Term Paper 2010

Cell Disruption

Rishabh [Type the company name] Term Paper 2010

Submitted To: Mr C.P. Pandia Lect. Bioprocess Engineering L.P.U

Submitted By: Rishabh Garg B.Tech Biotech Roll No: 33 Section: B7701

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Cell Disruption

ACKNOWLEDGEMENT

I would like to express my gratitude to all those who gave me the possibility to complete this term paper. I want to thank the Department of BIOTECHNOLOGY of LOVELY PROFESSIONAL UNIVERSITY for giving me permission to commence this term paper, to do the necessary research work and to use departmental data. I am furthermore thankful to Mr. C.P.Pandia, Lect. in Bioprocess Engineering 2 who gave and confirmed this permission and encouraged me to go ahead with my term paper. I am deeply indebted to my supervisor Lect.C.P.PANDIA , SPRINGER, WIKIPEDIA, SCIENCE DIRECT and GOOGLE whose help, stimulating suggestions and encouragement helped me in all the time of research for and writing of this term paper.

RISHABH GARG

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INDEX

S.No. 1. 2. 3. 4. 5.

Topic Introduction Mechanical Methods Non Mechanical Methods Applications References

Page No. 3 3 8 11 13

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Introduction

Cell disruption is a method or process for releasing biological molecules from inside a cell. A lot of biological molecules are inside the cell, and they must be released from it. This is achieved by cell disruption (lysis). Cell disruption is a sensitive process because of the cell wall's resistance to the high osmotic pressure inside them. Furthermore, difficulties arise from a non-controlled cell disruption, that results from an unhindered release of all intracellular products (proteins nucleic acids, cell debris) as well as the requirements for cell disruption without the desired product's denaturation. There are mechanical and non-mechanical cell disruption methods. The results of these methods are often evaluated in terms of the activity level of a cellular enzyme released to the disrupted suspension, combining the efficiency of the disrupting process with an estimate of the degree of cell disruption.

Mechanical Methods
Currently, intracellular products are released from microorganisms mainly by mechanical disruption of the cells. In this process, the cell envelope is physically broken, releasing all intracellular components into the surrounding medium. Equipment for cell disruption includes:

Cell disruption is needed for the extraction of intracellular products. The method used may vary depending on the type of cell and its cell wall composition. Irrespective of the method used, the main aim is that the disruption must be effective and the method should not be too harsh so that the product recovered remains in its active form. There are different methods available for cell disruption. In recent trends, intracellular products which are of commercial value are released from microorganisms mainly by

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disrupting the cells by mechanical methods which will be discussed in detail

Homogenizers.

This is the widely used method for large scale operations as well as lab scale. This method employs equipment called Homogenizer or Cell disruptor adapted from dairy industry which operates at extremely high pressures (upto 2500 bars). Cell disruptors and homogenizers are both positive displacement pumps each differs in the way that they create pressure on the sample and transfer it from pressurized chamber to another chamber which is at lower pressure. Homogenizers pressurize the sample in a chamber which is then released into a chamber of lower pressure through a homogenizing valve. Cell disruptors use a hydraulic force to accelerate the sample to high pressure and forcing them through a minute orifice to hit on a disruption head which is at a lower pressure. The basic homogenizer consists of a positive displacement pump which forces the cell suspension through the centre of the valve seat. Pressure can be controlled by adjusting the force imparted on the valve, which is controlled either pneumatically or hydraulically. As the cell suspension is pumped through a minute orifice at high pressure it causes a shear on the cell membranes. This is followed by the sudden release of the suspension with instant expansion. Disruption of the cell is accomplished at three stages causing the explosion thereby releasing its contents. 1. Impegment on the homogenizing valve 2. High turbulence and shear combined with compression produced in the minute gap 3. Sudden pressure drop upon release

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The main disruptive factor in this process is the pressure applied on the sample and consequent pressure drop across the valve. This causes the impact and shear stress on the cells making them to break which are proportional to the operating pressure.

