Module 6 Section 5: Cloning and Biotechnology

Question 1

What is cloning

Answer 1

Process of producing genetically identical cells or organisms from cells of an existing organism

Question 2

How can cloning be carried out

Answer 2

Cloning can occur naturally in some plants and animals
Can be carried out artificially instead

Question 3

What is vegetative propagation

Answer 3

Production of plant clones from non reproductive tissue (not the flower)
Type of asexual reproduction which produces clones
E.g. roots leaves and stems

Question 4

What are stolons/runners

Answer 4

Similar to rhizomes
Grow above ground on surface of soil
New shoots and roots can develop from nodes or form at the end of the stolon
E.g. strawberries

Question 5

Rhizomes

Answer 5

Stem structures that grow horizontally underground away from parent plant
Have nodes where new shoots and roots can develop
E.g. bamboo

Question 6

What are bulbs

Answer 6

Underground food stores
New bulbs can develop from original bulb and form new individual plant
E.g. onions

Question 7

What are tubers

Answer 7

Large underground food stores
Have eyes which can sprout and from a new plant
E.g. potatoes

Question 8

What are suckers

Answer 8

Large underground food stores
Have eyes which can sprout and from a new plant
E.g. elm trees

Question 9

Different types of plant cloning

Answer 9

Natural:
Plant cuttings
Vegetative propagation
Artificial:
Tissue culture
Micro propagation

Question 10

Techniques of artificial vegetative propagation

Answer 10

Cuttings
Grafting
Layering
All rely of formation of meristematic tissue from which plant organs can differentiate
Creates cultivars

Question 11

What is taking cuttings

Answer 11

Taking and growing a cutting from a stem/root/leaf

Question 12

Process of taking cuttings

Answer 12

Cuttings are part of the plant that is cut off of the parent plant.
Shoots with leaves attached are usually used.
New roots and leaves will grow from the cutting.
The shoot is cut at an angle.
A growth promoter may be used to help with the growth of the roots.

Question 13

What is grafting

Answer 13

Joining the shoot of one plant to the growing stem and root of another plant.

Question 14

Process of grafting

Answer 14

In grafting 2 plants are used to develop a new plant with combined traits from the 2 parent plants.
In grafting the scion is the above ground part of one plant.
The scion is attached to the stock which is the rooted part of the second plant.

Question 15

What is layering

Answer 15

Bending the stem of a growing plant downwards so it enters the soil and grows into a new plant.

Question 16

Process of layering

Answer 16

In layering a shoot of a parent plant is bent until it can be covered by soil.
The tip of the shoot remains above ground.
New roots and eventually a new plant will grow.
These plants can then be separated

Question 17

Method of producing a clone from a cutting

Answer 17

Use a scalpel or sharp secateurs to take a cutting, between 5 cm and 10 cm long, from the end of a stem of your parent plant between nodes (between 2 leaf joints)
Choose health stem
Cut stem at slant
Remove the leaves from the lower end of your cutting leaving just one at the tip.
Dip lower end of the cutting in rooting powder
This contains hormones that induce root formation.
Plant cutting in a pot containing a suitable growth medium (e.g. well-drained compost).
Provide cutting with a warm and moist environment by either covering the whole pot with a plastic bag or by putting it in a propagator (a specialised piece of kit that provides these conditions)
When your cutting has formed its own roots and is strong enough, plant it elsewhere to continue growing.

Question 18

Problems of the method to produce a clone from a cutting

Answer 18

Cannot produce many clones at once

Question 19

Process of growing an artificially cloned plant with tissue culture

Answer 19

Cells are taken from the original plant that’s going to be cloned (can also use explant)
Cells from the stem and root tips are used because they’re stem cells
Plant stem cells are totipotent: can develop into any type of cell
The cells are sterilised to kill any microorganisms (bacteria and fungi that compete for nutrients with the plant cells)
This would decreases their growth rate.
The cells are placed on a culture medium containing plant nutrients (glucose for respiration) and growth hormones (auxins)
When the cells have divided and grown into a small plant they’re taken out of the medium and planted in soil
Then develop into plants that are genetically identical to the original plant.

