Cloning and biotechnology

Clones are genetically identical copies. The term can apply to cells or whole organisms. Clones are produced by asexual reproduction in which the nucleus is divided by mitosis. Mitosis creates 2 identical copies of the DNA, which are then separated into 2 genetically identical nuclei before the cell divides to form 2 genetically identical cells. These cells may not be physically or chemically identical as, after division, they may differentiate to form 2 different types of cell.

Clones are formed in nature. Any organism that reproduces asexually will not produce clones of itself. For example, single-celled yeasts reproduce by budding and bacteria reproduce by binary fission. Both processes involve exact replication of DNA, so they cells produced are genetically identical. The offspring produced by cloning are genetically identical to the parent

• If the conditions for growth are good for the parent, then they will also be good for the offspring.
• Cloning is relatively rapid – so the population can increase quickly to take advantage of the suitable environmental conditions.
• Reproduction can be carried out, even if there is only one parent and sexual reproduction is not possible.

• The offspring may become overcrowded.
• There will be no genetic diversity (except that cause by mutation during DNA replication)
• The population shows little variation.
• Selection is not possible.
• If the environment changes to be less advantageous, the whole population is suscepitble.

The differentiation of many plant cells is not as complete as that in animals. Many parts of a plant contain cells that retain the ability to divide and differentiate into a range of types of cell. This means that plants are able to reproduce by cloning. Natural cloning involves a process called vegetative propagation. This is the process of reproduction through vegetative parts of the plants, rather than through specialised reproductive structure

Many plants grow horizontal stems that can form roots at certain points. These stems are called runners or stolens if they grow on the surface of the ground, and rhizomes if they are underground. Some rhizomes are adapted as thickened over-wintering organs from which one or more new stems will grow in the spring.

Suckers are new steps that grow from roots of a plant – these may be close to the base of an older stem or could be some distance away. In all cases, the original horizontal branch may die, leaving the new step as a separate individual.

Bulbs are an over wintering mechanism for many perennial monocotyledonous plants. Bulbs consist of an underground stem from which grow a series of fleshy leaf bases. There is also an apical bud, which will grow into a new plant in the spring. Often a bulb contains more than one apical bud and each will grow into a new plant e.g. a hyacinth bulb

Corms are often mistaken for bulbs. However, corms are solid rather than fleshy like a bulb. A corm is an underground stem with scaly leaves and buds. Corms remain in the ground over winter. In the spring the buds grow to produce one or more new plants. Croci and gladioli reproduce using corms.

The Kalanchoe plant reproduces asexually, as clones grown on the leaf margins. The immature plants drop off the leaf and take root.

Tubers are another type of underground stem. Potatoes are tubers. One potato will grow into one or more plants. Each new plant can then produce many new tubers (potatoes) later that year.

Animals do not clone as often as plants. There are, however, a few examples of natural clones. Mammals clone when identical twins are formed. This occurs when a fertilised egg (zygote) divides as normal, but the two daughter cells then split to become 2 separate cells. Each cell grows and develops into a new individual.

The easiest way to create clones is through making cuttings. To make a cutting, a stem is cut between 2 nodes. The end of the stem is then placed in moist soil. New roots will grow from the tissues in the stem – usually from the node, but they may grow from other parts of the buried stem. 

Some plants such as geraniums and blackberry will take root easily. Other plants may need further treatment. Dipping the cut stem into rooting hormone helps to stimulate root growth. It may also be helpful to wound or remove the bark from the cut end of the stem, as the=is encourages the plant to produce a callus. This can be useful to produce a large number of plants very rapidly. 

Cuttings can also be made successfully from other parts of the plant:

• Root cuttings, in which a section of root is buried just below the soil surface, and produces new shoots.
• Scion cuttings, which are dormant woody twigs.
• Leaf cuttings, in which a leaf is placed on moist soil. The leaves develop new stems and new roots. Some leaves may produce many new plants from one cutting.

Large-scale cloning by taking cuttings can be time-consuming and needs a lot of space. Also some plants do not respond well to taking cuttings. Many commercially grown houseplants are clones using tissue culture techniques. Tissue culture is a series of techniques used to grow cells, tissues or organs from a small sample of cells or tissue. It is 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. Tissue culture is widely used commercially to increase the number of new plants, in micropropagation.

