Cloning and biotechnology Flashcards
Clones
-Genetically identical copies of organisms/cells
-Produced asexually in which the nucleus is divided by mitosis
How are clones produced by mitosis
-Mitosis creates two genetically identical copies of the DNA
-The DNA is separated into two genetically identical nuclei
-Before the cell divides and creates two genetically identical cells
Why may the cells produced by mitosis not be identical
May not be physically/chemically identical as after division they may differentiate to form two different types of cell
How does yeast/ bacteria reproduce
Asexually by budding
Asexually by binary fission
Advantages of natural cloning
-If conditions of growth is good for the parent it will be good for the offspring
-Cloning is rapid so population can reproduce quickly to take advantage of the suitable environment conditions
-Reproduction can be carried out when sexual reproduction may not be possible
Disadvantages of natural cloning
-Offspring may become over crowded
-No genetic diversity ( except from mutations in the DNA)
-Population shows little variation
-Selection is not possible
-If environment changes to be less advantageous, the whole population is susceptible
Vegetative propagation
Natural cloning
Reproduction from vegetative parts of the plant - usually an over wintering organ rather than through specialised reproductive structures
Plant cloning
Differentiation of many plant cells is not as complete as in animals many parts of the plant contain cells with the ability to divide and differentiate into other types of cell
Examples of plant cloning by vegetative propagation
-Runner/ stolens/ rhizomes/ suckers
-Bulbs
-Corms
-Leaves
-Runners/ stolens / Rhizomes
Plants that grow horizontal stems that can grow roots at any point
-Grow on the surface of the ground (Runners/ stolens)
-Grow under ground (Rhizomes )
How are some Rhizomes adapted
Adapted as thickened overwintering organs from which one or more new stems will grow in the spring
Suckers
New stems that grow from the roots of the pant - may be close to the base of the older stem/ some distance away
-Original horizontal branch will die and leave the new stem as a separate individual
Bulbs
e.g. onions
-Overwintering mechanism
-Consists of an underground stem which grow a series of fleshy leaf bases
- Also an apical bud that will grow into a new plant in the spring
-Often bulb contains more than one apical bud and each will grow into a new plant
Corms
-Solid rather than fleshly like a bulb
-Underground stem with scaly leaves and buds
-Remain in the ground over winter
-In spring the buds grow to produce one or more plants
Leaves
Kalanchoe plant reproduces asexually as clones grow on the leaf margins
-Immature plants drop off the leaf and take root
Tubers
Another type of underground stem i.e. potatoes
-One potato will grow into one/more plants
-Each new plant can produce many new tubers (potatoes) later that year
Cloning in animals
Don’t clone as often as plants but there are some e.g. of natural cloning i.e. identical twins
How do identical twins form
Fertilised egg (zygote) divides as normal but the two daughter cells split to become two separate cells
-Each cell grows and develops into a new individual
What is the easiest way gardeners/growers make clones
Cuttings
-A stem is cut between the nodes
-Cut end of the stem is placed in moist soil
-New roots will grow from the tissue in the stem - usually from the node but may grow from other parts of the buried stem
What helps stimulate root growth from cuttings
Dipping the cut stem in rooting hormone / remove the bark from the cut end of the stem as this encourages the plant to produce a callus
Where else can a cutting be taken from a plant
-Root cuttings = section of the root is buried just below the soil surface and produces new shoots
-Scion cuttings = dormant woody twigs
-Leaf cuttings = leaf placed on moist soil ; leaves develop new stems and roots
What are the disadvantages of taking cuttings for natural cloning
Large scale cloning can be time time-consuming and take a lot of space
-Some plants don’t respond well to cuttings
Tissue culture
Growing new tissues, organs, plants from certain tissues cut from the sample plant
-Alternative to taking cuttings
-Carried out on a nutrient medium under sterile conditions
-Application of plant growth substances at the correct time can encourage the cells in the growing tissues to differentiate
What is tissue culture most used for
commercially used to increase the number of new plants in micropropagation
Micropropagation
Taking a small piece of plant tissue (meristem) and using plant growth substances to encourage it to grow and develop into a new plant
What are the six steps that micropropagation involves
(First step)
1) Suitable plant material is selected and cut into small pieces = explants
