60
C.B. Elias and J.B. Joshi
enzyme production is carried out using organisms which have strong or rigid
cell walls, as in the case of Gram positive bacteria such as yeast chemical
methods are normally inadequate for cell disruption. The other disadvantage of
this method is that the agents used for cell disruption such as solvents or
detergents are denaturing to proteins and hence cannot be used. The scale up of
these methods both enzymatically and chemically also pose difficulties in terms
of equipment and further recovery of enzyme and solvent.
Commonly used methods of mechanical disruption include high pressure
homogenisers, ultrasonic devices and bead milling. These systems have the
advantage that they can be scaled up using known principles and can be used for
almost any kind of microorganism. These features make mechanical disruption
a popular technique in industrial scale applications although one of the problems associated with these systems is the excessive amount of hydrodynamic
shear forces generated during the operation. This problem is mainly encountered during homogenisation and bead milling process. Cell disruption by
ultrasound is dependent upon the mechanical effects of a gaseous and/or
vaporous cavitation in the suspending medium. Ultrasonic vibrations cover
a range of frequencies extending upwards from 20 kHz. At high acoustic power
inputs microbubbles form at various nucleation sites in the fluid which grow
during the rarefaction phase of the sound wave. During the compression phase
the bubble contents are compressed to a minimum radius where the bubble
collapses releasing a violent shock wave which is propagated through the fluid.
In the collapse phase a large quantity of sonic energy is converted to mechanical
energy in the form of elastic waves which disintegrate into eddies. Cell disruption occurs due to the mechanical stresses arising from the shock waves of cavity
collapse as well as from flows and turbulence generated by the pulsating cavities.
Inactivation of enzymes has been observed during ultrasonication [53, 543.
However, excessive heat generated during conversion of sonic energy to mechanical energy and chemical effects such as ionisation and subsequent free radical
generation are reported as the main cause of enzyme inactivation [55]. Besides
these known effects damage due to the hydrodynamic flow field may also
occur [563.
The problem of denaturation of protein during cell disruption has serious
implications especially in the case of membrane associated proteins because the
shear forces which require to be applied to disrupt the rather strong microbial
cell walls can potentially disrupt the membrane-enzyme complex. These effects
have been studied in the progesterone 11 e hydroxylase complex. This enzyme is
intracellular and involves a cytochrome P450 enzyme and a NADPH cytochrome P450 reductase and is membrane associated. The effect of shear on this
enzyme complex during disruption has been reported for Rhizopus nigricans
[57, 58]. The cell grown in shake flasks and in stirred fermenter was carried out
in a concentric cylinder viscometer in order to disrupt cells under conditions
of defined shear. The shear rates used in the experiment ranged from
2870-6460 s- 1 for time periods up to 5 min. Both the magnitude of shear and its
duration have an effect on the activity of the enzyme system. Disruption of cells
C.B. Elias and J.B. Joshi
enzyme production is carried out using organisms which have strong or rigid
cell walls, as in the case of Gram positive bacteria such as yeast chemical
methods are normally inadequate for cell disruption. The other disadvantage of
this method is that the agents used for cell disruption such as solvents or
detergents are denaturing to proteins and hence cannot be used. The scale up of
these methods both enzymatically and chemically also pose difficulties in terms
of equipment and further recovery of enzyme and solvent.
Commonly used methods of mechanical disruption include high pressure
homogenisers, ultrasonic devices and bead milling. These systems have the
advantage that they can be scaled up using known principles and can be used for
almost any kind of microorganism. These features make mechanical disruption
a popular technique in industrial scale applications although one of the problems associated with these systems is the excessive amount of hydrodynamic
shear forces generated during the operation. This problem is mainly encountered during homogenisation and bead milling process. Cell disruption by
ultrasound is dependent upon the mechanical effects of a gaseous and/or
vaporous cavitation in the suspending medium. Ultrasonic vibrations cover
a range of frequencies extending upwards from 20 kHz. At high acoustic power
inputs microbubbles form at various nucleation sites in the fluid which grow
during the rarefaction phase of the sound wave. During the compression phase
the bubble contents are compressed to a minimum radius where the bubble
collapses releasing a violent shock wave which is propagated through the fluid.
In the collapse phase a large quantity of sonic energy is converted to mechanical
energy in the form of elastic waves which disintegrate into eddies. Cell disruption occurs due to the mechanical stresses arising from the shock waves of cavity
collapse as well as from flows and turbulence generated by the pulsating cavities.
Inactivation of enzymes has been observed during ultrasonication [53, 543.
However, excessive heat generated during conversion of sonic energy to mechanical energy and chemical effects such as ionisation and subsequent free radical
generation are reported as the main cause of enzyme inactivation [55]. Besides
these known effects damage due to the hydrodynamic flow field may also
occur [563.
The problem of denaturation of protein during cell disruption has serious
implications especially in the case of membrane associated proteins because the
shear forces which require to be applied to disrupt the rather strong microbial
cell walls can potentially disrupt the membrane-enzyme complex. These effects
have been studied in the progesterone 11 e hydroxylase complex. This enzyme is
intracellular and involves a cytochrome P450 enzyme and a NADPH cytochrome P450 reductase and is membrane associated. The effect of shear on this
enzyme complex during disruption has been reported for Rhizopus nigricans
[57, 58]. The cell grown in shake flasks and in stirred fermenter was carried out
in a concentric cylinder viscometer in order to disrupt cells under conditions
of defined shear. The shear rates used in the experiment ranged from
2870-6460 s- 1 for time periods up to 5 min. Both the magnitude of shear and its
duration have an effect on the activity of the enzyme system. Disruption of cells