Disruption Using Lytic Agents: Chemicals, Enzymes, and Mechanical Methods

1. Disruption using lytic agents Disruption process utilizing chemicals or enzymes as lytic agents are also used commonly, but tend to be expensive and also require removal of the lytic agent Chemicals as lytic agents EDTA: Treatment with EDTA is used to release periplasmic proteins from gram-negative bacteria as it disrupts the outer membrane of the bacteria by binding Mg2+ and Ca2+ ions that cross-link the lipopolysaccharide (LPS) molecules. Antibiotics: The common class of antibiotics such as penicillin or cycloserine inhibits peptidoglycan synthesis in growing cells, which are not able to maintain their osmotic pressure and hence disrupt. The assembly of peptidoglycon is also inhibited by Chaotropic agents such as gaunidine hydrochloride and urea, that disrupt water structure Note: Methods for gram-ngative bacteria and growing cells only

2. Disruption using lytic agents Chemicals as lytic agents Organic solvents and detergents: They cause dissolution of the lipids in the periplasmic membrane and the outer membrane. Detergents can be invariably used for solubilization of membrane proteins. Detergents like Triton X-100 is commonly used but other detergents like cholate and SDS are also used. Organic solvents like toluene, trichloroethane, chloroform and ether were found efficient in autolysis of yeast. Alkaline lysis: Effective but harsh. Alkali added to the cell suspension reacts with the cell walls and produces saponification of lipids in the cell walls.

3. Disruption using lytic agents Enzymes as lytic agents Lytic enzymes: Enzymes hydrolyse the walls of microbial cells, and when sufficient wall has been removed, the internal osmotic pressure bursts the periplasmic membrane allowing the intracellular components to be released. The best known lytic enzyme for bacteria is lysozyme (a carbohydrase) from hen egg white, which catalyzes the hydrolysis of β-1,4-glycosidic bonds in the pepetidoglycon layer of bacterial cell wall. Gram-positive bacteria more susceptible to enzymatic lysis than gram-negative. Glucanase used for yeast lysis Note: Combined mechanical, nonmechanical and lytic disruption provide efficient methods

4. Disruption of Animal and Plant Tissues • Absence of cell walls makes the disintegration of • mammalian tissue rather easy • Use of domestic homogenizer or industrial meat grinder • for cutting tissues • Colloid mill blender-type homogenizer for pilot or • industrial scale for finer grinding • Plant cell wall is rigid. Homogenization carried-out in cold • buffer with waring blender • Frozen and ground to dry powder • Phenolic compounds including tannins mix with the extract • and cause inactivation-use amberlite or PVP to remove • phenols

5. Extraction • Liquid-Liquid Extraction: Used to separate inhibitory fermentation • products such as ethanol, solvents, organic acids and antibiotics • Extraction requires the presence of two liquid phases • A multistep alternating aqueous-organic two phase systems are • used for antibiotic recovery • Solvents such as amylacetate or isoamylacetate are used • Provide both concentration and subsequent purification

6. Extraction The extraction of compound from one phase to the other is based on solubility differences of the compound in one phase relative to other. When the compound is distributed between two immiscible liquids, the ratio of the concentrations in the two phases is known as the distribution coefficient: Yl Kd = --------- XH Yl and XH are the concentrations of the solute in light and heavy phases, respectively. The light phase will be organic solvent and heavy phase will be fermentation broth

7. Extraction of penicillin • Typical penicillin broth contains 20 – 35 g antibiotic/liter • pKa values of penicillin 2.5 – 3.1 • Near pH 2.0 – 3.0 neutralization renders them extractable • by organic solvents because of more solubility in organic • solvent • Subsequent back extraction with aqueous phosphate buffer • (pH 5 – 7.5) increases penicillin concentration • Repeat the process • The penicillin is finally recovered as sodium penicillin • precipitate from a butanol-water mixture • Centrifugal Podbielniak extractors are used for the process

8. + _ _ + _ + + _ + _ + - + - _ + + - + _ + _ + _ + + _ _ + _ + _ _ + _ + + _ • Pricipitation • The distribution of charged and hydrophobic residues at the surface • of the protein molecule is the feature that determines solubility in various • solvents • The solubility behaviour of the protein can be changed drastically as the • solvent properties of water are manipulated, causing the protein to • precipitate out from the medium Hydrophobic patch

9. Precipitation: some important considerations The hydrophobic patches consist of the side chains of Phe, Tyr, Trp, Leu,Ile, Met, and Val. Acidic: Glu, Asp Basic: His, Lys, Arg