-Could be tiny pieces of leaf/stem/bud
-Meristem tissue used as free from virus infection
What are the six steps that micropropagation involves
(Second step)
2) Explants are sterilised using dilute bleach/alcohol
-Kills any bacteria/ fungi as these would thrive in the conditions required for the plant to grow well
What are the six steps that micropropagation involves
(Third step)
3) Explants placed on a sterile growth medium (usually agar gel) containing suitable nutrients i.e. glucose/ amino acids/ phosphates
-Gel also contains high concentrations of the plant growth substances auxin/cytokines
-Stimulates the cell of each explant to divide by mitosis to form a callus
What is a callus
A mass of undifferentiated totipotent cells
What are the six steps that micropropagation involves
(fourth step)
4) Once callus has formed it is divided to produce a larger number of small clumps of undifferentiated cells
What are the six steps that micropropagation involves
(fifth step)
5) Small clumps of cells are stimulated to grow, divide and differentiate into different plant tissues
=Achieved by moving cells to a diff growth media
Why do you need to move the cells to a diff growth media
(fifth step)
As each media contains diff ratio of auxin and cytokines
-First 100 auxin:1 cytokine stimulates roots to form
-Second 4 auxin: 1 cytokinin stimulates shoots to form
What are the six steps that micropropagation involves
(sixth step)
6) Once tiny plantlets have formed they are transferred to a greenhouse to be grown in compost/soil and acclimatised to normal growing conditions
Seed banks (tissue culture)
Use tissue culture techniques to store plants at a growth stage when they are not too large
-Technique important in the conservation of species whose seeds do not remain viable for long periods
Advantages of artificial cloning (1-3)
1) Rapid method of growing new plants rather than growing plants from a seed
2) Cloning can be carried out when sexual reproduction is not possible.
-Plants that have lost their ability to breed sexually can be re-produced i.e. commercially grown bananas
-Plants that are hard to be grown from seed can be reproduced i.e. orchids
3) Plants selected will all be genetically identical to the parent plant = display the same desirable characteristics such as high yield / disease resistance/ colour
Advantages of artificial cloning (4-6)
4) If original plant had unusual characteristics due to selective breeding / genetic modification then combination will be retained without the risk of losing it through sexual reproduction
5) New plants are all uniform in their phenotype = easier to grow and harvest
6) Using the apical bud (meristem) as an explant for tissue culture ensures the new plants are free from viruses
Disadvantages of artificial cloning (1-3)
1) Tissue culture labour is extensive
2) Expensive to set up the facilities to perform tissue culture successfully
3) Tissue culture could fail due to microbial contamination
In animals what does successful cloning begin with
Cells that are totipotent
Totipotent
Cells that can divide and differentiate into all types of cell found in an adult organism
-In animals theses are very early embryo cells
Reproductive cloning of animals may be useful for:
- Selective breeding/ genetic modification
-Genetically modified animals may develop unusual characteristics i.e. cows that produce less methane
Two main techniques to achieve reproductive cloning
-Embryo twinning: splitting an embryo to make two genetically identical embryos
- Somatic cell nuclear transfer (SCNT): A technique that involves transferring the nucleus from a somatic egg to an egg cell
Embryo splitting
1) A zygote is produced in vitro fertilisation from a male and female with desirable characterises
2) Zygote allowed to divide by mitosis to create a small ball of eggs
3) Cells are separated and allowed to continue to divide
4) Each small mass of cell is placed into the uterus of a surrogate mother
SCNT advantages
The only way to clone an adult
Advantage: phenotype known before the cloning starts
SCNT
1) Enucleation: egg cell obtained and nucleus is removed
2) Normal body cell (somatic cell) from the adult to be cloned is isolated and the nucleus is removed
3) Nucleus of adult cell fused with empty egg cell by an electric shock
4) Shock also triggers egg cell to start developing as if it has been fertilised
5) Cell undergoes mitosis to produce a small ball of cells
6) Young embryo placed in the uterus of a surrogate mother
Non-reproductive cloning
The production of cloned cells and tissues for purposes other than reproduction:
1) Therapeutic cloning
2) Cloning for scientific research
Therapeutic cloning
New tissues/ organs can be grown as replacements for people who are not well
Therapeutic cloning e.g.