Enzymes/Proteins are released at various rates depending on their cellular location. Proteins located in the periplasm are released faster whereas the proteins located within the cellular components are released at a slower rate. Unbound intracellular proteins may be released in a single pass whereas membrane bound enzymes or proteins may require several passes for reasonable yields to be obtained. The rate of cell disruption is directly proportional to the third power of the turbulent velocity of the product flowing through the homogenizer channel, which in turn is directly proportional to the pressure applied on the sample. The higher the pressure, the higher the release of cell contents per pass through the machine. The release of proteins in the cell disruption process can be explained by the following equation:

where, Pm Maximum amount of soluble protein PR Amount of soluble protein LOVELY PROFESSIONAL UNIVERSITY
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k - Temperature dependent release constant N Number of homogenizer passes P Operating pressure The operating parameters which affect the cell breaking efficiency of high-pressure homogenizers are as follows: - Operating Pressure - Process Temperature - Number of passes - Valve/Orifice design - Flow rate of the sample There are certain variables to be considered while designing a homogenizer/cell disruptor. They are: - type of homogenizing valve/orifice - operating pressure - stages of disruption - viscosity of the sample - temperature - type of the surfactant

High-pressure homogenizers are the best available means to mechanically disrupt nonfilamentous microorganisms on a large scale. The major disadvantage of homogenizer is the generation of heat during the process. The temperature rise can be prevented by cooling the sample to 40C and also providing proper cooling system for the equipment. While it is true that most of the proteins cannot withstand the high pressure inside the homogenizer, most proteins will be denatured by the temperature inside unless the device is cooled properly. Cell disruption will release degradative enzymes like Proteases along with the protein of interest. These enzymes can cause serious loss to enzyme activity by degrading our protein of interest. This loss can be minimized by cooling the sample before and after disruption. In addition, protease inhibitors like PMSF can be added along with the sample. LOVELY PROFESSIONAL UNIVERSITY
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Sonication
Another common laboratory-scale method for cell disruption applies ultrasound (typically 2050 kHz) to the sample (sonication). In principle, the high-frequency is generated electronically and the mechanical energy is transmitted to the sample via a metal probe that oscillates with high frequency. The probe is placed into the cell-containing sample and the high-frequency oscillation causes a localized low pressure region resulting in cavitation and impaction, ultimately breaking open the cells. Although the basic technology was developed over 50 years ago, newer systems permit cell disruption in smaller samples (including multiple samples under 200 L in microplate wells) and with an increased ability to control ultrasonication parameters. Disadvantages include:

Heat generated by the ultrasound process must be dissipated. High noise levels (most systems require hearing protection and sonic enclosures) Yield variability Free radicals are generated that can react with other molecules.

Bead method
Another common laboratory-scale mechanical method for cell disruption uses small glass, ceramic, zirconium, or steel beads and a high level of agitation by stirring or shaking of the mix. The method, often referred to as "beadbeating", works well for all types of cellular material - from spores to animal and plant tissues. At the lowest levels of the technology, beads are added to the cell or tissue suspension in a test tube and the sample is mixed on a common laboratory vortex mixer. While processing time is 3-10 times longer than that in specially machines (see below), it works for easily disrupted cells and is inexpensive. At the more sophisticated level, beadbeating is done in closed vials, centrifuge tubes, or sealed titer plates. The sample and the beads are vigorously agitated at about 2000 oscillations per minute in a specially designed clamp driven by a high energy electric motor. In some machines hundreds of samples can be processed simultaneously. To prevent degradation of RNA and proteins, some form of cooling is required because samples heat due to collisions of the beads. This can be accomplished by placing titer plates or vials in chilled aluminum blocks. [2]Another configuration suitable for larger sample volumes uses a rotor inside a sealed 15, 50 or 200 ml chamber to agitate the beads. The chamber can be
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surrounded by a cooling jacket. Using this same configuration, commercial machines capable of processing many liters of cell suspension are available. Disadvantages include:

Occasional problems with foaming and sample heating, especially for larger samples. Tough tissue samples such as skin or seeds are difficult to disrupt unless the sample is very small or has been pre-chopped into small pieces.

