Exploring Enzymes and Micro-Organisms

Exploring Enzymes and Micro-Organisms
All most all enzymes used commercially are obtained from
microorganisms. The microorganisms are grown or cultured and the
enzymes extracted or harvested from them.

Micro organisms are a good source of enzymes for the followingreasons:? They produced more enzyme molecules in relation to their body massthan most other organisms.? They are easy to manipulate genetically and can be subjected to genetransfer techniques.? Product yield can be increased by means of strain selection,mutation and by optimising growth conditions.?
Microorganisms are not influenced by climate and can be grown in suitable laboratories anywhere. This ensures independence of supply to political influence. ? Micro organisms can occupy a great variety of habitats and extreme conditions so their enzymes show an enormous range of ph and temperature characteristics. When the micro-organisms have produced enzymes it is possible to use the whole organism but isolated enzymes (enzymes which have been extracted from the micro-organism) are preferred for the following reasons.
When whole organisms are used some of the substrate is converted
to microbial biomass, using isolated enzymes is far less wasteful
Only one chemical process needs to be considered, so it much
easier to set up the optimum environmental conditions for that
process.
Only one enzyme is present so there will not be wasteful side
reactions
It is easier to isolate and purify the desired product.
There are a number of micro-organisms which are used to produce enzymes commercially and a few examples are: Micro-organism Uses Enzyme Aspergillus niger Preservative in drinks Catalase Aspergillus oryzae baillus subtils Prevents pipe blockage in brewing process by degrading glucan in beer improves clarity of beer Glucanase Bacillus subtilis aspergillus oryzae bacillus licheniforms Breakdown of starch to maltose and dextrines, removing stains from clothes flour improver desizing starch on textiles formation of glucose syrups Amylase A particular enzyme will be produced by many species of micro-organism. The best organism is selected by looking for the species which has some or all of the following properties:
Can grow rapidly
Can survive at a large range of ph, temperatures and
concentrations
Doesn?t produce toxins or offensive odours
Simple nutritional requirements
Once the particular micro-organism has been selected it needs isolation from other species as it is unlikely to be present on its own this can be done as follows:
You can use a temperature that only the specific micro-organism
survives at
You can put it into a medium that only it works at
You can put it into a ph that only that micro-organism survives at
And a concentration that only it survives at
To produce enzymes from a micro-organism the organisms need to be grown or cultured. The organisms are grown in growth or culture media. To produce microorganisms you only need to keep it at the temperature that microorganisms grow best at which usually is 370C. But this is a problem because this would then also grow other unwanted microorganisms. So this is why growth media is needed. Growth media is a specific media that can only facilitate that specific microorganism and so therefore all other unwanted microorganisms will be suppressed. Only the best microorganisms are isolated from a populationand then the best generation is cultured to present a microorganism on its own. This can be done when stock cultures master cultures these are then freeze dried. Culture media can be either liquid or solid. Liquid culture media is usually used to grow microorganisms such as yeast and it is commonly found in the form of a broth. This is used to make large amounts of microorganisms. Solid culture media is usually found in the form of an ager that?s usually used in small industries and in colleges (in this project we will be experimenting with solid agers). It?s really good if you want to make small quantities of microorganisms. Culture media can also be either complex or synthetic. Complexe media is a media that?s made of some unknown ingredients of chemical composition. This type of media is really good for growing wide ranges of microorganisms. This is because this culture is very rich in nutritional supplement to grow almost any microorganism. Synthetic media is media that is made of only chemical nutrients. Last is selective media that is when a media has been specially made for a specific micro-organism. When using liquid media bioreactors or fermentors are used. There are two main ways of using a bioreactor. The first one is continues culture that is when the process goes on 24 hours a day 7 days a week and is rarely stopped. It is in an open fermentor so nutrients can be added continuously and at a steady rate. The advantages of using continuous culture are:
Cells can be maintained at a constant physiological state because
the specific growth rate and the substrate concentration can be
set simply by setting the dilution rate
Continuous cultures can take advantage of cell immobilization
which allows the maintenance of high concentrations of cells in
the reactor at low substrate concentrations
Most downstream processing operations (apart from chromatography)
operate in a continuous manner. Continuous bioreactors thus fit in
will with overall operation of a bioprocess plant
Continuous reactors do not need to be shut down and cleaned as
regularly as a batch reactor and thus have a shorter ?turn-around?
time. This also reduces costs associated with cleaning and filling
of the reactor.
The above advantages give continuous bioreactors a greater productivity than batch bioreactors and thus continuous reactors can be smaller. The most important application of continuous cultures is in the wastewater treatment industry. The reasons for this are:
There is simply no choice as wasteflows are often so large
(measured in megalitres per day) that batch reactors could not
process the waste in time! Continuous cultures which are
considerably more efficient than batch reactors provide the
required efficiency.
The feed is ?full of microbes? and contamination is not a
consideration.
It should be noted though that pure culture continuous industrial scale bioreactor have been constructed and operated. The largest is the Pruteen plant constructed by ici in the 1970?s. The reactor used for this process was a continuous air-lift reactor. This reactor used methanol as the growth limiting substrate. The second way in using a bioreactor is batch culture. This is when the growth of micro-organisms in closed volume of medium no substances are added to the medium during culture except from oxygen. The organisms are grown in this medium until the conditions become unsuitable. Raw materials are added then they start to react, the whole process is monitored making sure that all of the conditions are kept within its normal conditions. Then when the process is completed the reactants are separated from the product and the process is repeated. When growing micro-organisms it is important to optimise conditions. The following factors are important and need optimising: ? The ph ? The temperature ? Concentration ? Nutrients ? Radiation ? Water ? Carbon dioxide The advantages of batch culture are: ? It is easy to set up and control the optimum conditions ? The reactor can be used for a lot of different processes which means that it has a larger target market ? If the culture gets contaminated that batch will be lost, but because they are doing small batches they will not lose that much money. On the other hand in continuous culture if the culture gets contaminated all of that culture will be lost and they will lose a lot of money. When growing bacteria in batch culture there are four phases of growth, which are:
Lag phase
Exponential phase
Stationary phase
Death phase
The lag phase is the first stage of the bacteria growth and there isn?t that much actual growth at this stage because the microorganisms are just getting used to the environment. So they need this time period to make enzymes which are needed to digest and use up the nutrients supplied by the medium. The exponential phase is the stage where the growth starts to progress and produce the amount of microorganisms quite rapidly. This process starts in around 2 hours and goes on for around 6 hours. The stationary phase is where the growth of the enzymes starts to slow down and begins to stop (hence stationary) this is due to the lack of nutrients which are vital. This then leads to a sudden build up of toxic waste. The death phase is when the microorganisms are starved of nutrients which are there to keep them alive and because they have used up all of the nutrients, they produce a lot of toxic waste which then stops the production of more microorganisms so which brings the reaction to a halt. The two growth phases following the exponential phase are together called the post exponential growth phase. During the exponential phase the growth of the microorganisms has just started and no nutriensta are added which encourages the growth process and no enzymes are being produced. This is the reason why the two growth phases after the exponential phase is called post exponential phase. When the micro-organism has finished producing the required enzymes the enzymes need extracting or harvesting from the liquid medium. This can be done by a filtration of the microorganisms from the media. Another way is by reducing the water of the media which is done by reverse osmosis. Unit 18 task 1B === Introduction ==== In this section I will be writing a report on the history washing powders. Plus I will also be writing about the recent and future developments of washing powders. I will also be writing a brief section on immobilisation. Recent and future developments of enzymes ========= The first ever washing powder research was started in 1913 by Dr Otto Rohm. He discovered it when he got a German patent in which he used to extract parts of pancreas to clean dirty clothes. So after that he?s company started to manufacture an enzyme detergent. It was composed of sodium carbonate and the pancreas extracts but this solution didn?t work. This was because the solution was very alkaline and therefore it reduced the reactivity of the enzymes. In the second world war there was a extreme shortage of soap, so further developments were made by a Dr called E. Jagg in Switzerland. He?s progress was limited because there wasn?t enough animal pancreases to go round because they were reserved for the production of insulin. So after the war in 1947 that products were made by using pancreatic trypsin with bile salts. After that it then went onto producing stain removing products in 1959, then in 1963 bio tech was launched and it was really successful as a pre-washer and soaker. Proteases will remove most protein-based stains such as egg and blood, at low temperatures of 30 o C -50 o C. but without the right enzymes more difficult stains will need higher temperatures to shift them. In 1989 amylase was added to biotech to make it more effective over a larger range of stains. Now some of the future developments with enzymes used in washing powders were since proteases was currently worked best at 55o C there is a big search for new enzymes with an optimum temperature of 20 o C - 30 o C. With genetic engineering it would be possible to transfer genes from less productive micro-organisms with suitable low temperature enzymes into more productive species. Very recent detergent enzymes have been developed to remove fatty stains from cloths at low temperatures. This caused a problem because it was hard to find a microorganism that produced high levels of lipase to make manufacture economically viable. Plus it had to work in very alkaline conditions. Soon a suitable lipase was found in a strain of fungus (Humicola) but the fungus didn?t produce high yields. So a gene for the lipase was transferred by genetic engineering into Aspergillus oryzae which is more easily cultured in a fermenter and gives high yields of the enzyme so it was perfect. Immobilization of enzymes is when literally an enzyme is attached to insoluble materials that act as a support for the enzyme. The enzyme can then be held in place during the reaction and then can be removed after the reaction plus it can be used again. The advantages of immobilisation are:
prevention of losses due to flushing away of enzyme
a more stable enzyme
the possibility to produce an enzymes with altered properties
it is cheap and quick
The disadvantages of immobilisation are
losses of enzyme activity can occur during the making of the beads
diffusion of substrates and products may be hampered by
partitioning of the enzyme in the immobilised layer
the enzyme may have a more constrained conformation in the
immobilised state, giving it a lower catalytic activity
may be a high initial investment for the immobilisation, compared
to free enzyme
Immobilisation was actually developed in the 1950s. The first commercial use of immobilised enzymes was by the Tanaka Seiyaku Company in Japan, where an immobilised aminoacylase was used to resolve amino acids into their L and D isomers. The first major commercial use of immobilization was in the 1970s, with the development of immobilised glucose isomerase for the preparation of high-fructose syrups from starches. Other uses of immobilised enzymes include:
Fumarase to turn turn fumaric acid into malic acid for food use
use in the pharmaceutical industry of penicillin amidase to
prepare 6-apa from native penicillins.
Use of nitrile hydratase to prepare acrylamide from acylonitrile
An area where enzyme stability and therefore immobilisation is important is in the industrial area of biosensors. This involves either immobilising an enzyme into an electrode tip or impregnating it in a strip. Another area is in the immobilisation of whole cells (picture above), both for analytical biochemistry and also possibly disease therapy. There are various methods for immobilizing enzymes. They can be absorbed on to insoluble matrix, such as collagen. Held inside a gel such as silica gel. And held within a semi-permeable membrane

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