-Skin can be grown in vitro to act as a graft for skin burns
-Cloned cells can repair damage to the spinal cord of a mouse and restore its capability to produce insulin in its pancreas
-Potential to grow whole new organs to replace diseased organs
What is the advantage of tissues grown from the patients own cells
Less chance of rejection from the body’s immune system
Cloning for scientific research
-Useful for research into the action of genes that control development and differentiation
-Also used to grow specific tissues/organs for use in tests on the effects of medicinal drugs
Arguments for artificial cloning in animals
1) Produces whole herd of animals with a high yield/ shows an unusual combination of characteristics i.e. silk in their milk (lambs)
2) Produces genetically identical individuals of high value characteristics
3) Identical embryos/ tissues are useful in assessing the effect of genes and hormones with no interference from different genotypes
4) Testing medicinal drugs on clones stops drugs being tested on humans/animals
5) Can produce cells genetically identical to the donor to repair tissue damage from accidents
6) Individuals from an endangered species can be cloned to increase numbers
Arguments against artificial cloning in animals
1) Lack of genetic variation may expose the herd to certain diseases/pests
2) Animals may be produced with little regard for their welfare, which may result in meat producing chickens that cannot walk (unethical)
3) Success rate of adult cloning is poor and its a lot more expensive then conventional breeding
Arguments against artificial cloning in animals
1) Lack of genetic variation may expose the herd to certain diseases/pests
2) Animals may be produced with little regard for their welfare, which may result in meat producing chickens that cannot walk (unethical)
3) Success rate of adult cloning is poor and its a lot more expensive then conventional breeding
4) Cloned animals may be less healthy and have shorter life spans
5) Ethical issues regarding how long the embryo survives and whether it is right to create a life and simply destroy it
6) If endangered species are cloned to increase numbers it does not help increase genetic diversity
Biotechnology
The use of living organisms/ parts of living organisms in industrial processes to produce food, drugs or other products
What are the four main areas, in which micro-organisms are used in biotechnology
1) Food
2) Pharmaceutical drugs
3) Enzymes
4) Other products
Microorganisms used in food production
-Ethanol in beer and wine (yeast)
-CO2 to make bread rise (yeast)
-Lactic acid to make bread/cheese (Lactobacillus)
Microorganisms used in pharmaceutical drugs
-Penicillin (penicillium fungus)
Microorganisms used in enzymes
-Protease/lipase in washing powder (bacteria)
Advantages of using micro-organisms in biotechnology
1) Cheap/ easy to grow
2) Production takes place at lower temperatures (saves on fuel and energy)
3) Production takes place at normal atmospheric pressure (safer than using chemical reactions which requires high pressure)
4) Production not dependent on climate (can take place anywhere with suitable resources and equipment)
5) Micro-organisms can be fed by products from other food industries i.e. starch, waste, water
6) Short life cycle/ reproduce quickly
What is the reaction vessel called
Fermenter: vessel used to grow populations of microorganisms
What are other organisms are used in biotechnology
Genetically modified animals to produce useful products
Other forms of biotechnology
-Gene technology
-Gene modification snd therapy
-Selective breeding
-Cloning by embryo splitting / micropropogation
-use of enzymes in industrial processes
-Immunology
What types of food are micro-organisms in
-Yogurt
-Cheese
-Baking
-Alcohol beverages
Yogurt what micro-organisms are involved
Milk that has undergone fermentation (lactobacillus bulgaricus) (Streptococcus thermophilus)
How is yogurt made
1) Bacteria convert lactose to lactic acid
2) Acidity denatures the milk protein causing it to coagulate
3) Bacteria partially digests the milk making it easy to digest
Fermentation also produces the flavour characteristics of yogurt
Coagulate
(of a fluid, especially blood) change to a solid or semi-solid state
Bacteria in yogurt as probiotics
L. acidophilus
-May benefit human health by improving digestion of lactose
-Aids gastrointestinal function and stimulating the digestive system
Cheese - how is it made (1)
1) Milk is pre-treated with a culture of bacteria (Lactobacillus) that can produce lactic acid from lactose
2) Once acidified the milk is mixed with rennet
3) Rennet contains enzyme Rennin which coagulates the milk protein (casein) in the presence of calcium ions
Cheese - how is it made (2)
What happens when the milk is mixed with Rennet?
