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Enzyme Purification Aulanni’am Biochemistry Laboratory Chemistry Department Brawijaya University

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Enzyme Purification Aulanni’am Biochemistry Laboratory Chemistry Department Brawijaya University Powered By Docstoc
					Enzyme Purification


         Aulanni’am
   Biochemistry Laboratory
    Chemistry Department
     Brawijaya University

        Aulani "Biokimia Enzim" Presentasi 9
    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.



                          Aulani "Biokimia Enzim" Presentasi 9
      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


                    Aulani "Biokimia Enzim" Presentasi 9
                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.




                  Aulani "Biokimia Enzim" Presentasi 9
                  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
                    Aulani "Biokimia Enzim" Presentasi 9
                      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


                        Aulani "Biokimia Enzim" Presentasi 9
                   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.



                     Aulani "Biokimia Enzim" Presentasi 9
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


                Aulani "Biokimia Enzim" Presentasi 9
                    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

                        Aulani "Biokimia Enzim" Presentasi 9
      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


                           Aulani "Biokimia Enzim" Presentasi 9
 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




                   Aulani "Biokimia Enzim" Presentasi 9
                 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
                        Aulani "Biokimia Enzim" Presentasi 9
          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

                Aulani "Biokimia Enzim" Presentasi 9
                        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
                         Aulani "Biokimia Enzim" cell debris or plant seed
        particularly well for animal 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
                  Aulani "Biokimia Enzim" Presentasi 9
                     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
                     Aulani "Biokimia Enzim" Presentasi 9
             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            Filter                              React precipitate
Prec calcium
with                         precipitate                               •ffff
                                                                 with sulfuric
carbonate                                                        acid




 Purified Citric                                  Filter
                                                  precipitate
      Acid
                          Aulani "Biokimia Enzim" Presentasi 9
          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
                                                                      Calcium Citrate as
    Contaminated Citric Acid                                          a precipitate plus
                               Aulani "Biokimia Enzim" Presentasi 9   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               Filter
a precipitate plus                                               Calcium Citrate,
                        Ex. Plate filter, Rotary                 biomass, water
contaminants            presses, rack-and-
                        frame presses
                          Aulani "Biokimia Enzim" Presentasi 9
           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



                                 CSTR                            Calcium sulfate as a
 Calcium Citrate,
                                                                 precipitate, free citric
 biomass, water
                                                                 acid, biomass, water

Kirk, 15                  Aulani "Biokimia Enzim" Presentasi 9
            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                           Filter
                                                                  Citric acid,
precipitate, free citric             Ex. Plate filter, Rotary     water
acid, biomass, water                 presses, rack-and-
                                     frame presses
                           Aulani "Biokimia Enzim" Presentasi 9
Kirk 15
               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. 7.5-
           8.8%

  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                  Aulani "Biokimia Enzim" Presentasi 9
  KICgroup 1
              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                     Citric
                                                                 Acid

                          Aulani "Biokimia Enzim" Presentasi 9
Aulani "Biokimia Enzim" Presentasi 9

				
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