1. Suitable plant material is selected and cut into small pieces. These are called explants. 

2. The explants are sterilised using dilute bleach or alcohol. This is essential to kill any bacteria and fungi, as these would thrive in the conditions supplied to help the plant grow well.

3. The explants are placed on a sterile growth medium (normally agar gel) containing suitable nutrients such as glucose, amino acids and phosphates, and hormones.

4. Once a callus has formed, it is divided to produce a larger number of small clumps of undifferentiated cells.

5. These small clumps of cells are stimulated to grow, divide and differentiate into different plant cell tissues. This is achieved by moving the cells to a different growth medium. Each medium contains different ratios of auxin and cytokinin. The first medium contains the ratio 100 auxin:1 cytokinin, and these stimulates roots to grown. The second medium contains the ratio 4 auxin:1 cytokinin, which stimulates shoots to form.

6. Once tiny plantlets have been formed, these are transferred to a greenhouse to be grown in compost or soil and acclimatised to normal growing conditions.

• Cloning is a relatively rapid method of producing new plants compared with growing plants from seed.
• Cloning can be carried out where sexual reproduction is not possible. Plants that have lost their ability to breed sexually can be reproduced, for example commercially grown bananas. Similarly, plants that are hard to grow from seed can be reproduced, for example orchids for the horticulture industry.
• The plants selected will all be genetically identical to the parent plant. They will therefore display the same desirable characteristics such as high yield, resistance to a common pest or disease, or a particularly colour of flower.
• If original plant had an unusual combination of characteristics due to selective breeding or genetic modification, then this combination can be retained without the risk of losing that combination through sexual reproduction.
• The new plants are all uniform in their phenotype, which makes them easier to grow and harvest.
• Using the apical bus (meristem) as an explant for tissue culture ensures the new plants are free from viruses.

• Tissue culture is labour intensive.
• It is expensive to set up the facilities to perform tissue culture successfully.
• Tissue culture can fail due to microbial contamination.
• All the cloned offspring are genetically identical and are therefore susceptible to the same pests and/or diseases. Crops grown in monocultures allow rapid spread of a disease or pest between the closely planted crop plants.
• There is no genetic variation, except that introduced by mutation.

Some invertebrate species such as greenfly and water fleas have evolved the ability to clone naturally. In other species it is a rare event. Therefore, most cloning of animals is artificial. 

Successful cloning starts with cells that are totipotent such cells can divide and differentiate into all types of cell found in the adult organism. In animals, the only truly totipotent cells are very early embryo cells.

Reproductive cloning can produce large numbers of genetically identical animals. Cloning may be useful for:

• Elite farm animals produced by selective breeding (artificial selection) or genetic modification. For example, a particularly good individual bull whose value is as a stud – supplying sperm for artificial insemination.
• Genetically-modified animals developed with unusual characteristics, for example, goats that produce spider silk in their milk and cows that produce less milk.

The 2 main techniques to achieve reproductive cloning are embryo twinning and somatic cell nuclear transfer (SCNT).

Mammals can produce identical offspring (twins) if an embryo splits very early in development. This process has given as rise to an artificial technique that has been in use since the 1970s.

1. A zygote (fertilised egg) is created by in vitro fertilisation (IVF).

2. The zygote is allowed to divide by mitosis to form a small ball of cells.

3. The cells are separated and allowed to continue dividing.

4. Each small mass of cells is placed into the uterus of a surrogate mother.

This technique has been used to clone elite farm animals or animals for scientific research. However, the precise genotype and phenotype of the offspring produced will depend upon the sperm and eggs used. Therefore, the phenotype will be unknown until the animals are born.

SCNT is the only way to clone an adult. The advantage is that the phenotype is known before cloning starts. This process was first performed successfully on a mammal in 1996, to produce Dolly the sheep.

