Transcript Fermentation: An Overview

Enzyme Purification
Aulanni’am
Biochemistry Laboratory
Chemistry Department
Brawijaya University
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Introduction to Purification
Many biological processes require a purification scheme to reduce the
fermentation broth to its pure final product.
Once citric acid is made in the production fermenter, the broth is
still highly contaminated.
There are many different types of purification methods (extraction,
filtration, coagulation, etc.) but for citric acid recovery, the most
commonly used method is precipitation with lime and sulfuric acid
and filtration to produce free citric acid.
The following tutoring will go through various purification techniques
and then take you through a typical purification scheme for citric
acid.
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Types of Purification Efforts
A purification scheme can be made up any
combination of the following isolation efforts





Extraction
Precipitation
Coagulation and Flocculation
Centrifugation
Filtration
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Extraction
Extraction is used to liberate a product of
microbial growth from the cells or cellular
constituents that served as the enzyme source
either by mechanical or non-mechanical means.
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Extraction
Mechanical Extraction
• Mechanical disruption of the cell is easy to achieve
on a small scale but can fail when used industrially
• High Pressure Homogenizer
- A positive displacement pump with an
adjustable valve, has been used to break
microorganisms like Aspergillus niger,
Escherichia coli, and Bacillus megatherium.
- When cell concentration is high, the spores or
mycelia from the microorganism can clog the
valve
• High Speed Ball Mill
- Used for release of proteins within yeast
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Extraction
Non-mechanical Extraction
• Desiccation
- Air drying that must be followed by buffer extraction
• Physical and Chemical Lysis
- Osmotic shock produced by an abrupt change in salt
concentration of the medium
• Solvent Extraction
- Liquid extraction of a product from soluble particles
within the cell
- Must choose solvent accordingly, and purification efforts
will follow to recover product from solvent
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Precipitation
Precipitation is a procedure where the addition of a
ionic solution to an ionic fermentation broth forms
insoluble particles, where the desired product is
usually contained within those particles.
Ionic fermentation broths usually consist of enzymes or
proteins.
The ways to precipitate out a product can vary from
simple pH and temperature changes to chemical
reactions involving metalions.
Precipitation reactions are carried out in reactors,
continuous and batch.
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Temperature and pH variation
Overall, most proteins and enzymes display
increased solubility with increased temperature,
but care must be taken to prevent loss of
product
By adjusting the pH, an enzyme’s polarity can be
lowered so that it has a zero net charge; at this
lowest polarity, the enzyme has low solubility in
an aqueous solution
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Precipitation
Precipitation by Organic Solvents
• By adding an organic solvent to an aqueous fermentation broth, the
dielectric constant will decrease causing the solubility to decrease
• Often used industrially because it’s inexpensive and simple
Precipitation by Metal Ions
• Metal salts with lower solubilities can formed by enzymes and
proteins
• Nucleic acids, which are present in microbial cells, must be
removed prior to this type of precipitation because they reduce the
resolution of separation
• Manganous salts can be used to selectively precipitate out those
nucleic acids
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Coagulation and Flocculation
Coagulation is defined for biological processes to be when small
particles directly adhere to each other, while flocculation is when
an agent acts as a bridge that joins particles together.
Coagulation and flocculation techniques are usually applied to
either whole cells, cell debris, or soluble proteins.
Whole Cells
• Many flocculation agents are used to separate products, such as
anionic and cationic ployelectrolytes, polyamines, alumina, and
synthetic polymers
• Less information is known about coagulants, but some studied
inorganic coagulants have been alum, ferric salts, and calcium salts
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Coagulation and Flocculation
Cell Debris and Proteins
• Coagulation and flocculation are useful techniques in
removing the cell debris that can be produced during
mechanical agitation
• Coagulation and flocculation can be used alternatively
to precipitation methods to remove enzymes
• The same agents for whole cell removal can be applied
to cell debris and protein removal
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Centrifugation
Centrifugation involves separation of liquids and particles
based on density. Centrifugation can be used to separate cells
from a culture liquid, cell debris from a broth, and a group of
precipitates. There are numerous types of centrifuges, but only a
few will be presented here.
Tubular Bowl Centrifuge
• Most useful for solid-liquid separation with enzymatic
isolation
• Can achieve excellent separation of microbial cells and
animal, plant, and most microbial cell debris in solution
Disc Bowl Centrifuge
• Widely used for removing cells and animal debris
• Can partially recover microbial cell debris and protein
precipitates
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Centrifugation
Perforate Bowl Basket Centrifuge
• Exception at separation of adsorbents, such as
cellulose and agarose
Zonal Ultracentrifuge
• Applied in the vaccine industry because it can
easily remove cell debris from viruses
• Can collect fine protein precipitates
• Has been used experimentally to purify RNA
polymerase and very fine debris in enzymes
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Filtration
Filters use a filter cloth or some porous material along
with applied pressure to push smaller particles
through the filter, thus
separating elements of the solution based on size.
