it flows thought he column, so the flow rate will
reduce. The reduced flow rate will again increase the contact time of the substrate with
the enzyme and produce more product due to more enzyme substrate complexes being
formed. The milk will be exposed to an increased total surface area of the smaller beads
so there will be more enzyme substrate complexes formed so increasing the yield.
Diagram to show that a smaller bead size (A) can pack closer together than a larger
bead size (B). This closer packing gives the smaller bead size a larger total surface
area.
A B
12
3.0.4 Concept An increase in temperature can increase the yield of product.
Explanation of the effect of temperature on yield of product.
At high temperatures the substrate will have higher kinetic energy and so more ESC will
form to make more product.
3.0.5 Immobilised enzymes have the following advantages when used in
industrial processes:
1. The immobilised enzyme does not contaminate the product.
2. The immobilised enzyme can be easily recovered and re-used so
reducing costs as enzymes are expensive.
3. Immobilise enzymes allow for a more rapid process.
4. More than one enzyme can be used at the same time.
5. The immobilised enzymes can be easily removed or added.
6. The immobilised enzymes can tolerate extreme conditions like higher
temperatures and extreme ph.
3.0.6 Concept The type of enzyme immobilisation used can affect the rate of
the enzyme catalysed reaction. If the enzyme is immobilised into alginate
beads the rate of the catalysed reaction can be slower than that achieve by
enzymes immobilised onto the surface of cellulose fibres.
Enzymes immobilised to cellulose
fibres are fully exposed to the
substrate so many ESC can form
so making the rate rapid.
Enzymes immobilised in alginate beads are
not as accessible to the substrate. For an
ESC to form the substrate will have to diffuse
into the alginate bead to reach the active site
of the enzyme. This will slow the rate down.
Enzymes less
accessible to
substrate
13
3.0.7 Concept Immobilised enzymes have a higher optimum temperature and
denature at higher temperatures because the3 inert matrix stabilised the
tertiary structure of the enzyme to prevent the breaking of hydrogen bonds.
Rat
e o
f re
acti
on
Temperature
Immobilised enzyme
Free enzyme
Optimum temperature
14
QUESTIONS ON SECTION 1 TO 3
1
15
16
2
17
3
18
19
4
20
21
22
5
23
6
24
25
26
ADDITIONAL QUESTIONS ON ENZYMES
1
27
28
29
30
2
31
32
3
33
34
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