Question 20

When is tissue culture used

Answer 20

Used to clone plants that don’t readily reproduce or are endangered or rare
e.g. British orchids.
It’s also used to grow whole plants from genetically engineered plant cells

Question 21

What is micropropagation and how is it done

Answer 21

When tissue culture is used to produce lots of cloned plants very quickly.
Cells are taken from developing cloned plants and subcultured (grown on another fresh culture medium)
Repeating this process creates large numbers of clones

Question 22

Where is micropropagation used

Answer 22

Used extensively in horticulture and agriculture
e.g. to produce fields full of a crop that has been genetically engineered to be pest-resistant.

Question 23

Difference between agriculture and horticulture

Answer 23

Both involve cultivating crops
Agriculture relates to farming
Such as crops for human use
Horticulture involves cultivating any plant for any purpose usually on a smaller scale

Question 24

Arguments for plant cloning

Answer 24

Desirable genetic characteristics are always passed on to clones
Doesn’t always happen when plants reproduce sexually.

Tissue culture allows plants to be reproduced in any season because the environment is controlled.

Less space is required by tissue culture than would be needed to produce the same number of plants by conventional growing methods.

It produces lots of plants quickly compared to the time it would take to grow them from seeds

Question 25

Arguments against plant cloning

Answer 25

Undesirable genetic characteristics are always passed on to clones.

Cloned plant populations have no genetic variability, so a single disease could kill them all.

Production costs of tissue culture are very high due to high energy use and the training of skilled workers
Unsuitable for small scale production.

Contamination by microorganisms during tissue culture can be disastrous and result in complete loss of the plants being cultured.

Preparing cuttings can be time consuming and needs a lot of space

Question 26

Method of split vein cutting

Answer 26

Remove complete leaf and score the large veins on the lower leaf surface using a scalpel
Put it on top of the growth medium with the broken veins facing down
Place leaf in warm moist environment
New plant should form from each break in the veins
When shoots have formed its own roots and is strong enough, plant it elsewhere to continue growing

Question 27

Natural types of animal cloning

Answer 27

Embryo twinning (twins)

Question 28

Artificial types of animal cloning

Answer 28

Artificial embryo twinning
Somatic cell nuclear transfer

Question 29

Process of embryo twinning

Answer 29

An egg is fertilised by a sperm as in a singleton birth.
Forms a zygote.
The single zygote undergoes a few cell cycles (mitotic divisions) to become an embryo (monozygotic)
At the embryo stage, the embryo splits in two
Two embryos that form are identical, with the same genotype and develop in utero together.
The result is the birth of identical offspring, always of the same gender, with identical phenotype.

Question 30

Why are non-identical twins not considered clones

Answer 30

Formed from separate eggs and sperm, they are not considered clones.

Question 31

What is artificial embryo twinning

Answer 31

The process of embryo twinning produces offspring that are clones of each other but not of their parents (similar to natural embryo twinning)

Question 32

Process of artificial embryo twinning

Answer 32

An egg cell is extracted from a female cow and fertilised in a Petri dish (can also be done by extracted early embryo from pregnant animal)
The fertilised egg is left to divide at least once, forming an embryo in vitro (outside a living organism).
The individual cells from the embryo are separated and each is put into a separate Petri dish.
Each cell divides and develops normally so an embryo forms in each Petri dish.
The embryos are then implanted into female cows (surrogate mothers)
The embryos continue to develop inside the surrogate cows, and eventually the offspring are born.
They’re all genetically identical to each other.

Question 33

What is somatic cell nuclear transfer (SCNT)

Answer 33

This involves replacing the haploid nucleus of an unfertilised egg with a diploid nucleus from an adult donor.

Question 34

Process of somatic cell nuclear transfer

Answer 34

A somatic cell (any cell that isn’t a reproductive cell) is taken from sheep A.
The nucleus is extracted and kept.
An oocyte (immature egg cell) is taken from sheep B.
Its nucleus is removed to form an enucleated oocyte.
The nucleus from sheep A is inserted into the enucleated oocyte (from sheep B)
It now contains the genetic information from sheep A.
The nucleus and the enucleated oocyte are fused together and stimulated to divide (e.g. by electrofusion where an electrical current is applied).
This produces an embryo.
The embryo is implanted into a surrogate mother and eventually a lamb is born that’s a clone of sheep A.

