Cell disruption of Escherichia coli using high-pressure systems

1st April 2013

Although some biological products are secreted from the cell or released during autolysis, the preparation of many others requires cell disruption to release intracellular material(1).

Testing the disruption of Escherichia coli has been performed at a confidential source to illustrate percentage soluble protein release and to give an overview of high-pressure cell disruption.

Cell disruption focuses on obtaining the desired product from within the cell, and it is the cell wall that must be disrupted to allow the contents of the cell out. The cell wall conveys its strength to the cell and can be formed from differing kinds of complex polysaccharides which are generally cross-linked by peptides to a degree and give different organisms varying levels of strength(2). In essence the objectives of cell disruption are as follows:

* To solubilise the maximum amount of the product present in the cell whilst still maintaining maximum biological activity.

* To avoid secondary alteration of the product that will render it useless eg denaturisation and oxidation,

* To limit the detrimental effects of the disruption stage on the following separation steps,

High-pressure cell disruption systems developed by Constant Systems Ltd use a hydraulic mechanism that acts on a piston seal within a cylinder to force the sample through a fixed orifice to a chamber of lower pressure. The sample is not released, but accelerated and forced through an orifice up to speeds of 550 m/sec and achieving pressures up to 40kpsi or 2700 Bar.

The electrically controlled hydraulic system and fixed orifice guarantee the disruption environment is repeatable between operating intervals. The precision high pressures ensure greater yielding breakage of the hardest microorganisms.

Standard high pressure Homogenisers, on the other hand, pressurise the sample in a chamber (via a crank shaft mechanism or compressed gas) and then release it through a manually or automatically controlled valve (homogenising valve) into another chamber. Traditionally these cannot go to extremely high pressures (40kpsi or 2700 Bar) necessary to disrupt hard cell walled micro organisms and due to the homogenising valve and pressure creation mechanism, variability over the pressure can have implications on per centage breakage and repeatability between operating intervals.

Constant Systems cell disrupter and standard built-in cooling jacket (Fig. 1). A cooling chamber whereby coolant is circulated through surrounds the entire disruption head, giving a large surface area for cooling exchange to take place. Temperature is measured by a thermocouple positioned at the outlet and is digitally displayed on the control panel so the operator has visibility throughout the process.

The disrupter was cleaned prior to use with a 2 per cent Virkon solution in order to prevent contamination from previous use. 250ml of 2 per cent Virkon solution was passed through the machine at 40kpsi. The system was then flushed with 250ml distilled water.

The sample was prepared from a 24-hour culture in Nutrient Broth (CM 67). This was then separated into 7 x 50ml aliquots in order to test at each pressure setting.

The pressure settings used were 15, 20, 25, 30, 35 and 40kpsi. The samples were passed through the machine separately starting with 40kpsi and working downwards through the pressures.

For this series of investigations a protein assay was used, this is widely recognised as a good measurement of cell disruption. The following equation gives the actual disruption per centage taking into account the background levels of protein before disruption:

Rp= Cf - Cb

Ct ­ Cb

Cf = Free protein.

Ct = Total protein.

Cb = Background protein.

The disruption profile for Escherichia coli indicated low protein release percentage up to a 20kpsi pressure setting, however a dramatic rise up to over 99 per cent was realized with the higher pressures (35-40kpsi). The difference in protein release between 35kpsi and 40kpsi is minimal therefore it is concluded that 35kpsi, with the Constant Systems Cell Disrupter, be the maximum pressure used for Escherichia coli.

For optimal cell lysis conditions, a high yielding and temperature controlled cell disruption stage needs to take place.

The high-pressure cell disrupter demonstrated superior proportions of soluble protein release from Escherichia coli. Importance here is placed on the ability to disrupt the organism and receive 100 percentage soluble protein release with one pass.

Testing has demonstrated pressure versatility of the cell disrupter. Being able to disrupt at specific pressure settings with a high degree of control has a significant impact on characterisation of pressure versus soluble protein release. As cell wall strengths differ (if only slightly) between growth mediums and length of growth cycles, having the ability to control different pressures accurately gives the operator an improved disruption tool when optimising the process.

References: 1. Foster, D Cell Disruption: Breaking Up Is Hard To Do, Biotechnology, 1992; 2. Coss, G M Investigating a Novel High Pressure Homogeniser for Producing Cell Disruption, Ph.D. Univeristy of Wales, Swansea, 1999; 3. Dictionary of Microbiology and Molecular Biology 3rd Edition, 2001.

Aidan Audouy is with Constant Systems Ltd, Low March, Daventry, Northants, UK.





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