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Cell Disruption Methods

1Steps in DSP:

Primary recoveryProduct PurificationRecovery of Intracellular Products:

It requires additional processing such as cell disruption , lysis , permeabilization , or extraction. eg: Intracellular polymers such as poly--hydroxybutyrate (PHB) may be recovered either by cell disruption or solvent extraction.Source: Principles of Fermentation Technology by Whittaker and Stanbury.Factors influencing product release2. Microorganism used:Cell wall propertiesCell Size.Cell density

Intracellular ProductsIntracellular productsrDNA intracellular productsGlucose isomeraseChymosin (yeast/E.coli)-galactosidaseInsulin (E.coli, mammalian)PhosphataseImmunoglobulinEthanol dehydrogenaseInterferons (mammalian) Dnase, RnaseHuman growth hormone (E.coli)NADH/NAD+Human serum albuminCell Wall Properties:Gram +ve has thick layer of peptidoglycan than Gram -veYeast has thick cell wallOuter Protein Mannan complexsInner -glucansFungi has thick cell wallGlycoproteins, Chitin, and -glucans

CELL DISRUPTIONObjectives: To extract biological products of interest that are not secreted from the cell.Cell Disruption: breaking the cell wallCell Lysis: Chemical treatments releasing the products.Goals ?Solubilize the product present in the cells with maximal biological activityAvoid secondary alteration of product, e.g., Denaturation, Proteolysis, Oxidation Minimize the problems associated with cell disruption which may affect further processing.Source: Bioprocessing Edited by G. StephanopoulosConditions For Cell Disruption:Preservation of product bioactivityBuffer (pH 6-8)Antioxidants(S-S bond breaking)Protease inhibitorsTemperature (2-40C)Methods Of Cell disruption:Physico-Mechanical Methods

1. Liquid Shear:Widely used in large scale enzyme purificationHow these Homogenizer devices works ?Cell suspension is forced under high pressure (up to 1500 bar) through a narrow discharge valve.followed by a pressure drop to atmospheric Cell disruption mechanisms: Impingement on the valve High liquid shear in the orifice Sudden pressure drop upon discharge - cavitation.

The liquid shear cell disruption is often associated with the cavitation phenomenon that involves formation of vapor cavities in liquid due to local reduction in pressure that could be affected by ultrasonic vibrations, local increase in velocity, and so forth.Impingement means Tocollideorstrikeagainstsomething:Shear force: Aforceactinginadirectionparalleltoasurfaceortoaplanarcrosssectionofabody13 Liquid Shear homogenizers:High pressure homogenizers (up to 1500 bar)French Press: laboratory levelTypical volumes few milliliters to a few hundred millilitersAPV Manton Gaulin-homogenizer :Pilot and production scale Used for yeast bacterial cells and fungal myceliumPressure - 550 kg/cm2 for 60% yeast suspensionHigh pressure cylinder with small orifice and needle valve at its base.Cell suspension is placed within the cylinder and pressurized using the plunger/Piston(10,000 50,000 psi)The suspension emerges through the orifice at very high velocity in the form of a fine jet.impact plate: the jet impinges further cell disruption

French Press

APV Manton Gaulin Homogenizer Slurry passes through a nonreturn valve and impinges against the operative valve under high pressureCells then pass through a narrow channel followed by a sudden pressure drop at the exit to the narrow orifice. The large pressure drop causes cavitation in the slurry and the shock waves so produced disrupt the cells.

cooling the slurry to between 0 and 4C to minimize loss in enzyme activitybecause of heat generation during the process16Effect of Operating Pressure on Disruption Efficiency

Laboratory high-pressure homogenizer : French Press

Source: frenchpressurecell.comhttp://www.google.co.in/imgres?q=homogenizer&hl=en&gbv=2&biw=1366&bih=533&tbm=isch&tbnid=TRSaGi1eo2xYPM:&imgrefurl=http://en.wikipedia.org/wiki/Homogenization_(chemistry)&docid=r6_fOQqRZSIeJM&imgurl=http://upload.wikimedia.org/wikipedia/commons/thumb/7/76/Homogenizing_valve.svg/400px-Homogenizing_valve.svg.png&w=400&h=361&ei=_Z2dTox2rq2JB_CcxMsJ&zoom=1http://www.phys.sinica.edu.tw/TIGP-NANO/Course/2010_Spring/Classnotes/AAC_Chapter%203%20Centrifugation%20English_20100325.pdf182. Solid shearPressure extrusion of frozen micro-organism(-250 C) through a narrow orifice.Cell disruption mechanisms: Combination of liquid shear and presence of ice crystals.Not suitable for materials sensitive to freezing and thawing.Eg: - Hughes press - Commercial equipment is called X press http://www.phys.sinica.edu.tw/TIGP-NANO/Course/2010_Spring/Classnotes/AAC_Chapter%203%20Centrifugation%20English_20100325.pdf19

