Enzyme Technology
Ultrasonic cell disruption
The treatment of microbial cells
in suspension with inaudible ultrasound (greater than about 18 kHz) results in
their inactivation and disruption. Ultrasonication utilises the rapid sinusoidal
movement of a probe within the liquid. It is characterised by high frequency (18
kHz - 1 MHz), small displacements (less than about 50 mm), moderate
velocities (a few m s−1), steep transverse velocity gradients (up to
4,000 s−1) and very high acceleration (up to about 80,000 g).
Ultrasonication produces cavitation phenomena when acoustic power inputs are
sufficiently high to allow the multiple production of microbubbles at nucleation
sites in the fluid. The bubbles grow during the rarefying phase of the sound
wave, then are collapsed during the compression phase. On collapse, a violent
shock wave passes through the medium. The whole process of gas bubble
nucleation, growth and collapse due to the action of intense sound waves is
called cavitation. The collapse of the bubbles converts sonic energy into
mechanical energy in the form of shock waves equivalent to several thousand
atmospheres (300 MPa) pressure. This energy imparts motions to parts of cells
which disintegrate when their kinetic energy content exceeds the wall strength.
An additional factor which increases cell breakage is the microstreaming (very
high velocity gradients causing shear stress) which occur near radially
vibrating bubbles of gas caused by the ultrasound.
Much of the energy absorbed by
cell suspensions is converted to heat so effective cooling is essential. The
amount of protein released by sonication has been shown to follow Equation
2.9.
The constant (k) is independent of cell concentrations up to high levels and
approximately proportional to the input acoustic power above the threshold power
necessary for cavitation. Disintegration is independent of the sonication
frequency except insofar as the cavitation threshold frequency depends on the
frequency.
Equipment for the large-scale continuous use of ultrasonics has been
available for many years and is widely used by the chemical industry but has not
yet found extensive use in enzyme production. Reasons for this may be the
conformational lability of some (perhaps most) enzymes to sonication and the
damage that they may realise though oxidation by the free radicals, singlet
oxygen and hydrogen peroxide that may be concomitantly produced. Use of radical
scavengers (e.g., N2O) have been shown to reduce this inactivation. As
with most cell breakage methods, very fine cell debris particles may be produced
which can hinder further processing. Sonication remains, however, a popular,
useful and simple small-scale method for cell disruption.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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