10. Interacting forces keeping protein soluble in water • 1. The polar interaction between protein and solvent • 2. The ionic interaction between protein and salt ion • 3. The repulsive force between protein and protein • 4. The repulsive force between protein and small aggregate

11. Modes of Precipitations • Protein precipitants include inorganic cations and anions NH4+, K+, Na+, • SO42-, PO43-, Cl-, Br-, NO3- etc for salting out • Bases or acids, H2SO4, HCl, NaOH for isolectric precipitation • Organic solvents such as ethanol, acetone, methanol, n-propanol • Non-ionic polymers like PEG and polyelectrolytes like PEI, PAA, carboxy • methyl cellulose • Heat and pH induced perturbations

12. Precipitation Protein Solution Unstable protein solution Aggregate (floc) Uniform precipitate after adding precipitant formation particles

13. Salting in Salting out Protein solubility 0 0.5 2.0 3.0 4.0 Salting In and Salting Out • All proteins require some counter-ions (i.e. salt) to be soluble in aqueous media. Therefore, protein solubility increases with  salt concentration at low ionic strength. • At higher ionic strength, protein solubility generally decreases with  salt concentration due to reducing the activity of water and neutralization of surface charge. • Each protein has a distinct solubility profile as a function of salt concentration defined by: • Log S(mg/ml) = A - m(salt concentration) • where A is constant dependent on temp. & pH and m is constant dependent on the • salt employed.

14. Precipitation Salt precipitation Saturated concentration of ammonium sulphate for protein solution ~ 4.05 M Protein fractionation by salt: e.g., 0 – 30%; 30 – 60%; 60 – 80% Grams ammonium sulfate to be added to 1 liter of protein solution a) At M1 molar, to take it to M2 molar g = 533(M2 – M1)/4.05 – 0.3 M2 b) At S1% saturation, to take it to S2% saturation g = 533(S2 – S1)/100 – 0.3 S2 Note: After salt precipitation the salt is removed by dialysis or desalting columns for further application in purification

15. Ammonium Sulfate Nomogram

16. Precipitation: Practical Considerations Trial Fractionation with Ammonium Sulfate Percent Percent Percent saturation enzyme protein Purification range precipitated precipitated factor First trial 0 – 40 4 25 40 – 60 62 22 2.8 60 – 80 32 32 1 80 supernatant 2 21 Conclusion: Enzyme precipitated more in 40-60% than in 60-80%; try 45 -70% Second trial 0 – 45 6 32 45 – 70 90 38 2.4 70 supernatant 4 30 Conclusion: Good recovery, but purification factor not as good as in first trial; if purity important, try 48 – 65% Third trial 0 – 48 10 35 48 - 65 75 25 3.0 65 supernatant 15 40

17. Salt Precipitation: some important considerations • Most effective salts are those with multiple-charged anions such as • sulfate, phosphate and citrate • For cations, monovalent ions are used NH4+ > K+ >Na+ • Potassium salts are ruled out on solubility grounds except potassium • phosphate which however, produces higher density in the solvent than • protein aggregate- difficulty in centrifugation • Sodium sulfate not highly soluble at lower temperature, citrate cannot be • used below pH 7.0, produces strong buffering action • Phosphates are less effective • Finally one salt has all the advantages and no disadvantage (except if • required to operate at high pH): Ammonium sulphate

18. Salt Precipitation: some important considerations • Salt never precipitates all the protein, but just reduces its solubility • If the starting material has a enzyme concentration of 1 mg/ml; reduction • in solubility to 0.1mg/ml means 90% precipitation • On the other hand if the starting material has a concentration of 0.1 mg/ml • no precipitation will occur • So precipitation is not an absolute property of the enzyme concerned, but • will depend on both the properties of other proteins present (coprecipitation) • and the protein concentration in the starting solution • The addition of salts increase the density of the medium and thus brings • densities close to the densities of protein aggregates in the solution. • thus high speeds and longer times are required for centrifugation.

19. Salt Precipitation: some important considerations - The effect of protein purity on ammonium sulfate precipitation of proteins

20. Salt precipitation • Different types of salts effect the solubility of proteins to different extents. Most widely used in protein fraction are sulfate salts, particularly ammonium sulfate (NH4)2SO4. 340 68 -66 18 kdal • In general, the larger the protein, the lower the salt concentration required to precipitate it.

21. Salt removal by dialysis • Dialysis membranes are available with pore sizes from very small (1,500 MW cut off) to very large (50-100 kDa cut off). • Also available in conical shapes for use in the centrifuge to both desalt and concentrate protein.