1. An egg cell is obtained and its nucleus is removed, known as enucleation.

2. A somatic cell from the adult to be cloned is isolated and may have the nucleus removed.

3. The complete adult somatic cell or its nucleus is fused with the empty eggs cell by applying an electric shock.

4. The shock also triggers the egg cell to start developing, as though it had just been fertilised.

5. The cell undergoes mitosis to produce a small ball of cells.

6. The young embryo is placed into the uterus of a surrogate mother

Therapeutic cloning 

• New tissues and organs can be grown as replacement parts for people who are not well:
• Skin can be grown in vitro to act as a graft over burned areas.
• Clones cells have been used to repair damage to the spinal cord of a mouse and restore the capability to produce insulin in the pancreas.
• There is the potential to grow whole new organs to replace diseased organs.
• Tissues grown from the patient’s own cells will be genetically identical and so avoid rejection, which is a problem when transplanting donated organs.

Cloning for scientific research

Cloned genetically identical embryos can be used for scientific research into the action of genes that control development and differentiation. They can also be used to grow specific tissues or organs for use in tests on the effect of medicinal drugs.

• Can produce a whole herd of animals with a high yield or showing an unusual combination of characteristics (such as producing silk in their milk).
• Using genetically identical embryos and tissues for scientific research allows the effects of genes and hormones to be assessed with no interference from genotypes.
• Produces genetically identical copies of very high value individuals retaining the same characteristics.
• Testing medicinal drugs on cloned cells and tissues avoids using animals or people for testing.
• Can produce cells and tissues genetically identical to the donor, for use in repairing damage caused by disease or accidents.
• Individuals from an endangered species can be cloned to increase numbers.

• Lack of genetic variation may expose the herd to certain diseases and pests. Animals may be produced with little regard for their welfare, which may have undesirable side effects such as meat-producing chickens than cannot walk.
• The success rate of adult cell cloning is very poor and the method is a lot more expensive than conventional breeding. Clones animals may be less healthy and have shorter life spans.
• There are ethical issues regarding how long the embryo survives and whether it is right to create a life simply to destroy it.
• Doesn't increase genetic diversity. 

• Microorganisms are cheap and easy to grow.
• Production process takes place at lower temperature than would be required to make the molecules by chemical engineering, this means that fuel is saved and the cost is reduced.
• Can take place at normal atmospheric pressure.
• The production process is not dependent on climate.
• The microorganism can be fed by-products from other food industries but starting materials often have to be pre-treated, which adds to the cost.
• Microorganisms have a short life cycle and reproduce quickly. Some microorganisms may reproduce as often as every 30 minutes under ideal conditions (by binary fission). Therefore, a large population can grow very quickly inside the reaction vessel (fermenter).
• Microorganisms can be easily genetically modified. This allows very specific production processes to be achieved.
• There are fewer ethical considerations to worry about in using microorganisms.
• The products are often released from the microorganism into the surrounding medium. This makes the product easy to harvest.
• The product is more pure and easy to isolate than in conventional chemical engineering processes. This means lower downstream processing costs.

Microorganisms are not the only organisms used in biotechnology. Genetically-modified mammals such as sheep, goats and cows can be used to produce useful proteins. In some mammals the proteins are incorporated into the milk and can be easily harvested. For example, goats have been genetically modified to possess the gene for spider silk secrete it into their milk. In other cases the protein may be secreted into the blood. For example, cows have been genetically modified to synthesise human antibodies, which can be isolated from their blood.

In recent years the term ‘biotechnology’ has come to mean using organisms in production processes. However, biotechnology also encompasses the following processes:

• Gene technology
• Genetic modification and gene therapy
• Selective breeding
• Cloning by embryo-splitting and micropropagation
• The use of enzymes in industrial processes
• Immunology

Yoghurt is milk that has undergone fermentation by Lactobacillus bulgaricus and Streptococcus thermophillus. The bacteria convert lactose to lactic acid. The acidity denatures the milk protein, causing it to coagulate. The bacteria partially digest the milk, making it easy to digest. Fermentation also produces the flavours characteristics of yoghurt.

Other bacteria, such as L. acidophilus, L. subsp. casei and Bifidobacterium, may be added as probiotics – bacteria which may benefit human health by improving digestion of lactose, aiding gastrointestinal function and stimulating the immune system.

Milk is usually pre-treated with a culture of bacteria (Lactobacillus) that can produce lactic acid from the lactose. Once it is acidified, the milk is mixed with rennet. Rennet contains the enzyme rennin (chymosin), which is found in the stomachs of young mammals. Rennin coagulates the milk protein (casein) in the presence of calcium ions:

• Kappa-casein, which keeps the casein in solution, is broken down. This makes the casein insoluble. 
• The casein is precipitated by the action of calcium ions, which bind the molecules together. 