Question 35

Uses of artificial cloning

Answer 35

Scientists use cloned animals for research purposes
e.g. to test new drugs on cloned animals. They’re all genetically identical, so the variables that come from genetic differences (e.g. the likelihood of developing cancer) are removed.

Cloning can be used to save endangered animals from extinction by cloning new individuals.

Cloning can also be used in agriculture so farmers can increase the number of animals with desirable characteristics to breed from, e.g. cow with high milk production could be cloned.

Animals that have been genetically modified to produce a useful substance that they wouldn’t normally produce (e.g. a beneficial protein in their milk) could be cloned to produce lots of identical animals that all produce the same substance.

Scientists only want the cloned embryonic stem cells.
These cells are harvested from young embryos and have the potential to become any cell type so could be used to replace damaged tissues in a range of diseases, e.g. heart disease, spinal cord injuries, degenerative brain disorders like Parkinson’s disease.
If replacement tissue is made from cloned embryonic stem cells that are genetically identical to the patient’s own cells, it won’t be rejected by their immune system.

Question 36

Arguments for animal cloning

Answer 36

Desirable genetic characteristics are always passed on to clones (e.g. high milk production in cows) which doesn’t always happen with sexual reproduction.

Infertile animals can be reproduced.

Increasing the population of endangered species helps to preserve biodiversity.

Animals can be cloned at any time — you wouldn’t have to wait until a breeding season to get new animals.

Cloning can help us develop new treatments for disease, which could mean less suffering for some people.

Question 37

Arguments against animal cloning

Answer 37

Animal cloning is very difficult, time-consuming and expensive.

There’s no genetic variability in cloned populations, so undesirable genetic characteristics are always passed on to clones.
This means that all of the cloned animals in a population are susceptible to the same diseases (single disease could wipe them all out)

Some evidence suggests that clones may not live as long as natural offspring.
Some think this is unethical.

Using cloned human embryos as a source of stem cells is controversial.
The embryos are usually destroyed after the embryonic stem cells have been harvested — some people believe that doing this is destroying a human life.

Question 38

What is reproductive cloning

Answer 38

If the embryo is implanted into the uterus of a surrogate, a new cloned organism will develop

Question 39

What is therapeutic cloning

Answer 39

Embryonic cells can be induced to differentiate to create specific tissues or organs for transplantation.

Question 40

What is biotechnology

Answer 40

Biotechnology is the industrial use of living organisms to produce food, drugs and other products

Question 41

Why are microorganisms usually used for biotechnology

Answer 41

Their ideal growth conditions can be easily created
Microorganisms will generally grow successfully as long as they have the right nutrients, temperature, pH, moisture levels and availability of gases (e.g. some need oxygen).
Grow rapidly under the right conditions due to short life cycle so products can be made quickly.
They can be grown on a range of inexpensive materials — makes them economical to use.
They can be grown at any time of the year.
Occupy very little space

Question 42

How are enzymes used in biotechnology

Answer 42

Enzymes use can be contained within cells of organisms (intercellular)
Enzymes that aren’t contained in cells (isolated enzymes) can also be used
Some enzymes used are naturally secreted by microorganisms (extracellular enzymes) (may also have to be extracted)

Question 43

Why use naturally secreted enzymes over extracted enzymes

Answer 43

Naturally secreted enzymes are cheaper as it can be expensive to extract enzymes from cells

Question 44

How are microorganisms used to brew beer

Answer 44

To make beer, yeast (e.g. Saccharomyces cerevisiae) is added to a type of grain (barley) and other ingredients.
The yeast respires anaerobically using the glucose from the grain and produces ethanol and CO2
When anaerobic respiration produces ethanol, the process is called fermentation.

Question 45

How are microorganisms used to bake bread

Answer 45

Yeast is the organism that makes bread rise.
The CO2 produced by fermentation of sugars in the dough makes sure it doesn’t stay flat.
Many flat breads, like tortillas, are made without yeast.

Question 46

How are microorganisms used to make cheese

Answer 46

Add rennet to the milk.
Enzyme chymosin (in the rennet) clots the milk
Lactic acid bacteria is added which converts lactose in the milk into lactic acid
This makes it turn sour and solidify.
The liquid portion (whey) is removed from the curd by cutting, stirring & heating.
The curd is then pressed into moulds and the different treatments while making the curd determine the characteristic flavour and texture of the cheese.

