Enzyme Purification

1. Enzyme Purification Aulanni’am Biochemistry Laboratory Chemistry Department Brawijaya University Aulani "Biokimia Enzim" Presentasi 9

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

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

10. 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

11. 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

12. 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

13. 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

14. 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 well for animal cell debris or plant seed debris Aulani "Biokimia Enzim" Presentasi 9

15. 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

16. 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

17. 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. Prec React citric acid with calcium carbonate Filter precipitate • ffff React precipitate with sulfuric acid PurifiedCitric Acid Filter precipitate Aulani "Biokimia Enzim" Presentasi 9

18. 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 Kirk 15 Calcium Carbonate, CaCO3 CSTR Calcium Citrate as a precipitate plus contaminants Contaminated Citric Acid Aulani "Biokimia Enzim" Presentasi 9

19. 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-and-frame presses Calcium Citrate, biomass, water Aulani "Biokimia Enzim" Presentasi 9

20. 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. Kirk, 15 Sulfuric Acid, SO4 CSTR Calcium sulfate as a precipitate, free citric acid, biomass, water Calcium Citrate, biomass, water Aulani "Biokimia Enzim" Presentasi 9

21. 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. Kirk 15 Filter Ex. Plate filter, Rotary presses, rack-and-frame presses Calcium sulfate as a precipitate, free citric acid, biomass, water Citric acid, water Aulani "Biokimia Enzim" Presentasi 9

22. 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 KICgroup 1 Aulani "Biokimia Enzim" Presentasi 9

23. 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

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