Chapter 22


Natural cloning

Asexual reproduction is a form of cloning and it results in offspring produced by mitosis known as clones, which are genetically identical to both the parent organism and each other. Vegetative propagation occurs in mant species of flowering plants, a structure forms which develops into a fully differentiated plant, which may be propagated from the stem, leaf, bud or root and it eventually becomes independant from its parent. Vegetative propagation often involves perennating organs which enable plants to survive adverse conditions, as they contain stored food and can remain dormant in the soil, allowing survival from one growing season to the next, for example:

  • Bulbs - eg daffodils, where the leaf bases swell with stored food from photosynthesis and buds form internally to develop into new shoots and new plants in the next grwoing season.
  • Runners - eg strawberry plants, where a lateral stem grows away from the parent plant and roots develop where the runner touches groun, and a new plant develops and the runner withers leaving the new individual independant.
  • Rhizomes - eg marram grass, a rhizome is a specialised horizontal stem running underground often swollen with stored food, buds develop and form new vertical shoots to grow new plants.
  • Stem tubuers - eg potatoes, where the tip of an underground stem becomes swollen with stored food to form a tuber or storage organ, buds on these develop to produce new shoots.
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Using natural clones in horticulture

Natural plant cloning is exploited in horticulture by farmers and gardeners to produce new plants, splitting up bulbs, removing young plants from runners and cutting up rhizomes all increase plant numbers cheaply and the new plants have exactly the same genetic characteristics as their parents, it is also possible to take cuttings of many plants and apply rooting hormones to the base of cuttings to encourage the growth of new roots. Propagation from cuttings has several advantages over using seeds as it is much faster and ensures a high quality crop, the main disadvantage is the lack of genetic variation in the offpsring should a new abootic facter appear. Many of the world's important food crops are propagated by cloning such as bannanas and sweet potatoes, coffee and tea bushes from cuttings. Sugar cane is one of the fastest growing crops and is usually propagated by cloning by taking short lengths of cane and buried in shallow trenches.

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Micropropagation using tissue culture

Micropropagation is the process of making large numbers of genetically identical offspring from a single parent plant using tissue culture techniqies which is used when a desirable plant doesn.t readily produce seeds, doesn't respond well to natural cloning, has been genetically modified or selectively bred with difficulty, or is required to be pathogen free by growers. The basic principles of micropropagation and tissue culture are:

  • Take a small sample from the plant you want to clone, the meristem tissue from shoot tips or axial buds is often dissectd in sterile conditions to avoid contamination.
  • The sample is immersed in sterilising agents such as bleachm ethanal or sodium dichloroisocyanurate which doesn't need to be rinsed off, making it more liekly to be sterile.
  • The material removed from the plant is called the explant, which is placed in a sterile culture medium containing a balance of plant hormones to stimulate mitosis, and the cells proliferate to form a mass of identical cells called a callus.
  • The callus is then divided up into clumps which are transferred to a new culture medium containing a different medium of hormones and nutrients to stimulate the development of tiny genetically identical plantlets.
  • The plantlets are planted to geow in compost where they grow into small plants.
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Advantages and disadvantages of micropropagation

Arguments for micropropagation:

  • Micropropagation allows for the rapid production of large numbers of plants with known genetic makeup which will yield good crops.
  • Culturing meristem tisease produces disease free tissue.
  • It makes it possible to produce viable numbers of plants after GMO of plant cells.
  • It provides a way of producing very large numbers of new plants which are seedless and therefore sterile to meet consumer tastes.
  • It provides a way of growing plants which are naturally relatively infertile or difficult to grow.
  • It provides a wat of reliably increasing the numbers of rare endagered plants.

Arguments against micropropagation:

  • It produces monoculture of plants which are all succeptable to the same abiotic factors.
  • It is relatively expensive and requires skilled workers.
  • The explants and plantlets are vunerable to infection during the production process.
  • If the source material is infected with a virus, all the clones will also be infected, and in some cases a large number of plants are lost during the process.
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Natural animal cloning

Cloning in invertebrates: Natural cloning in invertebrates can take several forms, some animals such as starfish can regenerate entire animals from fragments of the original if they are damaged, hyrda produce small buds on the side of their body which develop into genetically identical clones, and many insects can reproduce offspring without mating. 