Question 47

Where is chymosin in rennet obtained from for cheese making

Answer 47

Used to be from the lining of calves stomachs
Chymosin can be obtained from yeast cells that are genetically modified

Question 48

How are microorganisms used to make yoghurt

Answer 48

A starter culture of Lactobacillus bulgaricus and Streptococcus thermophilus bacteria are introduced to pasteurised milk.
The bacteria use sugars in the milk to respire and produce lactic acid as a waste product.
Lactic acid denatures the proteins in the milk, causing them to coagulate (stick together). This produces the thick texture and sour taste of yoghurt.
Flavours can be added at this stage to produce flavoured yoghurt

Question 49

How are microorganisms used to produce penicillin

Answer 49

In times of stress, fungi from the Penicillium genus produce an antibiotic, penicillin, to stop
bacteria from growing and competing for resources.
Penicillin is one of the most common antibiotics used in medicine, so we produce it on a massive scale.
The fungus (usually Penicillium chrysogenum) is grown under stress in industrial fermenters
and the penicillin produced is collected and purified to be used in medicine.

Question 50

How are microorganisms used to produce insulin

Answer 50

Insulin is a hormone that’s crucial for treating people with Type 1 diabetes.
Insulin is made by genetically modified bacteria, which have had the gene for human insulin production inserted into their DNA.
These bacteria are grown in an industrial fermenter on a massive scale and the insulin produced is collected and purified

Question 51

How are microorganisms used in bioremediation

Answer 51

This is using organisms (usually microorganisms) to remove pollutants, like oil and pesticides, from contaminated sites.
Commonly, pollutant-removing bacteria that occur naturally at a site are provided with extra nutrients and enhanced growing conditions to allow them to multiply and thrive.
These bacteria break down the pollutants into less harmful products, cleaning up the area.
E.g. bioremediation using bacteria has been used to clean up oil spills at sea.

Question 52

How are microorganisms used as a source of protein and give an example

Answer 52

This is called single celled protein which can act as a food source
E.g. the fungus Fusarium venenatum which is used to make the meat substitute Quorn

Question 53

Advantages of using microorganisms to produce food

Answer 53

Microorganisms used to make single-cell protein can be grown using many different organic substrates, including waste materials such as molasses (a by-product of sugar processing).
Production of single-cell protein could actually be used as a way of getting rid of waste products.

Microorganisms can be grown quickly, easily and cheaply.
Production costs are low because microorganisms have simple growth requirements, can be grown on waste products and less land is required in comparison to growing crops or rearing livestock.

Microorganisms can be cultured anywhere if you have the right equipment, reproduction not dependent on weather or breeding cycles
This means that a food source could be readily produced in places where growing crops and rearing livestock is difficult (e.g. very hot or cold climates).
This could help tackle malnutrition in developing countries.

Single-cell protein is often considered a healthier alternative to animal protein as it has little fat and high protein

Can be made to taste like anything

Question 54

Disadvantages of using microorganisms to produce food

Answer 54

Ideal growth conditions can also grow unwanted bacteria
These contaminations can be dangerous or spoil the food so lots of effort must be used to control for contamination

People may not like the idea of eating food that has been grown using waste products.

Single-cell protein doesn’t have the same texture or flavour as real meat.

If single-cell protein is consumed in high quantities, health problems could be caused due to the high levels of uric acid released when the large amounts of amino acids are broken down (risk of gout)

People may have concerns about eating GM food

Need have additives to get flavour

Question 55

What does biotechnology use

Answer 55

Cultures of microorganisms
A culture is a population of one type of microorganism that’s been grown under controlled conditions.

Question 56

Where are cultures grown in biotechnology

Answer 56

Fermentation vessels
Can obtain lots of the microorganism (e.g. for production of single-celled protein) or to collect lots of a useful product that the microorganism makes.