Detergent methods
Detergent-based cell lysis is an alternative to physical disruption of cell membranes, although it is sometimes used in conjunction with homogenization and mechanical grinding. Detergents disrupt the lipid barrier surrounding cells by disrupting lipid:lipid, lipid:protein and protein:protein interactions. The ideal detergent for cell lysis depends on cell type and source and on the downstream applications following cell lysis. Animal cells, bacteria and yeast all have differing requirements for optimal lysis due to the presence or absence of a cell wall. Because of the dense and complex nature of animal tissues, they require both detergent and mechanical lysis to effectively lyse cells. In general, nonionic and zwitterionic detergents are milder, resulting in less protein denaturation upon cell lysis, than ionic detergents and are used to disrupt cells when it is critical to maintain protein function or interactions. CHAPS, a zwitterionic detergent, and the Triton X series of nonionic detergents are commonly used for these purposes. In contrast, ionic detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and function. SDS, an ionic detergent that binds to and denatures proteins, is used extensively for studies assessing protein levels by gel electrophoresis and western blotting.

Ball Mills.
In a ball mill, cells are agitated in suspension with small abrasive particles. Cells break because of shear forces, grinding between beads, and collisions with beads. The beads disrupt the cells to release biomolecules. The kinetics of biomolecule release by this method is also a first-order process.

Blenders
(high speed or Waring), the french press, or even centrifugation in case of weak cell walls, also disrupt the cells by using the same concepts. LOVELY PROFESSIONAL UNIVERSITY
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Disadvantages of Mechanical Disruption Mechanical disruption methods suffer from several drawbacks. Because cells are broken completely, all intracellular materials are released. Therefore, the product of interest must be separated from a complex mixture of proteins, nucleic acids, and cell wall fragments. Released nucleic acids may increase the viscocity of the solution and may complicate subsequent processing steps and especially chromatography, in case of laboratory experiments. The cell debris, produced by mechanical lysis, often consists of small cell fragments, making the solution diffi cult to clarify. Complete product release often requires more than one pass through the disruption device, exacerbating the problem by further reducing the size of the fragments. These are difficult to remove by conti nuous centrifugation, because the throughput of the device is inversely related to the square of the particle. diameter. Filtration is complicated by the gelatinous nature of the homogenate and by its tendency to foul membranes Furthermore, mechanical methods expose the cells, and hence the extracted product in very harsh conditions. While it is now generally accepted that most proteins can tolerate the and the high pressure inside an homogenizer or a ball mill, most will be denaturated by the heat generated unless the device is sufficiently cooled.

NON MECHANICAL METHODS

Another way to disrupt the cells is to cause their permeabilization. This can be acheived by numerous methods. The most important are :

Chemical Permeabilization.: Many chemical methods have been employed in order

to extract intra cellular components from micro organisms by permeabilizing the outerwall barriers. It can be achieved with organic solvents that act by creation of canals through the cell membrane: toluene, ether,phenylethyl alcohol DMSO, benzene, methanol, chloroform Chemical permeabilization can also be achieved with antibiotics, thionins, surfactants (Triton, Brij, Duponal) chaotropic agents, and chelates. A very important case is EDTA (chelating agent) widely used for permeabilization of Gram negative microorganisms. Its effectiveness is a result of its ability to bond the divalent cations of Ca++, Mg++. The last ones stabilize the structure of outer membranes, by bonding the lipopolysaccharides to each other. Once these cations are removed from the EDTA, the lipopolysaccharides are removed, resulting in increased permeability areas of the outer walls Chaotropic agents, such as urea and guanidine are capable of bringing some hydrophobic compounds into aqueous solutions. They accomplish this by disrupting the structure of water, making it a less hydrophilic enviro nment and weakening the hydrophobic interactions among solute molecules.