Cloning in vertebrates: The main form of vertebrate cloning is the formation of monozygotic twins where the early embryo splits to form two seperate ones. Some female amphibians and reptiles produce offspring when no mate is available, the offspring are often male rather than female, so although they are not clones all their genetic matierial arises from the mother.

Artifical cloning in animals: It is relatively easy to produce artificial clones of some invertebrates such as fragmentation of a starfish, it is harder with vertebrates but some methods are now used in the production of high quality farm animals and the developement of genetically engineered animals for farming.

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Artificial twinning

After an egg is fertilised it divides to form a ball of cells, each of which is totipotent, in artifical twinning the splitting of the early embryo is done manually into more than two pieces and results in a number of identical offspring. The stages of artifical twinning are:

  • A cow with desirable traits is treated with hormones so she super ovulates releasing more mature ova than normal. The ova may be fertilised naturally or by artificial insemination by a bull with good traits, the early embroys are gently flushed out of the uterus, or the mature eggs can can be removed and fertilised in the lab.
  • Usually when the cells are still totipotent the cells of the embryo are split to produce several smaller embryos, each capable of producing a healthy calf.
  • Each of these is grown in the lab for a few days before implanting them into surrogate mothers, the embryos develop normally and are bprn naturally so a number of identical clones is produced by different mothers.

This technology makes it possible to greatly increase the numbers of of offspring produced by the animals with the best genetic stock, embryos may be frozen allowing the success of a particular embryo to be assessed and if the the stock is good the rest can be implanted.

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Somatic cell nuclear transfer

Artifical twinning clones an embryo, however it is now possible to clone an adult animal by taking the nucleus from an adult somatic cell and transferring it to an enucleated egg cell (which has had its nucleus removed). A tiny electric shock is used to fuse the egg and nucleus to stimulate the cel to divide and form an embryo that is a clone of the original adult. This process is known as somatic cell nuclear transfer (SCNT), which is simple in theory but in practise there are many difficulties so the technique is still not widely used:

  • The nucleus is removed from a somatic cell of an adult animal and the nucleus is removed from a mature ovum harvested from a different female of the same species  - it is enucleated.
  • The nucleus from the adult somatic cell is placed into the enucleated ovum and given a mild electric shock so it fuses and begins to divide. The embyro developed is transferred into the uterus of a third animal where it develops to term. The new animal is a clone of the animal frim which the original somatic cell is derived, although the mtDNA will come from the egg cell.

This process is also known as reproductive cloning because live animals are the end result, there are problems that can arise from SCNT, but scientists are constantly improving the technique, and can be used in fields such as farming and genetic engineering to grow organs for transplants.

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Arguments for and against animal cloning

Arguments for animal cloning: Artifical twinning enables high-yielding farm animals to produce many more offspring than normal reproduction, it enables the success of the male animal at passing on desirable genes to be determined. SCNT enables GM embryos to be replicated and to develop, giving many embryos from one engineering procedure and is important in the the production of theraputic human proteins in the milk of GMO farm animals. SCNT also enables scientists to clone specific animals, and has the potential to enable rare, endagered or even extinct animals to be reproduced from stored tissue.

Arguments against animal cloning: SCNT is very inefficient and often takes many eggs to produce a single cloned offspring, malformed offspring can often be produced, or animals produced by clones have a shortened lifespan, and has veen relatively unsuccessful in increasing the populations of rare organisms, and the idea of restoring extinct organisms is becoming increasingly unconvincing that it will be possible.

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Cloning humans

  • Scientists have reproduced clones of primates by artificial twinning but it is proving very difficult to produce a SCNT clone of a primate.
  • Part of the problem seems to be the spindle proteins needed for cell division in primates are sited very close to the nucelus, so the removal of the nucleus for the production of an enucleated ovum also destroys the mechanism by which the cell divides, which isn't a problem with many other mammals because the spindle proteins are more dispersed in the cytoplasm.
  • In addition the synchronisation of the stage of the embryo and the state of the reproductive organs of the mother have to be exactly attuned im primates whereas there seems to be more flexibility in some other mammals.
  • In recent years scientist have finally produced embryonic stem cell lines by SCNT, so may be possible to develop these potentially important theraputic cells from humans.
  • In most countries there is strict legislation to prevent the reproductive cloning of humans, but a modified version of SCNT has the potential to produce human embryonic stem cells from an adult to be used in stem cell treatment, and is called theraputic cloning, and could potentially be used to to grow replacement organs which won't trigger an immune response in a patient.
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Biotechnology involves applying biological organisms or enzymes to the synthesis, breakdown, or transformation of materials in the service of people, from the production of cheese or wine to molecular technologies using DNA manipulation to produce genetically engineered organisms to synthesise drugs such as insulin and antibiotics, or use in biological systems to remove soil and water pollution in processes known as bioremediation. The most commonly used organisms in biotechnology are fungi, particularily the yeasts, and bacteria, which are useful for new biotechnology based around genetic manipulation. Most biotechnology involves using biological catalysts in a manufacturing process and the most stable, convenient and effective form of the enzymes is often a whole microorganism, microorganisms are ideal for a variety of reasons:

  • There are no welfare issues to consider, all that is needed is optimum conditions and there is a massive range of microorganisms capable of carrying out many chemical reactions.
  • Genetic engineering allows artificial manipulation of microorganisms to carry out reactions they would normally, and they have a short life cycle and a rapid growth rate, so with the right conditions huge quantities of microoganisms can be produced, and nutrient requirements are often simple/cheap, and GMO means they can utilise marerials that would usually be waste. The conditions needed for grwoth are often low temperatures, a supply of oxygen and removal of waste gases as they provide their own catalysts.
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Indirect food production

Microorganisms are widley used in biotechnological processes to make food such as bread and yoghurt, here the microorganisms have an indirect effect and it is their actions on other food that is important. There are generally few disadvantages to using microorganisms indirectly ro produce human food, but conditions need to be ideal for both the microorganisms and so the food being produced doesn't go off or cause diseases so the process have to be sterile:

  • Baking: Active yeast mixed with sugar and water to stimulate anaerobic respiration and carbon dioxide results in the bread rising, and the yeast cells are killed when the bread is cooked.
  • Brewing: Yeast respires anaerobically to produce ethanol througyh malting and fermentation to break down sugars that yeast enzymes can use, before the yeast is inhibited by falling pH.
  • Cheese making: Bacteria feed on the lactose in milk changing the texture and taste through pasturisation and breaking down or milk sugars.
  • Yoghurt making: Bacteria produce extracellular polymers that give yoghurt a smooth thick texture through fermentation and pasturisation.
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Direct food production

Single cell proteins (SCP) are being developed to prevent potential protein shortages around the world, most well known one is Fusarium venatatum which is a single celled fungus used to make Quorn which is grown in large fermenters using glucose syrup as a food source and the protein is then bound with albumin and compressed. Other  microorganism proteins have been less successful despite the fact that proteins from algae, bacteria and yeasts can be produced quickly and easily.

Advantages of using microorganisms to produce food: Microorganisms reproduce fast and produce proteins faster than animals and plants, they have a high protein content with little fat, they can use a variety of waste materials and can be genetically modified to produce the protein required. Production of microorganisms is not dependant on weather or breeding cyclrd, and there are no welfare issues when growing them.

Disadvantages of using microorganisms to produce food: Some microorganims can also produce toxins if the conditions are not optimum, the microorganism has to be seperated from the nutirent broth, sterile conditions are essential, the proteins have to be purified to ensure they contain no contaminants, it has little flavour and many people have concernes about using GM organisms and eating GM food.

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The first effective antibiotic was penicillin which nw uses P.chrysogenum which needs high oxygen and nutrient levels to grow well, along with a maintained temperature pH, semi continuius batch processing is used. The process uses relatively small fermenters as it is difficult to maintain high oxygen levels in large bioreactors, the mixture is continuously stirred to keep it oxygenated, a rich nutrient medium is needed, and contains a buffer to maintain a pH of about 6.5.

In the past insulin was extracted from the pancreas of animals meaning the supply of insulin was erratic because it depended on the demand for meat, and some people were allergic to animal insulin and it was often impure. The developemnt of GMO bacteria revolutionised insulin production. The bacteria are grown in a fermenter and downstream processing results in a constant supply of pure human insulin. 

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In bioremediation microorganisms are used to break down pollutants and contaminants in the soil or in the water, there are different approaches to bioremediation:

  • Using natural organisms - many microorganisms naturally break down organic materia producing carbon dioxide and water, soil and water pollutants are often biological such as sewage and crude oil, so if naturally occuring microorganisms are supported then will break down and neutralise many of the contaminants, such as added nutrients too oill spillages to promote microbial growth.
  • GM organisms - scientists are trying to develop GM bacteria which can break down or accumulate contaminants which they would not usually encounter, such as bacteria that can remove mercury from water.
  • Plants - there are some pollutants such metals which can be removed by plants taking them into their sap through hyperaccumulation which some can do through bioleaching.