The resulting solid, called curd, is separated from the liquid component (whey) by cutting, stirring and heating. The bacteria continue to grow, producing more lactic acid. The curd is then pressed into moulds. 

Treatment while making and pressing the curd determines the characteristics of the cheese. Flavour is determined during the later ripening and maturing processes. The cheese can be given additional favour by inoculation with fungi such as Penicillium to produce blue' cheese.

Bread is a mixture of four, water and salt with some yeast (which is a single-celled fungus, Saccharomyces cereviside). Bread-making processes have three key steps: 

1. Mixing - the ingredients are mixed together thoroughly by kneading. This produces dough.  

2. Proving/fermenting -  the dough is left in a warm place for up to three hours while the yeast respires anaerobically. This produces carbon dioxide bubbles, causing the dough to rise. 

3. Cooking - the risen dough is baked. Any alcohol evaporates during the cooking process.

Alcoholic beverages are also the product of the anaerobic respiration of yeast (S. cerevisiae). Wine is made using grapes that naturally have yeasts on their skin. Grapes contain the sugars fructose and glucose. When the grapes are crushed, the yeast uses these sugars to produce carbon dioxide and alcohol.

Ale or beer is brewed using barley grains that are beginning to germinate. This process is called malting. As the grain germinates it converts stored starch to maltose, which is respired by the yeast. Anaerobic respiration again produces carbon dioxide and alcohol. Hops are used to give a bitter taste to the liquid.

More recently, microorganisms have been used to manufacture protein that is used directly as food. The microorganism used most frequently is the fungus Fusarium venenatum. The fungal protein or mycoprotein is also known as single-cell protein (SCP). The best known example of a mycoprotein is Quorn, which was first produced in the early 1980s. It is marketed as a meat substitute for vegetarians and a healthy option for non-vegetarians, as it contains no animal fat or cholesterol.

There is huge potential in SCP production using such microorganisms as Kluyveromyces, Scytalidium and Candida. These fungi can produce protein with a similar amino acid profile to animal and plant protein. They can grow on almost any organic substrate, including waste materials such as paper and whey (curdled milk from which the curds have been removed). 

• Production of protein can be many times faster than that of plant or animal 
• The biomass produced has very high protein content (45-85%)
• Production can be decreased or increased according to demand 
• There are no animal welfare issues. 
• The microorganisms provide a good source of protein. 
• The protein contains no animal fat or cholesterol. 
• The microorganisms can easily be genetically modified to adjust the amino acid content of the protein. 
• SCP production could be combined with removal of waste products 
• Production is independent of seasonal variations. 
• Not much land is required.

• Some people may not want to eat fungal protein or food that has been grown on waste. 
• Isolation of the protein - the microorganisms are grown in huge fermenters and need to be isolated from the material on which they grow 
• The protein has to be purified to ensure it is uncontaminated. 
• Microbial biomass can have a high proportion of nucleic acids which must be removed. 
• The amino acid profile may be different from traditional animal protein - and particularly it can be deficient in methionine 
• Infection - the conditions needed for the microorganisms to grow are also ideal for pathogenic organisms. Care must be taken ensure the culture is not infected with the wrong organisms. 
• Palatability - the protein does not have the taste or texture of traditional protein sources.

Commercial drug production uses large stainless steel containers called fermenters, in which the growing conditions can be controlled to ensure the best possible yield of the product. The conditions must controlled include: 

• temperature -  too hot and enzymes will be denatured, too cool and growth will be limited 
• nutrients available - microorganisms require nutrients to grow and synthesise the product. Sources of carbon, nitrogen, minerals and vitamins are needed 
• oxygen availability - most microorganisms respire aerobically 
• pH - enzyme activity and hence growth and synthesis are affected by extremes of pH 
• concentration of product - if the product is allowed to build up, it may affect the synthesis process. 

A fermenter must first be sterilised using superheated steam. It can then be filled with all the components required for growth and supplied with a starter culture of the microorganism to be used. The culture will be left to grow and synthesise the products.