1) Kappa- casein (keeps casein in solution) is broken down. Casein insoluble
2) Casein is precipitated by the action of Calcium ions, which binds the molecules together
Cheese - how is it made (3)
Curd
1) Resulting solid: curd is separated from the liquid component by stirring and heating
2) Bacteria continues to grow and produces more lactic acid
3) Curd is then pressed into moulds
Cheese - how is it made (4)
Treatment while pressing the curd
This determines the characteristics of the cheese
Flavour determined by the later ripening and maturing process
Cheese - how is it made (4)
How is extra flavour added
Inoculation with fungi (e.g. penicillium) to produce ‘blue’ cheese
Bread - what is it (1)
A mixture of flour; water; salt; yeast (Single celled fungus)
Bread - how is it made (2)
1) Mixing - ingredients are mixed thoroughly together by kneading
2) Proving/fermenting - Dough is left in a warm place for up tot three hours whilst the yeast respires anaerobically. Produces CO2 which causes the bread to rise
3) Cooking - the risen dough is baked. Any alcohol evaporates during the cooking process
Alcoholic beverages
The product of the anaerobic respiration of yeast (S .cerevisiae)
Wine - How is made
Made using grapes that have natural yeasts in skin
-Grapes contain sugars/ fructose/ glucose
When grapes are crushed the yest, uses these sugars to produce CO2 and alcohol
Ale/ beer - How is it made?
Brewed by barley grains that are beginning to germinate (malting)
-As grain germinates it converts starch to maltose, which is respired by the yeast
-Anaerobic respiration produces CO2 and alcohol
-Hops used to give a bitter taste to the liquid
Single celled protein (SCP)
Fungal protein / mycoprotein used in food
-Quorn
-Can produce protein with a similar amino acid profile to animal/plant protein
-Grow on almost any organic substrate, including waste materials such as paper and whey (curdled milk from which the curds have been removed)
Advantages of using micro-organisms in food (1)
1) Production of protein faster to produce than animal/plant protein
2) Biomass produced has a high protein content (48-85%)
3) Production can be increased/decreased accordingly
4) No animal welfare issues
5) Micro-organisms - good source of protein
Advantages of using micro-organisms in food (2)
6) Micro-organisms genetically modified to adjust the amino acid content of a protein
7) SCP production can be combined with the removal of waste products
8) Production independent of weather
9) Not much land required
Disadvantages of using micro-organisms in food (1)
1) Some people may want to eat fungal food/ food grown on waste
2) Isolation of the protein - micro-organisms grown in huge fermenters and need to be isolated from the material on which they grow
3) Protein has to be purified to ensure it is uncontaminated
4) Microbial biomass can have a high proportion of nucleic acids which must be removed
Disadvantages of using micro-organisms in food (2)
5) Amino acid profile may be different from traditional animal protein - particularly deficient in methionine
6) Infection - conditions needed for micro-organisms to grow are also ideal for pathogenic organisms. Care must be taken to ensure the culture is not infected with the wrong organisms
7) Palatability - protein does not have the taste/ texture of traditonal protein sources
What are the two types of growth medium a microorganism is grown in a laboratory
-A broth kept in bottles/ tubes
-Agar melted and poured into petri dishes
Agar
A polysaccharide of galactose obtained from seaweed, which is used to thickened the medium into gel
What does typical nutrient agar contain
peptones (from enzymatic breakdown of gelatine)
-Yeast extracts
-Salt
-Water
-Glucose/blood
Aseptic technique
Sterile technique’s used in culturing and manipulating microorganisms
-Used to reduce the likelihood of contaminating the medium with unwanted bacteria/fungi
Standard procedure for an aseptic technique
1) Wash hands
2) Disinfect the working area
3) Have a Bunsen burner nearby to heat the air
4) As you open the vessel, pass the neck of the bottle over the flame to prevent bacteria in the air from entering the bottle. The bottle should also be flamed as it is closed
5) Do not lift the lid of the petri dish off completely - just open it enough to allow the introduction of the new desired micro-organism
6) Any glassware/metal equipment should be passed through the flame before and after contact with the desired micro-organism
Why in an aseptic technique do you need a Bunsen burner nearby
Causes the air to rise and prevents air borne micro-organisms settling
-Also creates an area around it of sterile air in which the microbiologist can work
What are the three key steps you must follow when growing micro-organisms on agar plates
1) Sterilisation
2) Inoculation - introduction of micro-organisms to the sterile medium
3) Incubation
Sterilisation
Sterilised by heating in an autoclave at 121 degrees for 15 mins.