Often bioremediation takes place on the sit of the contamination or sometimes material is removed for decontamination, in most cases natural microorganisms outperform GM ones, but it may be possible for them to be more effective in the future as our ability to manipulate genetic material increases.

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Culturing microorganisms

Culturing microorganisms involves growing a large enough amount that they are visible for scientific experiments and use in medicine, the correct health and safety procedures must be met in order to lower risk of mutation resulting in the developement of a pathogenic strain and to prevent contamination with pathogenic microorganisms from the environment. The microorganisms to be cultured needn nutrients as well as the right conditions for temperature, pH and oxygen/ The food provided is known as the nutrient medium and can be in a liquid form (broth) or a solid form (agar). Some microorganisms need a precise balance of nutrients but often the medium is simply enriched with a good protein source, which allows samples containing a very small number of organisms to multiply rapidly. The nutrient medium mist be kepy sterile until it is reading to use through aseptic techniques.

Inoculating broth: Make a suspension of bacteria to be grown, mix with known volume of sterile nutrient medium, stopper flask and incubate at suitable temperature and shake regularily to aerate the broth providing oxygen for the growing bacteria.

Inoculating agar: Sterilise wire loop, dip into bacterial suspension and then drag across agar, and close petri dish lid, but don't seal it so oxyegn can enter preventing growth of anaerobic bacteria, incubate at a suitable temperature.

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The growth of bacterial colonies

Bacteria can reproduce very rapidly, undergoing asexual reproduction every 20 minutes in optimum conditions, however in a close system limited nutrients and a build up of waste products acts as a brake on reproduction and growth, logarithmic numbers are used to represent the billions of descendants from the initial organism. There are 4 stages to the standard growth curve:

  • Lag phase - bacteria are adapting to their new environment and are growing and synthesising the enzymes they need and are not yet reproducing at their maximum rate.
  • Exponential phase - this is where the rate of bacterial reproduction is close to or at its theoretical maximum.
  • Stationary phase - this occurs when the total growth rate is zero, as the number of new cells formed from binary fission is cancelled out by the number of cells dying.
  • The decline or death stage comes when reproduction has almost ceases and the death rate of cells is increasing.
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Factors preventing exponential growth of bacteria

  • Nutrients available - initially there is lots of food but as the numbers of microorganisms multiply exponentially it is used up, and after some time the nutrient concentration will become insufficient to support further growth and reproduction unless more nutrients are added.
  • Oxygen levels: - as the population rises, so does the demand for oxygen for respiration sp oxygen levels can become a limiting factor.
  • Temperature - the enzyme-controlled reactions within the microorganism are affected by the temperature of the culture medium, as a low temperature will slow down growth and reproduction and a high temperature will denature the enzymes, killing the microorganisms.
  • Build up of waste - as bacterial numbers rise the build up of toxic material may inhibit further growth and can even poison or kill the culture.
  • Change in pH - as carbon dioxide produced by the respiration of the bacterial cells increases, the pH of the culture falls until a point where the low pH affects enzyme activity and inhibits population growth.
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Industrial culturing of microorganisms

Sometimes substances are wanted which are formed as an essential part of the normal functioning of the microorganism, for example ethanol, amino acids and enzymes. These are known as primary metabolites . In some circumstances organisms produce substances which are not essential for normal growth but are still used by plants, such as pigments or the toxic chemicals plants produce to protect themselves against attack from herbivores. The organism, in the short term, will not suffer without them, these chemicals are known as secondary metabolites and examples include penicillin and many other antibiotics. Once a microorganism has been chosen as has the ideal bioreactor, it can be produced either by batch or continuous fermentation:

  • Batch fermentation is where the microorganisms are inoculated into a fixed volume of medium and as growth takes place nutrients are used up as both new biomass and waste products build up. As the culture reaches the stationary phase overall growth ceases but biochemical changes to form the desired product often occur, and the process is stopped before the death phase and the products are harvested, and the whole system is sterilised for the next batch.
  • Continuos fermentation is where microorganisms are inoculated into a sterile growth medium which is continually added onces the culture reaches its exponential point of growth. Culture broth is continually removed keeping the culture volume inside the bioreactor a constant. 
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Industrial culturing of microorganisms II