Some products are synthesised by the microorganism during normal metabolism when they are actively growing. These are called primary metabolites. Such products are continually released from the cells and can be extracted continuously from the fermenting broth. The broth is topped up with nutrients as these are used by the microorganisms. Some of the broth is removed regularly to extract the product and remove cells from the broth - otherwise the population becomes too dense. This is known as continuous culture and keeps the microorganism growing at a specific growth rate.

Other products are produced only when the cells are placed under stress, such as high population density or limited nutrient availability. These are called secondary metabolites and are produced mostly during the stationary phase of growth. Here, the culture is set up with a limited quantity of nutrients and allowed to ferment for a specific time. After this time, the fermenter is emptied and the product can be extracted from the culture. This is known as batch culture.

Asepsis is ensuring that sterile conditions are maintained. The nutrient medium would also support the growth of unwanted microorganisms which would reduce production, because the unwanted microorganisms: 

• compete with the cultured microorganisms for nutrients and space 
• reduce the yield of useful products 
• spoil the product. 

They may also: 

• produce toxic chemicals 
• destroy the cultured microorganisms and their products. 

In processes where foods or medicinal chemicals are produced, all products must be discarded if contamination by unwanted organisms occurs.

Florey and Chain devised the process to successfully mass produce penicillin through fermentation by the fungus Penicillium chrysogenum. Modern strains of the fungus have been selectively bred to be more productive than the early strains. Penicillin is a secondary metabolite - it is only produces once the population has reached a certain size. Therefore, penicillin is manufactured by batch culture. 

• The fermenter is run for six to eight days. The culture is then filtered to remove the cells. 
• The antibiotic is precipitated as crystals by the addition of potassium compounds. The antibiotic may be modified by the action of other microorganisms or by chemical means. 
• The antibiotic is mixed with inert substances and prepared for administration in tablet form, as a syrup or in a form suitable for injection.

Insulin is widely used to treat Type 1 diabetes. It was previously extracted from the pancreas of animals such as cattle or pigs sent sent for slaughter. Insulin from slaughtered animals is not identical human insulin and so is less effective than human insulin, and expensive to extract. 

In 1978, synthetic human insulin was developed by genetically modifying a bacterium. The gene for human insulin was combined with a plasmid to act as a vector, so the gene could be inserted into the bacterium Escherichia coli. The resulting genetically-modified bacterium enabled the production of vast quantities of human insulin at relatively low cost. Insulin is manufactured by continuous culture. 

Bioremediation is the use of microorganisms to clean the soil and underground water on polluted sites. The organisms convert the toxic pollutants to less harmful substances. The idea really started when Ananda Chakrabarty modified a Pseudomonas bacterium in 1971, enabling it to break down crude oil, and he proposed that it could in treating oil spills. Solvents and pesticides can also be used be treated using bioremediation. Bioremediation involves stimulating the growth of suitable microbes that use the contaminants as a source of food. It requires the right conditions for the growth of microorganisms which are:

• Available water
• A suitable temperature 
• Suitable pH. 

Where conditions are not quite suitable, they may be modified by the addition of suitable substances. In some cases, additional nutrients such as molasses may be needed to ensure the microorganisms can grow effectively. It may also be necessary to pump in oxygen for aerobic bacteria. Where conditions cannot be made in situ, the soil may be dug up and moved to be treated ex situ. 

• Uses natural systems
• Less labour/equipment is required 
• Treatment in situ
• Few waste products 
• Less risk of exposure to clean-up

However, bioremediation is only suitable for certain products; heavy metals such as cadmium and lead cannot be treated. 

Microorganisms will grow on almost any material that provides the carbon compounds for respiration and a source of nitrogen for protein synthesis. However, in the laboratory, microorganisms are usually grown in one of two types of growth medium:

A soup like liquid called a broth, kept in bottles or tubes

A set jelly-like substance called agar, which is melted and poured into petri dishes. 

Typical nutrient agar contains peptones (from the enzymatic breakdown of gelatine), yeast extract, salts and water; it may also contain glucose or blood. 