-This kills all living organisms, including any bacteria/ fungal spores
-When the medium has cooled sufficiently to handle, it is poured into sterile petri dishes and left to set
-Important to leave the lid on the petri dish to prevent infection
How is the high temperature in the autoclave achieved
By boiling water at high pressure inside the autoclave
After use of the autoclave how is equipment sterilised
By heating
Inoculation
introduction of micro-organisms to the sterile medium
-Streaking
-Seeding
-Spreading
-Cotton swab
Streaking (Inoculation)
A wire inoculating loop is used to transfer a drop of liquid medium onto the surface of the agar
-The drop is drawn out into a streak by dragging the loop across the surface
-Be careful not to break the surface of the agar
Streaking (Inoculation) steps
1) Heat inoculating loop in blue flame and allow to cool briefly
2) Remove cap from the broth culture and flame mouth of bottle
-Dip cool sterile inoculating loop in broth
-Flame and recap the bottle
3) Spread a streak of culture over surface of agar and cover with a lid. Reheat inoculating loop in blue flame
4) Use adhesive tape to hold lid on petri dish and incubate at 25 degrees
Image inoculation streaking
Seeding (Inoculation)
A sterile pipette can be used to transfer a small drop of liquid medium to the surface of the agar or to the petri dish before the agar is poured in
Spreading (Inoculation)
A sterile glass spreader may be used to spread the inoculated drop over the surface of the agar
A small cotton swab (Inoculation)
Can be moistened with distilled water and used to collect the micro-organisms from a surface and then carefully wiped over the surface of the agar medium
Incubation
-Petri fish must be labelled and the top taped to the bottom using two strips of adhesive tape
-Petri dish is then placed in a suitable warm environment such as an incubator
-Then placed upside down as this prevents drops of condensation falling onto the surface of the agar (also prevents the agar medium from drying out too quickly)
-Suitable temperatures will depend on the type of organism being grown
Why must incubation you be careful that the petri dish is not sealed completely
Can lead to a selection of anaerobic bacteria that may be pathogenic
Incubation - when cultures are examined after
24-36 hours
-Do not open the petri dish
-Bacteria grow into visible colonies that may be shy/dull
-Colonies may be a wide range of different colours
-Each colony results from a single bacterium
What will different types of fungi look like
Filamentous fungi grow into a mass of hyphae which may be circular but the mass is not shiny and look like cotton wool
Single celled fungi grow as circular colonies
Using a liquid medium (a broth)
Clear and turns cloudy when bacteria have grown
-Used to increase the number of micro-organisms before transferring to the agar plates / counting / identification
-Aseptic techniques described above should be applied when using a broth
-Can be used to investigate population growth
Using a liquid medium (a broth) population growth
Used to measure the growth rate of micro-organism
-Sterile broth is inoculated and the population size is measured at regular intervals during incubation
-Population size can be measured by transferring a small sample to an agar plate and incubating the agar culture - each individual microorganism will produce a visible colony
Serial dilutions
When using a broth there may be too many colonies which merge together which makes the count impossible
-Serial dilution reduces the population density
How to produce a serial dilution
At each step the broth is diluted by a factor of 10
-Take 1cm3 of broth and add 9cm3 of distilled water and repeat
-When recording pop density need to multiply count by dilution factor and vol added to plate
closed culture
-Required for population growth
-Refers to a population in which all the conditions are set at the start and there is no exchange with the external environment
-The growth will follow a predictable pattern
How are the conditions in a closed culture different from batch production
Certain substances such as oxygen may be added to keep the population growing until the nutrients are used up
Lag phase
Early phase pop size does not grow quickly as pop to small and adjusting to environment
This may involve:
-Taking up water
-Cell growth
-Activating certain genes
-Synthesising specific proteins (enzymes(
