aulani "biokimia enzim" presentasi 9 enzyme purification aulanni’am biochemistry...

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

Enzyme Purification Enzyme Purification

Aulanni’amAulanni’amBiochemistry LaboratoryBiochemistry LaboratoryChemistry DepartmentChemistry DepartmentBrawijaya UniversityBrawijaya 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.


Types of Purification EffortsTypes of Purification Efforts

A purification scheme can be made up anycombination of the following isolation efforts

Extraction Precipitation Coagulation and Flocculation Centrifugation Filtration


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.


ExtractionMechanical 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


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


PrecipitationPrecipitation 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.


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


PrecipitationPrecipitation 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


Coagulation and FlocculationCoagulation 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


Coagulation and FlocculationCell 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


CentrifugationCentrifugation 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


CentrifugationPerforate 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


FiltrationFilters 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 well for animal cell debris or plant seed 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


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


Purification of Citric AcidA 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.

•ffffPrecReact citric acid with calcium carbonate

Filter precipitate

React precipitate with sulfuric acid

Filter precipitate

Purified Citric Acid


Purification of Citric AcidThe 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 toneutralize 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

Kirk 15

Contaminated Citric Acid

CSTR Calcium

Citrate as a precipitate plus contaminants

Calcium Carbonate, CaCO3


Purification of Citric AcidThe 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-and-frame presses

Calcium Citrate, biomass, water


Calcium Citrate, biomass, water

Purification of Citric AcidTo 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.

Kirk, 15

CSTR

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

Sulfuric Acid, SO4


Purification of Citric AcidIn 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.

Kirk 15

Filter

Ex. Plate filter, Rotary presses, rack-and-frame presses

Citric acid, water

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


Purification of Citric AcidFurther PurificationCitric 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 36.6ºCKirk 16,17 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.

Citric Acid

Purification

aulani "biokimia enzim" presentasi 9 enzyme purification aulanni’am biochemistry...

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