• Wash you hands. 
• Disinfect the working area. 
• Have a Bunsen burner operating nearby to heat the air. This causes the air to rise and prevents air-borne microorganisms settling. It also creates an area around it if sterile air in which the microbiologist can work. 
• As you open a vessel, pass the neck of the bottle over the flame to prevent bacteria in the air entering the bottle. The bottle should also be flamed as it is closed. 
• Do not lift the lid of the Petri dish completely - just open it enough to allow introduction of the desired microorganism. 
• Any glassware or metal equipment should also be passed through the flame before and after contact with the desired microorganisms. 

Microorganisms can grow almost anywhere. The key is to ensure that they grow on the medium used and that you grow the desired microorganism rather than others that have infected the medium by mistake. There are 3 stages to growing microorganisms on agar plates:

• Sterilisation 
• Inoculation 
• Incubation 

The nutrient agar medium and any equipment to be used must be sterilised. The medium is sterilised by heating in an autoclave at 121C for 15 minutes (the high temperature is achieved by boiling water under high pressure inside the autoclave). This kills all living organisms, including any bacterial or fungal spores. When the medium has cooled sufficiently to handle, it is poured into sterile Petri dishes and left to set. It is important that the lid is kept on the Petri dish to prevent infection. All equipment used from this point onwards must be sterilised by heating. 

Inoculation is the introduction of microorganisms to the sterile medium. This can be achieved in a number of ways:

• Streaking - a wire inoculating loop is used to transfer a drop of liquid medium into the surface of the agar. The drop is drawn out into a streak by dragging the loop across the surface. Take care not to break the surface of the agar. 
• Seeding - 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 - a sterile glass spreader may be used to spread the inoculated drop over the surface of the agar. 
• A small cotton swap or cotton bud can be moistened with distilled water and used to collect microorganisms from a surface and then carefully wiped over the surface of the agar medium.

The Petri dish must be labelled and the top taped to the bottom using two strips of adhesive tape - be careful not to seal the Petri dish completely, as this can lead to selection of anaerobic bacteria which may be pathogenic. The Petri dish is then placed in a suitable warm environment such as an incubator. It should be placed upside down as this prevents drops of condensation falling onto the surface of the agar. It also prevents the agar medium from drying out too quickly. 

Cultures can be examined after 24-36 hours. Do not open the Petri dish. Bacteria grow into visible colonies, which may be shiny or dull. Some colonies are round with entire edges, while other can have crenated edges. The colonies can also be a range of different colours. Each colony results from a single bacterium. 

Filamentous fungi grow into a mass of hyphae, which may also be circular, but the mass is not shiny and often looks like cotton wool with fluffy aerial hyphae. Single celled fungi (yeasts) grow as circular colonies. 

All Petri dishes must be completely sterilised after use and before disposal. Thoroughly wash your hands after handling a Petri dish, as any moisture coming out of the dish could be a source of infection. 

A liquid broth is initially clear but will turn cloudy when bacteria have grown. A liquid broth can be useful to increase the numbers of microorganisms before transferring to agar plates for counting or identification. Similar aseptic techniques can be applied when using a broth. 

A liquid broth can be used to measure the growth rate of a microorganism. A sterile broth is inoculated and the population size measured at regular intervals during incubation. The population size can be measured by transferring a small sample to an agar plate and incubating the agar culture. It is essential to reduce the population density, this can be done by serial dilution. 

Lag phase - The population does not grow quickly. This is because population is still small,and because the organisms are adjusting to their genetic new environment. This may involve taking up water, cell growth, switching on certain genes and synthesising specific proteins (enzymes).

Log (exponential) phase- In the log phase, the organisms have adjusted to their environment. They each have the enzymes needed to survive. Each individual has sufficient nutrients and space to grow rapidly and reproduce. The population doubles in size with each generation. In some microorganisms, this can be as frequently as once every 20-30 minutes. 

Stationary phase - Eventually the increasing numbers of organisms used up the nutrients and produce increasing amounts of waste products such as carbon dioxide and other metabolites. The rate of population growth declines and the number of individuals dying increases until the reproduction rate equals the death rate. This is the stationary phase, where there is no population growth. 

Death (decline) phase - The nutrients run out and the concentration of waste products may become lethal. More individuals die than are produced and the population begins to fall. Eventually all the organisms will die. 

Primary metabolites produces during the normal activities of the microorganism will be collected from a fermenter during the log phase. In a fermenter, the population is not kept in a closed culture, but the conditions are maintained for optimal growth. 

