name: option group: enzymes 2

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

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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.

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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).

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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.

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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

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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.

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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.

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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

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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

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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

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QUESTIONS ON SECTION 1 TO 3

1

26

ADDITIONAL QUESTIONS ON ENZYMES

1

name: option group: enzymes 2

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