Question 57

What are the two main methods of culturing microorganisms

Answer 57

Batch fermentation
Continuous fermentation

Question 58

How is batch fermentation used to culture microorganisms

Answer 58

This is where microorganisms are grown in individual batches in a fermentation vessel
When one culture ends it’s removed and then a different batch of microorganisms is grown in the vessel.
This is known as a closed culture

Question 59

How is continuous fermentation used to culture microorganisms

Answer 59

Where microorganisms are continually grown in a fermentation vessel without stopping.
Nutrients are put in and waste products taken out at a constant rate.

Question 60

Why are the conditions inside a fermentation vessels kept constant

Answer 60

This maintains conditions that are optimum for growth which maximises yield of microorganisms and desired products

Question 61

How is pH regulated inside the fermentation vessel and how does it maximise yield

Answer 61

Constantly monitored by a pH probe and kept at the optimum level

Allows enzymes to work efficiently, so the rate of reaction is kept as high as possible

Question 62

How is temperature regulated inside the fermentation vessel and how does it maximise yield

Answer 62

Kept constant by a water jacket that surrounds the entire vessel.

Allows enzymes to work efficiently, so the rate of reaction is kept as high as possible

Question 63

How is access to nutrients regulated inside the fermentation vessel and how does it maximise yield

Answer 63

Paddles constantly circulate fresh nutrient medium around the vessel.

Ensures that the microorganisms always have access to their required nutrients.

Question 64

How is volume of oxygen regulated inside the fermentation vessel and how does it maximise yield

Answer 64

Sterile air is pumped into the vessel when needed.

Makes sure that the microorganisms always have oxygen for respiration.

Question 65

How is the vessel kept sterile inside the fermentation vessel and how does it maximise yield

Answer 65

Superheated steam sterilises the vessel after each use.

Kills any unwanted organisms that may compete with the ones being cultured.

Question 66

Label the fermentation vessel

Answer 66

Question 67

What is metabolism

Answer 67

Sum of all chemical reactions in an organism
Produces: new cells, chemicals (hormones, enzymes), waste products

Question 68

What is the metabolic rate

Answer 68

The rate that the chemical reactions are carried out by an organism

Question 69

What are primary metabolites

Answer 69

Substances produced by an organism as part of its normal growth
They are produced most of the time so their concentrations match the population size
E.g: proteins (amino acids/enzymes), nucleic acids, ethanol and lactate

Question 70

What are secondary metabolites

Answer 70

Substances produced by organisms that are not part of its normal growth
Rarer than primary metabolites
Concentrations don’t match population size
E.g: antibiotics in fungi, codeine, morphine

Question 71

When are secondary metabolites produced

Answer 71

May only be produced when microbe is well-established in the growth medium.

Question 72

What is a closed culture and what is the pattern of how microorganism population changes over time in the closed culture

Answer 72

A closed culture is when growth takes place in a vessel that is isolated from the external environment
Extra nutrients are not added and waste products are not removed from the vessel during growth.
Population follows a standard growth curve

Question 73

What are the stages of growth on a standard growth curve

Answer 73

1)Lag phase
2)Exponential/log phase
3)Stationary phase
4)Decline/death phase

Question 74

What is the lag phase

Answer 74

Population size increases slowly because the microorganisms have to make enzymes and other molecules before they can reproduce.
This means the reproduction rate is low.

Question 75

What is the log/exponential phase

Answer 75

Population size increases quickly because the culture conditions are at their most favourable for reproduction (lots of food and little competition).
The number of microorganisms doubles at regular intervals.
Rate of reproduction is at theoretical maximum

Question 76

What is the stationary phase

Answer 76

Population size stays level because the death rate of the microorganisms equals their reproductive rate (binary fission)
Microorganisms die because there’s not enough food and poisonous waste products build up.

Question 77

What is the decline/death phase

Answer 77

Population size falls because the death rate is greater than the reproductive rate.
This is because food is very scarce and waste products are at toxic levels.

