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Producción de enzimas

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Production of Enzymes
Learning objective:
• To be able to explain why it is more
efficient to use isolated enzymes than
whole cells.
• To be able to explain how
contamination of end products is
eradicated.
• To be able to describe how this
process occurs.
Uses of enzymes
• commercial uses- e.g. biological
washing powders
• medical uses- e.g. biosensors,
therapeutic enzymes, drugs
• industrial uses - e.g. bioconversion,
fruit juice extraction, sweeteners
Enzymes are being used more and more for
industrial bioconversion i.e. making a
chemical product using purified enzymes
rather by pure chemical methods (e.g. citric
acid production) or using whole cells (e.g.
yeast in brewing).
• Considerations when selecting a strain:
– Does it do what is required
– Is it safe
– Is it cost effective
• Enzymes may be intracellular or
extracellular. What is the advantage of
extracellular production?
– Already outside cell
– Limited number secreted so easier to
isolate
– More robust so less likely to be broken
down by heat of chemicals
• Why are intracellular enzymes more difficult
to isolate than extracellular ones?
• Because they are inside the cell, first the cell
has to be broken open then the enzyme
separated from the mixture of all the cellular
contents
• Why is it more efficient to use isolated
enzymes than whole cells.
• Isolated enzymes are usually more efficient
in biotechnology than whole cells because
enzyme concentration is higher and no
unwanted enzymes are present
Enzyme production
Surface and submerged techniques:
Surface = enzyme produced on the
surface of a solid medium
Submerged = the mould or bacterium
producing the enzyme is grown
throughout a liquid medium
Advantages and disadvantages?
Submerged – more yield as growth
throughout but aeration necessary
• The maximum enzyme production is usually
in stationary phase of microbe growth, so a
batch or fed-batch process are usually used.
• The medium must be chosen to stimulate the
microbe into synthesising the correct
enzyme.
• For example to stimulate a microbe to
synthesise amylase enzymes, a medium with
starch but no sugars is used.
• What type of medium would you use to
stimulate a microbe to synthesise a
protease?
• A medium with proteins but no amino
acids is used.
• Microbes are encouraged into the log
phase initially with a medium with a lot of
protein
• This encourages rapid increase in the
number of cells, but not much protease is
produced.
• Cells are then introduced into the
fermentation vessel and allowed to grow
for a further 1-8 days.
• The medium now has very little protein in
it. Why?
• The microbe must produce a lot of
protease because as the enzyme leaves
the cell it doesn’t immediately come
into contact with protein that it can
break down – more protease produced
to maximise the amount of amino acids
from the small amount of protein.
Down stream processing
The remaining mixture contains
enzymes, waste materials, nutrients
and cells
The enzyme is extracted by downstream
processing
The nature of the downstream processing
depends on two considerations:
• Whether enzyme is intracellular or
extracellular
• How pure the final product needs to be.
Industrial enzymes can be quite crude, but
medicinal enzymes must be extremely pure.
The purer the enzyme, the more complex the
downstream processing, and the more
expensive it is.
Extracellular
enzyme
Break open cells by
grinding or ultra-sonics
Filter
Intracellular
enzyme
Cell biomass (useful
waste product)
Enzyme in
solution
Concentrate by evaporation at
low temperature and pressure or
by osmosis
Crude enzyme in solution
eg protease in chemical
industry
Precipitate
Powdered crude enzyme eg
pectinase
Chromatography
Pure enzyme for medicine
eg glucose oxidase
Immobilised enzymes
• When purified enzymes are used to make large
quantites of another product, downstream
processing can be difficult and expensive.
• Immobilising enzymes is cheaper.
• Enzyme molecules are attached to a support
matrix rather than free in solution. They still
function properly but can be kept separate
from the reactants and the products.
• Immobilised enzymes are usually used in
continuous flow-through re-actors, which have
a low volume.
• Isolated enzymes can be immobilised
so that they do not contaminate the end
product and can be used again and
again
Methods of Immobilisation
Entrapment
• The enzyme is entrapped within an inert
matrix, such as alginate, silica or collagen,
and cannot be washed out.
• The substrate and product molecules can
diffuse in and out of the matrix, but this
diffusion may limit the rate of the reaction.
• This is the most gentle method of
entrapment, and does little damage to the
enzymes.
Adsorption
• The enzyme molecules are attached by weak
physical forces to a support matrix, such as
glass beads or carbon particles.
• This does not chemically modify the enzyme
molecules, but the adsorption process may
cause the enzymes to loose their shape and
therefore their activity.
• The molecules may also become detached
during the bioconversion reaction.
Cross-linking
• Enzyme molecules are chemically crosslinked by covalent bonds using
glutaraldehyde:
E=CH–CH2–CH2–CH2– CH=E.
This only works for some enzymes, but is very
successful.
Used in Biosensors
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