Log (exponential phase)
Organisms have adjusted to new environment
-Each have enzymes needed to survive / sufficient nutrients / growth space
-Population size doubles with each generation
-This can be frequently as once every 20-30 mins
Stationary phase
Eventually the increasing number of the organisms use up the nutrients and produce increasing amounts of waste products such as CO2 and other metabolites
-Rae of population growth declines and the number of individuals dying increase until the reproduction rate equals the death rate
-Stationary phase - no population growth
Death phase
Nutrients run out and the concentration of waste products may become lethal
-More individuals die then are produced and the population begins to fall
-Eventually the organisms will die
Growth of population in a closed culture image
Primary metabolites
Primary metabolites produced during the normal activities of the micro-organism will be collected from a fermenter during the log phase
-In a fermenter the population is not kept in a closed culture but conditons are maintained for optimum growth
Fermenter
Commercial drug production can be controlled to ensure the best possible yield of the product
What must be controlled in the fermenter
1) Temperature - too hot the enzymes will denature and growth will be limited
2) Nutrients available - microorganisms require nutrients to grow and synthesise the product. Sources of carbon, nitrogen, minerals and vitamins are needed
3) Oxygen availability - most microorganisms respire aerobically
4) pH - enzyme activity and hence growth and synthesis are affected by extreme of pH
5) Concentration of product - if product is allowed to build up it may affect the synthesis of the product
How is a fermenter sterilised
Using superheated steam
What are the different parts of the fermenter
1) Pressure vent prevents any gas build up
2) Air inlet - sterile air provides oxygen in aerobic fermenters
3) Mixing blades
4) Water jacket inlet - allows circulation of water around the fermenter to regulate temperature
5) Outlet for draining the fermenter
6) Air outlet often in a ring - air bubbles out from outlets mix with culture
7) Electronic probes to measure pH/O2 / temperature
8) Water jacket outlet
9) Inlet for the addition of nutrients
10) Motor - rotates the blades so culture is mixed evenly
What are all inlets and outlets fitted with
Filters to prevent contamination
Primary metabolites
Products synthesised by the microorganisms during normal metabolism
-Continually released from the cells and can be extracted continuously from the fermenting broth
-Broth is topped up with nutrients as these are used by the microorganisms
- Some of the broth is removed - otherwise population becomes too dense
What kind of culture is primary metabolites
Continuous culture - keeps the microorganisms at a specific growth rate
Secondary metabolites
Products produced when the cells are placed under stress such as high population density or limited nutrient availability
-Produced mainly in stationary phase
-Culture is set up with a limited quantity of nutrients and allowed to ferment for a specific time
-Fermenter is then emptied and the product can be extracted from the culture
What kind of culture does secondary metabolites produce
Batch culture
Asepsis
Ensures sterile conditions are maintained
-Nutrient medium would also support the growth of unwanted microorganism which would reduce production
Asepsis - how would the growth of unwanted microorganisms reduce production
-Competition with the cultured microorganisms for nutrients and space
-Reduce the yield for useful products
-Spoil the product
-Produce toxic chemicals
-Destroy the cultured microorganisms and their products
Penicillin
Produced by mass fermentation of a fungus
-Secondary metabolite
-Produced by batch culture
Production of penicillin
1) Fermenter is run for 6-8 days
2) Culture is filtered to remove the cells
3) Antibiotic is precipitated as crystals by the addition of potassium compound
-Antibiotic may be modified by the action of other microorganisms or by chemical means
4) The antibiotic is mixed with inert substances and prepared for administration in tablet from / syrup for injection
Insulin
Obtained from a genetically modified bacterium by combing the human insulin gene with a plasmid to act as vector then inserting it into the E-coli
-Enabled the production of insulin by continuous culture
Bioremediation