Semi-continuous X-press Operating with a sample temperature of - 35CIdeal for microbial products which are temperature labile.90% disruption with a single passage of S. cerevisiae using a throughput of 10 kg yeast cell paste h -13. Agitation with abrasivesGrinding cells with Abrasives : Grind and smash cells Simple Mortar and PestleBall mill/Bead mill/ Dyno mill.Abrasives: Glass beads, Kieselguhr , Silica, Alumina, Zirconium Oxide and Titanium Carbide .Disintegrator: contain a series of rotating discs and small beads (0.5 to 0.9 m diameter).Reason for Cell rapture: Shear Forces/ Grinding Between Beads/ Collisions with Beads

Ball Mill: Ball mill containing series of rotating discs and small beads. Heat dissipation overcome by cooling jacket.Ideal for disruption of Yeast, Spores, Microalgae, Fungi.85% disintegration of 11% w/v suspension of S. cerevisiae was achieved with a single pass (Mogren et al., 1974)

Dyno-Mill Multi-Lab23Ball mill:

4. Ultrasonication Another liquid-shear methodHigh frequency vibration (20kHz/s) at the tip of an ultrasonication probe cavitations cell disruption.Ultrasonic vibrator generate high frequency wavesTransducer - converts waves into mechanical oscillations by Titanium Probe.

http://www.phys.sinica.edu.tw/TIGP-NANO/Course/2010_Spring/Classnotes/AAC_Chapter%203%20Centrifugation%20English_20100325.pdf25 Ultrasonication Mechanism: Cavitation followed by shock wavesHigh frequency formation of tiny bubbles bubbles collapse releasing mechanical energy (shockwave) ~ thousands atm pressureFrequency: 25 kHzDuration depends on cell type, sample size and cell concentrationBacterial cells (E. coli) 30-60 sYeast cells 2-10 minutesUsed in conjunction with chemical methodsCell barriers are weakened by small amounts of enzymes or detergents energy reduced Ultrasonication Rods are broken readily than cocciGram Negative easily than Gram Positive.Not effective for molds.Power requirements - high, need for cooling because of large heating effect, short working life for probes.Laboratory scale method- high cost.

Factors Effecting Ultrasonication:

Source: Purification and Analysis of Recombinant Proteins edited by Ramnath Seetharam, Satish K. Sharma5. Freeze - ThawingEmploys slow freezing and thawing of concentrated cell paste.Ice crystals formed disrupt the cells.Little commercial acceptanceSlow, limited release of cell componentsSeveral cycles may be required.High costeg: -glucosidase from Saccharomyces cerevisiae.Chemical MethodsDamage the lipoproteins of the cell membrane.

1. Detergents

CHAPS: (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate)Detergents are amphipathic molecules, meaning they contain both a nonpolar "tail" having aliphatic or aromatic character and a polar "head".31DetergentsTriton X-100 + Guanidine HCL( Chaotrophic agent) widely used for release of cellular proteins.CTAB is widely used in the isolation of DNA from plants.Detergents May cause protein denaturation/ precipitation require removal before further purification.SDS removal was achieved by adsorption onto Zeolite Y

CHAOTROPIC AGENTS

In molecular cloning, chaotropic agents are used primarily to destroy the three-dimensional (3D) structure of proteins. The most powerful of the chaotropic agents are guanidinium isothiocyanate and guanidinium chloride which denature proteins to a random coil state. It appears that progressively more guanidinium salt binds to protein as denaturation proceeds.

The first guanidinium salt used was a chloride which was used as a deproteinization agent during the isolation of RNA. Guanidinium chloride is strong enough to inhibit ribonucleases, but it it not strong enough to extract RNA from tissues that are rich in RNAse. Guanidinium isothiocyanate is a stronger chaotropic agent and contains potent cationic and anionic groups that form strong hydrogen bonds. Guanidinium isothiocyanate can be used in the presence of a reducing agent to break protein disulfide bonds and in the presence of a detergent to disrupt hydrophobic interactions.

These agents can also be used to renature proteins. For example, overexpression of heterologous proteins inE. colican result in the formation of protein aggregates known as inclusion bodies. Solubilization with 6M guanidine hydrochloride or 8M Urea will dissociate the protein aggregates and completely denature the protein. The chaotropic agent can then be slowly removed by dilution or dialysis to refold the protein into its native conformation.