Continuous culture enables continuous balanced growth as waste, products and microorganisms are constantly removed. Both methods of operating a bioreactor can be adjusted to ensure a maxiumum production of biomass or primary/secondary metabolites, and most systems are adapted for maxiumu yield through batch or semi-continuous cultivation. Continuous cultivation is largely used for the production of single celled proteins and in some water treatment processes. All bioreactors produce a mixture of nutrient broth, microorganism, metabolites and waste products. The useful part of the mixture has to be seperated out by downstream processing, which is one of the most difficult parts of the whole bioprocess, and the percentage of the overall cost due to downstream processing varies from 15-40%.

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Controlling bioreactors

Whether a bioreactor is a simple container containing microbial broth or an aseptic fermenter, it is very important to control and manipulate the growing conditions to maximise the yield of product:

  • Temperature: If the temperature is too low the microorganisms will not grow quickly enough but if the temperature gets too high the enzymes will start to denature, so bioreactors often have cooling systems linked to  sensors to maintain optimum conditions using negative feedback.
  • Nutrients and oxygen: Oxygen and nutrient medium can be added in controlled amounts to the broth when probes or sample tests indicate that levels are dropping.
  • Mixing things up: Inside a bioreactor there are large volumes of thick viscous liquid due to the growth of microorganisms, so simple diffusion is not enough to ensure that all the microorganisms recieve enough food and oxygen , so bioreactors need a mixing mechanism so they are stirred continuousluy.
  • Asepsis: If a bioprocess is contaminated by microorganisms form the air, or from the workers it can seriously affect the yield, so most bioreactors are sealed aseptic units. If a process involves genetically engineered organisms it is a legal requirement that they should be contained within the bioreactor and not be released into the environment.
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Isolated enzymes

Advantages of using isolated enzymes in biotechnology rather than the whole organism:

  • Less wasteful - whole microorganisms use up substrate growing and reproducing, producing biomass rather than product, whichn isolated enzymes do not.
  • More efficient - isolated enzymes work at much higher concentrations than is possible when they are a part of the whole microorganism.
  • More specific - no unwanted enzymes are present, so no wasteful reactions occur.
  • Maximise efficiency - isolated enzymes can be given conditions for maximum product formation, which may differ from those needed from the growth of the whole microorganism.
  • Less downstream processing - pure product is produced by isolated ezymes whereas microorganisms give a variety of products in the final broth, making isolation more difficult.
  • Most of the isolated enzymes used in industrial processes are extracellular enzymes produced by microorganisms, making them easier and cheaper to use than extracellular enzymes.
  • Extracellular enzymes are secreted, making them easy to isolate and use.
  • Each microorganism produces relatively few extracellular enzymes, so it is easy to identify the required enzyme compared to the hundreds of intracellular enzymes produced by cells.
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Isolated enzymes II

  • Extracellular enzymes tend to be more robust than intracellular enzymes as conditions outside the cell are less tightly controlled than conditions in the cytoplasm, so extracellular enzymes are adapted to cope with greater variations in pH and temperature than intracellular ones.

However despite the advantages of using extracellular enzymes intracellular enzymes are still used as iosltaed enzymes as there is a bigger range of intracellular enzymes so in some cases they provide the ideal enzyme for a process, and in this cases the benefits of using an intracellular enzyme outweigh the disadvantages for more expensive extraction and the need for more tightly controlled conditions. Examples of intracellular enzymes used as isolated enzymes in industry include glucose oxidase for food preservation and penicillin acylase for converting natural penicillin into more effective semoi-synthetic drugs. 

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Immobilised enzymes

Isolated enzymes are more efficient than whole organisms, but using free enzymes is often very waseful, enzymes are not cheap to produce  and at the end of the process they cannot usually be recovered. Increasingly enzymes used in industrial processes are immobilised - attached to an inert support system over which the substrate passes and is converted to product. This is a case of technology mimicking nature - enzymes in cells are usually are usually bound to membranes to carry out their repeated cycles of catalysis. Because immobilised enzymes are held stationary during the catalytic process, they can be recovered from the reaction mixture and be reused, and the enzymes do not contaminate the end product so less dowstream processing is needed, making the process more economical.