The use of microorganisms to clean the soil and underground water on polluted sites
-Organisms convert toxic pollutants to less harmful substances
-Can break down crude oil/ solvents/ pesticides
-Stimulates the growth of microbes that use the contaminants as a food source
What are the right condition’s for microorganisms to grow (bioremediation)
-Available water
-Suitable pH
-Suitable temperature
What happens if conditions are not suitable (bioremediation)
Can be modified with the addition of certain substances
e.g. molasses/ pump O2 into aerobic bacteria
-Can be dug up and removed to be treated ex situ if conditions are not suitable in situ
Advantages of bioremediation
-Uses natural systems
-Less labour/equipment is required
-Treatment in situ
-Few waste products
-Less risk of exposure to clean up personnel
Disadvantages of bioremediation
Only suitable for certain products: heavy metals and lead cannot be treated
Immobilised enzymes
An enzyme that is held in place and not free to diffuse through the solution
-Taken out of suspension and held so that they do not mix freely with the substrate
-Amplifies biotechnological processes
Why are non-immobilised enzymes not beneficial for biotechnological processes
Enzymes are not used up in the reaction and remain in the suspension when the reaction has been completed
-This means enzyme and product must be isolated which could be expensive
Advantages of immobilised enzymes
- Extraction costs are lower as enzymes do not mix with the product
-The enzyme can easily be re-used
-Enzymes are surrounded by an immobilising matrix, which protects them from extreme conditions such as temperature and pH
Disadvantages of immobilised enzymes
-Setting up the enzyme process is more expensive
-IE are usually less active then free enzymes making the reactions slower
Methods used to immobilise enzymes
Bound to a surface or trapped so cannot enter substarte solution
-Adsorption
-Covalent bonding
-Entrapment
-Membrane separation
Adsorption
-Bound to a supporting surface by hydrophobic interactions and ionic links
A: Enzymes are bound and AS exposed
D: AS might be slightly distorted by interactions
D: Bending is not always strong - enzymes can become detached and leak into the reaction mixture
Adsorption supporting surfaces
Clay ; porous ; carbon ; glass beads
Covalent bonding
-Enzymes bound to the supporting surface by strong covalent bonds
-Bonding used a cross-linking agent which may link them in a chain
A: Enzymes are less likely to detach and leak into the mixture
D: Expensive/ distort the enzyme AS which reduces activity
Entrapment
Enzymes are trapped in a matrix that does not allow free movement
- Calcium alginate often used
A: Unaffected and fully effective
D: Only suitable when enzymes and substarte are small as most be able to diffuse in and out
Membrane separation
Enzymes separated by a partially permeable membrane
-Must be small (entrapment) - may limit reaction rate
Immobilised enzymes diagram
Industrial uses of immobilised enzymes
-Glucose isomerase
-Penicillin acylase
-Lactase
-Aminoacylase
-Glucoamylase
-Nitrile hydratase
Glucose isomerase
-Converts glucose to fructose
-Used widely in syrup production
-Used to produce high fructose corn syrup (HFCS) this is often used in ‘diet foods’ as same sweetness but less sugar
Penicillin acylase
Semi-synthetic penicillin’s means that some penicillin resistant microorganisms are not resistant
Lactase
Lactose to glucose and galactose by hydrolysis
-Lactose-free milk
Aminoacylase
A hydrolase used to produce pure samples of L-amino acids by removing the aceyl group from the nitrogen of an N-acyl-amino acid
L-amino acids are used as the building blocks for synthesis of pharmaceutical compounds
Glucoamylase
Converts dextrins to glucose
-During hydrolysis of starch, short polymers of glucose (dextrins) are formed
-Glucoamylase can be used to digest sources of starch like corn
Used in a wide range of fermentation processes including the conversion of the starch pulp to alcohol used to produce gasohol - alternative fuel for motor vehicles
Nitrile hydratase
Converts nitriles to amides, including acrylonitrile to acrylamide
Acrylamide can be polymerised to form polyacrylamide, which is a plastic used as a thickener
polyacrylamide (Nitrile hydratase)
Used in the treatment of water - helps stick small contaminants together so that they can easily be precipitated
-Also used to make the gel for electrophoresis