Question 78

How to find out how many organisms will be present in a population after a certain number of divisions during the exponential/log phase

Answer 78

Only during exponential phase
N = N₀ x 2ⁿ
N: number of individuals present in population
N₀: initial number of cells
n: number of divisions

Question 79

How to culture microorganisms in a lab

Answer 79

Can be grown on agar plate - sterile petri dish containing agar jelly
Microorganisms transferred to the plate from a sample (bacteria in broth) using a sterile wire inoculation loop or sterile pipette and spreader
Spread using zig zag steak across the plate
Don’t break surface of agar jelly
Replace lid and hold it down with tape (don’t seal completely so oxygen can get in)
Incubate the plates and allow microorganisms to grow
Nutrients can be added to the agar to help improve the growing conditions

Question 80

How to sterilising inoculation loop

Answer 80

Hold it over a Bunsen flame until red hot
Don’t let it touch any surfaces to avoid contamination

Question 81

How to make an inoculation broth

Answer 81

Make a suspension of the bacteria to be grown.
Mix a known volume with the sterile nutrient broth in the flask.
Stopper the flask with cotton wool to prevent contamination from the air.
Incubate at a suitable temperature, shaking regularly to aerate the broth providing oxygen for the growing bacteria.

Question 82

What are the aseptic techniques used when culturing microorganisms

Answer 82

Regularly disinfect work surfaces to minimise contamination.

Work near a Bunsen flame.
Hot air rises, so microorganisms in the air should be drawn away from culture.

Sterilise the instrument used to transfer cultures before and after each use
e.g. sterilise a wire inoculation loop by passing it through a hot Bunsen burner flame for 5 seconds.
This will kill any microorganisms on the instrument.

Pre-sterilised plastic instruments should only be used once and then safely discarded.

If you’re using broth, briefly pass the neck of the broth container through a Bunsen burner flame just after it’s opened and just before it’s closed
This causes air to move out of the container, preventing unwanted organisms from falling in.

Minimise the time that the agar plate is open and put the lid on as soon as possible.
This reduces the chance of airborne microorganisms contaminating the culture.

Work in an inoculation cabinet (a chamber that has a flow of sterile air inside it).

Sterilise all glassware before and after use,
e.g. in an autoclave.

Wear a lab coat and, if needed, gloves.

Tie long hair back to prevent it from falling into anything.

Question 83

What are the limiting factors that can prevent exponential growth in a culture of bacteria

Answer 83

Nutrients available: food available will be used up as population multiplied which limits growth and reproduction unless more nutrients added

Oxygen: large populations demand more respiratory oxygen which can become limiting

Temperature: too high temperatures speed up reactions and can denature enzymes and kill microorganisms and too cold temperatures slow down growth and reproduction

Build up of waste: large populations produces lots of waste which can poison and kill population if levels get too high

Change in pH: CO2 from respiration increases and the pH can fall to a point where the low pH affects enzyme activity and inhibits growth

Question 84

Advantages of using batch production

Answer 84

Requirements are added and left to ferment
Easy as minimum attention required
Once sterilised can be used for variety of other processes
If contamination occurs only single batch lost
Inlet/outlet pipes unlikely to be blocked
Useful for production of secondary metabolites eg. penicillin

Question 85

Disadvantages of using batch production

Answer 85

Growth rate slower as nutrient levels decrease with time
Less efficient, as fermenter has ‘down time’

Question 86

Advantages of using continuous production

Answer 86

Steady input of nutrients into fermenter and steady harvest from it so growth rate higher
No down time so more efficient
Smaller vessels can be used as output continuous so less space needed for good yield
Useful for production of primary metabolites e.g. proteins/enzymes for growth

Question 87

Disadvantages of using continuous production

Answer 87

If contamination occurs huge volumes may be lost
Set up can be more difficult and maintenance of growing conditions can be difficult to maintain

Question 88

How can enzymes be immobilised

Answer 88

Encapsulated in jelly-like alginate beads which act as a semi permeable membrane

Trapped in silica gel matrix

Covalently bonded to cellulose or collagen fibres

Question 89

What are immobilised enzymes

Answer 89

Enzymes that are attached to an insoluble material so they can’t become mixed with the products

Question 90

Why do enzymes need to be immobilised

Answer 90

Isolated enzymes used in industry can become mixed in with the products of a reaction
Products then need to be separated from the mixture which can be complicated and costly
This can be avoided in large scale production by using immobilised enzymes

Question 91

Why use isolated enzymes

Answer 91

Less wastelul - whole microorganisms use up substrate growing and reproducing, producing biomass rather than product.
Isolated enzymes do not.

More efficient - isolated enzymes work at much higher concentrations than is possible when they are part of the whole microorganism.