It is important to note that chaotropic salts strongly absorb at 230nm. This can be an issue when using chaotropic agents in preparation of DNA and RNAin using a A260/A280ratio to assess the purity of the preparation. The presence of chaotropic salts will artificially lower this ratio.

32DetergentsPullulanase is an enzyme which is bound to the outer membrane of Klebsiella pneumoniae. The cells were suspended in pH 7.8 buffer and 1% sodium cholate was added. The mixture was stirred for 1 hour to solubilize most of the enzyme (Kroner et al., 1978)2. Osmotic shockSudden change in the salt concentration.Effect on microbial cells is normally minimalProcedure Allow the cells to equilibrate in a high sucrose medium(20%) Then rapidly diluting away the sucrose. Endosmosis- Cell RaptureUsed for :Extraction of luciferase enzyme from Photobacterium fischeri.

3. Alkali treatmentAlkaline lysis using hydroxide and hypochlorite is an effective and cheap method.It acts by saponifying the cell-wall lipidsExtremely harsh technique so the product must be resistant to degradation at high pH( 11.5-12.5 for 20-30 mins)Uses:Recovery of polyhydroxy alkanoates (PHA, biodegradable polymer) from E. coliExtraction of L-asparaginase.Recombinant Growth Hormone by NaOH ( pH 11)

Organic solvents:Methanol, ethanol, Isopropanol, butanolToluene: dissolves membrane phospholipids poresFreeze drying followed by acetone, butanol treatment- cell wall disruption.Organic Solvents:

Permeabilization:EDTA : widely used for Gram negative microorganisms. Binds to the divalent cations of Ca2+ , Mg2+ that stabilize the structure of outer membranes.DMSOChemical permeabilization by antibiotics:Penicillin or Cycloserine- interferes with cell wall synthesisEnzymatic Cell Lysis:

Source: Separation Processes in Biotechnology By Juan A. AsenjoPros & Cons of Enzyme Lysis:Specific reactionSelective release of product from selected locationRelease of cloned intracellular products.Low energy consumptionSmall risk of product damageHarmless to environment

High cost Removal of lysozyme (enzyme) from the productPresence of other enzymes (proteases) in lysozyme samples

Lysozyme:It Breaks -1,4- glycosidic linkage between NAM & NAG in peptidoglycan.Effective for Gram positive Gram negative Lysozyme + EDTALysozyme on its own cannot disrupt bacterial cells since it does not lyse the cell membrane. Combination of lysozyme and a detergent /osmotic shock

Lysozyme:Advantages: No specialized equipment necessary.Potential for selective release of productcleaner lysateDisadvantages: Not always reproducible. Enzyme Stability can be an issue. Can be expensive to scale upLysis of Yeast Cell:Yeast cell Glucanase & Mannanase + Proteases+ Chitinase

Cell lysisProduct renaturationSource: Separation Processes in Biotechnology By Juan A. AsenjoDisruption of bacterial cells releases large amounts of nucleic acids which increase the viscosity of the broth, often producing viscoelastic behavior. To ease further purification, the nucleic acids are usually removed by precipitation (e.g., with manganous sulfate, streptomycin or polyethyleneimine) [ 11; alternatively, viscosity may be reduced by enzymatic digestion of nucleic acids or high-shear processing in high-pressure homogenizers [ 191. Another alternative for eliminating nucleic acid polymers is heat shock treatment prior to disrupting the cells. Heat shock treatment would typically require rapid heating to at least 64C and a holding time of 20-30 minutes.

44Enzymatic lysisPlant cells :Cellulase & PectinaseAlgae: Cellulases

Process of lysis :

Quantifying Cell DisruptionMicroscopy (optical or SEM)Intact vs. broken cellDifferential staining - Methylene blue dye exclusionautomatic cell counting using a hemocytometerParticle size analyzers to (e.g., Coulter counters)ViscositySpectrophotometryProtein (260 nm)Nucleic acid(280 nm)Turbidity (550 nm)

SEM micrographs of S. aureusSummaryCell disruption methodsMechanicalChemical Cell membrane disruption normally use chemical methodsCell wall disruption normally use physical methodsHigh-pressure homogenization is the most suitable for bacterial broths.Bead mills are more widely used for fungal cultures.

48References: Principles of Fermentation Technology by Whittaker and Stanbury.Comprehensive Biotechnology by Murray and Moo Young.Bioprocess Engineering basic Concepts by Shuler and Kargi.Purification and Analysis of Recombinant Proteins edited by Ramnath Seetharam, Satish K. SharmaIndustrial microbiology an introduction by Michael. J Waites and others.