Secondary metabolites are produced in the stationary phase. The population must be kept in a closed culture and the metabolites can be collected at the end of the stationary phase or during the decline phase. 

Some biotechnological processes can be simplified by taking the enzymes out of the microorganisms. Enzymes are not used up in the reaction and remain in suspension when the reaction has been completed. In an industrial process, this means that the product must be isolated from the enzymes before use. This could be expensive. Immobilised enzymes are taken out of suspension and held so that they do not move freely with the substrate. This has the following advantages:

• Enzymes do not mix with the product, so extraction costs are lower.
• The enzymes can easily be reused.
• A continuous process is made easier, as there are no cells requiring nutrients, reproducing and releasing waste products.
• The enzymes surrounded by the immobilising matrix, which protects them from extreme conditions – so higher temperatures or a wider pH range without causing denaturing.

However, setting up the immobilised enzyme process is more expensive, and immobilised enzymes are usually less active that free enzymes, making the process slower.

Enzyme molecules are bound to a supporting surface by a combination of hydrophobic interactions and ionic links. Suitable surfaces include clay, porous carbon, glass beads and resins. The enzyme molecules are bound with the active site exposed and accessible to the substrate. However, the active site may be slightly distorted by the additional interactions affecting enzyme activity. The bonding forces are not always strong, and enzymes can become detached and leak into the reaction mixture.

Enzyme molecules are bonded to a supporting surface area such as clay using strong covalent bonds. The enzymes are bonded using a cross-linking agent, which may also link them in a chain. The production of covalent bonding can be expensive and can distort the enzyme active site, reducing activity. However, the enzymes are much less likely to become detached and leak into the reaction mixture.

Enzyme molecules are trapped in a matrix that does not allow free movement. The enzyme molecules are unaffected by entrapment and remain fully active. However, the substrate molecules must diffuse into the entrapment matrix, and the product molecules must be able to diffuse out. The method is therefore suitable only for processes where the substrate and product molecules are relatively small. Calcium alginate beads are often used in schools to immobilise enzymes by entrapment. Industrial processes may also use a cellulose mesh.

Enzyme molecules are separated from the reaction mixture by a partially permeable membrane. As in entrapment, the substrate and product molecules must be small enough to pass through the partially permeable membrane by diffusion. This access to the enzymes may limit the reaction rate.

• It converts glucose to fructose.
• It is probably the most widely used enzyme because of the number of applications of the syrup produced.
• Used to produce high fructose corn syrup (HFCS), which is much sweeter than sucrose. HFCS is often used in diet foods, as less sugar needs to be added for the equivalent sweetness. It may also be used as a sweetener in foods for diabetics. HFCS is cheaper than sucrose and so is widely used in the food industry to replace sucrose – especially in soft drinks, but also in many processes foods such as breakfast cereals, jam, ice cream, yoghurt and even sliced ham

It is also known as penicillin amidase. Formation of semi-synthetic penicillins, such as amoxicillin and ampicillin, which were first developed during the 1960s. Some penicillin resistant microorganisms are not resistant to these semi-synthetic penicillins. 

• Converts lactose to glucose and galactose by hydrolysis.
• Used to produce lactose-free milk. 
• Milk is an important source of calcium, which is needed for strong bones and teeth. People with insufficient calcium in their diet are more likely to develop weak bones or osteoporosis. It is therefore important that the people who are lactose intolerant are given calcium supplements or lactose-free milk. 

• A hydrolase used to produce pure samples of L-amino acids by removing the acyl group from the nitrogen of an N-acyl-amino acid. 
• L-amino acids are used as the building blocks for synthesis of a number of pharmaceutical and agrochemical compounds. They may also be used as additives for human food and animal feed stuffs. 

Converts dextrins to glucose. During the hydrolysis of starch, short polymers of glucose (dextrins) are formed. Hydrolysis by glucoamylase can convert these dextrins to glucose. Glucoamylase can be immobilised on a variety of surfaces and used to digest sources of starch such as corn and cassava. 

The enzyme is used in a wide range of fermentation processes, including the conversion of starch pulp to alcohol used to produce gasohol - an alternative fuel for motor vehicles. It is also used within the food industry to make high fructose corn syrup.