Maximise product formation - isolated enzymes can be given ideal conditions for maximum product formation, which may differ from those needed for the growth of the whole microorganism.

More specific - no unwanted enzymes present, so no wasteful side reactions take place.

Less downstream processing - pure product is produced by isolated enzymes.
Whole microorganisms give a variety of products in the final broth, making isolation of the desired product more difficult and therefore expensive.

Question 92

How are immobilised enzymes used in industry and why is this helpful

Answer 92

Attached to inert support system
Substrate passes through and is converted to product
Enzymes can be recovered from reaction mixture and reused again as they are held stationary
Means they do not contaminate end process

Question 93

Methods for immobilising enzymes

Answer 93

Entrapment
Adsorption
Covalent bonding
Membrane separation

Question 94

What is entrapment

Answer 94

Enzymes trapped in gel bead.
Reaction rates may be reduced as substrate must penetrate trapping medium (active site less easily available).
The product molecules must diffuse out of the gel bead matrix.
Only suitable for small substrate and product molecules that diffuse easily.

Question 95

What is adsorption

Answer 95

Enzymes bind to immobilising support (glass beads, clay) by hydrophobic interactions and ionic links.
Leakage can occur as bonds are not strong and they can become detached.
High reaction rates as active sites remain unchanged and exposed for accessibility to the substrate.

Question 96

What is covalent bonding

Answer 96

Enzymes are bonded to surfaces using a cross linking agent such as gluteraldehyde to link enzyme to insoluble clay.
Binding strong (little leakage - detachment) but some covalent bonding can distort the shape of the active site.

Question 97

What is membrane separation

Answer 97

Enzymes are separated by partially permeable membrane.
The substrate and product molecules must be small enough to move across membrane to access the active site.

Question 98

Advantages of using immobilised enzymes in industry

Answer 98

Columns of immobilised enzymes can be washed and reused
This reduces the cost of running a reaction on an industrial scale because you don’t have to keep buying new enzymes.

The product isn’t contaminated with the enzymes
No money or time is spent separating them out.

Recycling enzymes allows for continuous processes.

Immobilised enzymes are more stable than free enzymes
They’re less likely to denature (become inactive) in high temperatures or extremes of pH

Question 99

Disadvantages of using immobilised enzymes in industry

Answer 99

Extra equipment is required which can be expensive to buy

More expensive to buy than free enzymes, so coupled with the equipment costs, they’re not always economical for use in smaller-scale production

The immobilisation of the enzymes can sometimes lead to a reduction in the enzyme activity because they can’t freely mix with their substrate
This means that the rate of reaction is sometimes lower

Question 100

How are immobilised enzymes used in the production of semi-synthetic penicillins

Answer 100

Semi synthetic penicillin is produced which have the same antibiotic properties as natural penicillin, but are effective against penicillin resistant organisms
Immobilised penicillin acylase enzyme is used in their production

Question 101

How are immobilised enzymes used in the conversion of dextrins to glucose

Answer 101

Glucose and glucose syrup can be used to sweeten and thicken foods
Glucose can be derived from starchy foods, such as corn and potatoes, with the help of immobilised enzymes
Starch breaks down into dextrins (carbohydrate products), which are then broken down into glucose by the immobilised enzyme glucoamylase

Question 102

How are immobilised enzymes used in the conversion of glucose to fructose

Answer 102

Fructose is a sugar that’s much sweeter than glucose
Used as a sweetener in food so less sugar is needed to obtain same level of sweetness in food
Immobilised isomerase is used to convert glucose to fructose on an industrial scale

Question 103

How are immobilised enzymes used in the production of pure samples of L-amino acids

Answer 103

Immobilised aminoacylase is used for the industrial production of pure samples of L-amino acids which can be used for many purposes in the production of animal and human food and dietary supplements
Amino acids can be in the L or D form but most amino acids used in the body need to be the L form
When amino acids are chemically synthesised the L forms must be separated from the D forms
This is done using amino acylase

Question 104

How are immobilised enzymes used in industry

Answer 104

The immobilised enzymes are contained within a column through which the substrate is filtered in solution.
As the substrate runs through the column, enzyme-substrate complexes are formed and products are produced.
These products then flow out of the column, leaving the enzymes behind to catalyse the reaction again