Filtration for biological materials is generally
completed using batch filtration, rotary drum
filtration, or ultrafiltration methods.
Batch Filtration
• Usually performed under constant pressure with a pump
that moves the broth or liquor through the filter
• Filter cake will build-up as filtration proceeds and
resistance to broth flow will increase
• The filter press is the typical industrial version of a
batch vacuum filter, using a plate and frame arrangement
• Can be used to remove cells, but does not work
particularly wellAulani
for
animal
cell debris
or plant seed
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Enzim" Presentasi
9
debris
Filtration
Batch Filtration
• Usually performed under constant pressure with
a pump that moves the broth or liquor through
the filter
• Filter cake will build-up as filtration proceeds
and resistance to broth flow will increase
• The filter press is the typical industrial version
of a batch vacuum filter, using a plate and
frame arrangement
• Can be used to remove cells, but does not work
particularly well for animal cell debris or plant
seed debris
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Filtration
Rotary Drum Filtration
• Solution is vacuumed upward where it crosses a filter
septum removed by a positive displacement pump
• Filter cake is removed after each rotation to give a fresh
surface for filtration
• Rotary vacuum filters can be used to efficiently remove
mycelia, cells, proteins, and enzymes, though a filter aid
or precoat of the septum may be necessary
Ultrafiltration
• Utilizes a membrane to separate particles that are much
larger than the solvent used
• Successful removal occurs in the partical size range of 10
solvent molecular diameters to 0.5 μ
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Purification of Citric Acid
A typical method used for purification of citric acid from a
fermentation broth involves two major purification techniques:
precipitation and filtration. The following schematic displays a
generic citric acid purification scheme. The scheme will be
discussed in detail in the next few slides.
React citric acid
Prec
with calcium
carbonate
Purified Citric
Acid
Filter
precipitate
React precipitate
with sulfuric
•ffff
acid
Filter
precipitate
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Purification of Citric Acid
The citric acid broth from the production fermenter is highly
contaminated by leftover biomass, salts, sucrose, and water. First,
the citric acid must be reacted with calcium carbonate to
neutralize the broth and form the insoluble precipitate calcium
citrate. Calcium citrate contains about 74% citric acid. The
stoichiometric equation is as follows:
CaCO3 + Citric Acid → CO2+ Calcium Citrate
Calcium Carbonate, CaCO3
CSTR
Contaminated Citric Acid
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Calcium Citrate as
a precipitate plus
contaminants
Purification of Citric Acid
The calcium citrate is then washed, heated, and filtered to remove
any number of the contaminants. Depending on the specific
design of the purification scheme, filters can be placed before the
first reaction with calcium carbonate, in series between the two
precipitation reactions or in any other combination that works.
Also, it is important to choose the best kind of filter for what is
being removed. For simplicity, the filters here will remove larger
contaminants first (sucrose and salts) and the smaller
contaminants later.
Calcium Citrate as
a precipitate plus
contaminants
Filter
Ex. Plate filter, Rotary
presses, rack-andframe presses
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Calcium Citrate,
biomass, water
Purification of Citric Acid
To crack the calcium citrate precipitate, sulfuric acid is needed.
The temperature of this reaction should stay below 60ºC. The
reaction will produce free citric acid and a new precipitate,
calcium sulfate, which will need to be removed later. The
stoichiometric coefficients for this reaction are all one.
Sulfuric
Acid, SO4
Calcium Citrate,
biomass, water
Kirk, 15
CSTR
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Calcium sulfate as a
precipitate, free citric
acid, biomass, water
Purification of Citric Acid
In this filter, the calcium sulfate is washed away from the citric
acid and the leftover biomass is removed. Again, the
contaminants that were present in the fermentation broth can be
removed by additional filtration means, such as microfiltration or
ultrafiltration.
Calcium sulfate as a
precipitate, free citric
acid, biomass, water
Kirk 15
Filter
Ex. Plate filter, Rotary
presses, rack-andframe presses
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Citric acid,
water
Purification of Citric Acid
Further Purification
Citric acid can be produced in two forms – monohydrate and
anhydrous. These forms may require additional purification steps
to reach the desired purity.
1. Monohydrate
• contains one water molecule for every citric acid
molecule
• Requires repeated crystallization until water content is approx. 7.58.8%
2. Anhydrous
• Processed to remove all water from end product
• Prepare by dehydrating the monohydrate citric acid product at a
temperature above 36.6ºC
Kirk 16,17
KICgroup 1
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Purification Conclusion
Once the product has been brought to the desired purity, it would
be sent to packaging and distribution.
In summary, there are many different methods and types of
equipment that make up a purification scheme. Some of the
more common types or purification were discussed in the first
half of this tutorial. The second half of the tutorial dealt with a
specific citric acid purification scheme that utilizes precipitation
and filtration to recover free citric acid from a contaminated
fermentation broth.
Purification
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Citric
Acid
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