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Mechanical Permeabilization. One method is osmotic shock. While cells exposed

to slowly varying extracellular osmotic pressure are usually able to adapt to such changes, cells exposed to rapid changes in external osmolarity, can be mechanically injured. This procedure is typically conducted by first allowing the cells to equilibrate internal and external osmotic pressure in a high sucrose medium, and then rapidly diluting away the sucrose. The resulting immediate overpressure of the cytosol is is assumed to damage the cell membrane. Enzymes released by this method are believed to be periplasmic, or at least located near the surface of the cell.

Enzymatic method
The use of enzymatic methods to remove cell walls is well-established for preparing cells for disruption, or for preparation of protoplasts (cells without cell walls) for other uses such as introducing cloned DNA or subcellular organelle isolation. The enzymes are generally commercially available and, in most cases, were originally isolated from biological sources (e.g. snail gut for yeast or lysozyme from hen egg white). The enzymes commonly used include lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mann ase etc. Disadvantages include:

Not always reproducible.

In addition to potential problems with the enzyme stability, the susceptibility of the cells to the enzyme can be dependent on the state of the cells. For example, yeast cells grown to maximum density (stationary phase) possess cell walls that are notoriously difficult to remove whereas midlog growth phase cells are much more susceptible to enzymatic removal of the cell wall.

Not usually applicable to large scale.

Large scale applications of enzymatic methods tend to be costly and irreproducible. The enzyme must be removed (or inactivated) to allow cell growth or permit isolation of the desired material. In addition to the choice of detergent, other important considerations for optimal cell lysis include the buffer, pH, ionic strength and temperature.

Other Permeabilization Techniques. Basic proteins, such as protamine, or the

cationic polysaccharide chitosan can permeabilize yeast cells. Similarly, mammalian cells can be permeabilized by exposure to several natural substances such as streptolysin or even

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viruses. Electrical discharges have also been used to permeabilize mammalian cells in order to study secretion by exocytosis.

Lysis
For easily disrupted cells such as insect and mammalian cells grown in culture media, a mild osmosis-based method for cell disruption (lysis) is commonly used. Quite frequently, simply lowering the ionic strength of the media will cause the cells to swell and burst. In some cases it is also desirable to add a mild surfactant and some mild mechanical agitation or sonication to completely disassociate the cellular components. Due to the cost and relative effort to grow these cells, there is often only a small quantity of cells to be processed, and preferred methods for cell disruption tend to be a manual mechanical homogenizer, nitrogen burst methods, or ultrasound with a small probe. Because these methods are performed under very mild conditions, they are often used for subcellular fractionation studies. For cells that are more difficult to disrupt, such as bacteria, yeast, and algae, hypotonic shock alone generally is insufficient to open the cell and stronger methods must be used, due to the presence of cell walls that must be broken to allow access to intracellular components. These stronger methods are discussed below.

Solvent Use
A method was developed for the extraction of proteins from both pathogenic and nonpathogenic bacteria. The method involves the treatment of cells with sodium dodecyl sulfate followed by extraction of cellular proteins with acetone. This method is simple, rapid and particularly well suited when the material is biohazardous.[1] Simple and rapid method for disruption of bacteria for protein studies. S Bhaduri and P H Demchick Disadvantages include:

Proteins are denatured

The 'cell bomb'

Another laboratory-scale system for cell disruption is rapid decompression or the "cell bomb" method. In this process, cells in question are placed under high pressure (usually nitrogen or other inert gas up to about 25,000 psi) and the pressure is rapidly released. The rapid pressure drop causes the dissolved gas to be released as bubbles that ultimately lyse the cell. Disadvantages include:

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Only easily disrupted cells can be effectively disrupted (stationary phase E. coli, yeast, fungi, and spores do not disrupt well by this method). Large scale processing is not practical. High gas pressures have a high risk of personal hazard if not handled carefully.