Advantages of using immobilised enzymes:

  • Immobilised enzymes can be re-used and they can easily be seperated from the reactants and products of the reaction they are catalysing so reduce  processing which is cheaper.
  • More reliable - there is a high degree of control over the process as the insoluble support provides a stable microenvironment for the immobilised enzymes.
  • Greater temperature tolerance - immobilised enzymes are less easily denatured by heat and work at levels over a much wider range of temperatues, making  bioreactors easier to run.
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Immobilised enzymes III

  • Ease of manipulation - the catalytic properties of immobilised enzymes can ne altered to fit a particular process more easily than free enzymes, and the ability to keep bioreactors running continuously for long periods without emptying and cleaning them keeps running costs low.

Disadvantages of using immobilised enzymes:

  • Reduced efficiency - the process of immobilsing an enzyme may reduce its activity rate.
  • Higher inital cost of matyerials - immobilised enzymes are more exepensive than free enzymes, but don't need to be replaced frequently.
  • Higher intial cost - systems needed and bioreactors are more expensive to set up than traditional fermenters so there is an inital investment cost.
  • More technical issues - reactors which use immobilised enzymes are more complex than simple fermenters, so more things can go wrong.
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Process of immobilisation

  • Surface immobilisation - adsorption to inorganic carriers like carbon nanotubes or cellulose, it is simple/cheap to do, can be used with many processes, and enzymes are very accesible to substrate and the active site is virtually unchanged, but they can be lost from the matrix easily.
  • Surface immobilisation - covalent bonding with amino or OH groups, or ionic bonding with polysaccarides, cost varies, enzymes are strongly bound so unlikely to be lost, they are very accessible to substrate, pH and substrate concentration often hvae little effect on enzyme activity, but active sites may be modified during the process and become less effective.
  • Entrapment - in a matrix or gelatin, widely applicable to different processes, but may be expensive, can be difficult to entrap, the effect on activity varies, and diffusion of the substrate to and from the active site can slow and hold up the reaction.
  • Entrapment - membrane entrapment in microcapsules or behind a semi-permeable membrane, simple to do, small effect on enzyme activity, widely applicable, but is quite expensive and diffusion of the substrate to and from the active site can slow and hold up the reaction.
  • In some cases whole microorganisms are immobilised rather than just the enzymes, which has the same advantages but avoids the time consuming process of extracting the enzyme, but microorganism do need more of a controlled environment.
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Using immobilised enzymes

  • Immobilised penicillin acylase is used to make semi-synthetic penicillins, as many bacteria have developed resistance to naturally occuring penicillins so they are no longer very effective, fortunately many are still vunerable to semi-synthetic variations so penicillin acylase is very important in treating infections.
  • Immobilised glucose isomerase is used to produce fructose from glucose, which is sweater and often used as a sweetener in food industries, and this enzyme is used to isomerise cheap starch produced glucose into marketable fructose.
  • Immobilised lactase is used to produce lactose free milk for people who are lactose intolerant. Immobilised lactase hydrolyses lactose to glucose and galactose, removing lactose from milk.
  • Immobilised aminocyclase is used to produce pure samples of L-amino avids used in the production of pharmaceuticals, organic chemicals, cosmetics and food.
  • Immobilised glucoamylase, which can be used to complete the breakdown of starch to glucose syrup, amyalse enzymes break down starch into short chain dextrin polymers, and the final breakdown of these to glucose is catalysed by this enzyme.
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Using immobilised enzymes II

  • Immobilised nitrile hydratase is an enzyme with an increasing role in the plastics industry, acrylamide is an important compound used in the production of many plastics and is made by hydrating acrylonitrile, which traditionally was done with sulfuric acid and copper catalyst, but the conditions required were extreme and therefore expensive along with it producing by products making the yield poor. Using immobilised nitrile hydratase the conversion takes place under more moderate conditions so the process is cheaper, and has a much higher yield and no unwanted by products are produced.

Immobilised enzymes and microbial cells are increasingly important both as diagnostic tools in medicine, and the manufacture of drugs such as the immobilised Rhizopus arrhizus fungi used to produce the steroid drug cortisone. Biosensors are used in accurate monitoring of blood and unrin levels of substances such as glucose, urea, amino acids and lactic acid, as well in the monitoring of waste treatment and water analysis. They are based on an electrochemical sensor in close proximity to an immobilised enzyme. The enzymes react with a specific substrate and the chemicals produced are detected by the sensor. Because the size of the response is related to the concetration of the substrate these sensors can be very sensitive and accurate in their measurments.

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