Applications
1. Quorum sensing in biotechnology Many unicellular microorganisms use small signaling molecules to determine their local concentration. The processes involved in the production and recognition of these signals are collectively known as quorum sensing (QS). This form of cell-cell communication is used by unicellular microorganisms to co-ordinate their activities, which allows them to function as multi-cellular systems. Recently, several groups have demonstrated artificial intra-species and inter-species communication through synthetic circuits which incorporate components of bacterial QS systems. Engineered QS-based circuits have a wide range of applications such as production of biochemicals, tissue engineering, and mixed-species fermentations. They are also highly useful in designing microbial biosensors to identify bacterial species present in the environment and within living organisms. In this review, we first provide an overview of bacterial QS systems and the mechanisms developed by bacteria and higher organisms to obstruct QS communications. Next, we describe the different ways in which researchers have designed QS-based circuits and their applications in biotechnology. Finally, disruption of quorum sensing is discussed as a viable strategy for preventing the formation of harmful biofilms in membrane bioreactors and marine transportation.

2. Discovery of small molecule inhibitors of JAK3 kinase enzymes have become increasingly important as the target of many disease modification drug discovery programs. Disruption of JAK3 function results in quantitative and qualitative deficiencies in both B- and T-cell compartments of the immune system of JAK3 deficient mice and development of severe combined immunodeficiency in humans with the JAK3 genetic aberration. JAK3 plays a specific role in immune function and lymphoid development and it only resides in the hematopoietic system, thus the rationale for selective targeting. Inhibitors of JAK3 have shown utility in many different autoimmune disorders, including allograft rejection during transplantation, acute lymphoblastic leukemia, Type 1 diabetes, rheumatoid arthritis and allergic and asthmatic diseases

3. Protein display onto nano-sized bacterial magnetic particles for receptor analysis Magnetic particles offer vast potential in ushering new techniques, especially in biomedical applications, as they can be easily manipulated by magnetic force. Magnetotactic bacteria synthesize nano-sized biomagnetites, otherwise known as bacterial magnetic particles LOVELY PROFESSIONAL UNIVERSITY
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(BacMPs) that are individually enveloped by a lipid bilayer membrane. BacMPs are ultrafine magnetite crystals (50-100 nm diameters) with uniform morphology produced by Magnetospirillum magneticum AMB-1. Based on our elucidations on the molecular mechanism of BacMP formation in M. magneticum AMB-1, functional nanomaterials have been designed. Through genetic engineering, functional proteins such as enzymes, antibodies, and receptors were successfully displayed onto BacMPs.

REFERENCES 1. J.E. Bailey & D.F. Ollis Biochemical Engineering Fundamentals, 2nd edition Mc GrawHill, 1987. 2. T.J. Naglak, D.J. Hettwer, H.Y. Wang Chemical permeabilization of cells for intracellular product release in SEPARATION PROCESSES IN BIOTECH nology, MARCEL DEKKER INC, NY 1990. 3. J. Bulock & B. Christiansen, BASIC biotechnology, Academic Press, 1989. 4. J Brookman, Mechanism of cell Integration in High Pressure Homo genizer, Biotechnol. Bioeng., (1974), Vol. 16, p 371-383. 5. H. Felix, ANALYTICAL BIOCHEMISTRY (1982) Vol. 107, p. 207-214. 6. H Felix, PERMEABILIZED CELLS, Anal. Biochem. (1982) Vol. 120, p. 211-234. 7. M. Follows, P.J. Hetherington, P. Dunnil, Release of Enzymes from Baker's Yeast By Disruption In an Industrial Homogenizer, Biotechnol. Bioeng., (1971), Vol. 13, p.549-560 ^ Demchick PH and Koch AL (1983). "The permeability of the wall fabric of Escherichia coli and Bacillus subtilis". Appl Environ Microbiol 46 (4): 9413

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