Role of Hydrodynamic Shear on Activity and Structure of Proteins
61
at a shear rate of 5020 s- 1 resulted in less activity than at 4300 s- 1. Further it
was observed that at all the shear rates studied there was a reduction in the
activity even after 3 min.
Experiments carried out using the cell free extract of the enzyme complex
showed that treating the enzyme complex to shear rates between 720 and
2870 s- 1 for a short time of 4 min led to an increase in the enzyme activity. After
that there was a decline in the activity over a further exposure period of 40 min.
Glycerol was added to the medium to increase the shear stress at a given shear
rate, and showed that the enzymatic activity decreased with an increase in the
shear stress. In these studies experiments were also carried out under conditions
where air was excluded from the viscometer by sealing. The comparison of
activity results in the systems wherein air was present and absent showed
distinctly that air enhanced the subsequent decline of enzyme activity after the
initial increase in activity seen after 4 min. These results are similar to those
obtained with soluble enzymes wherein the presence of an air-liquid interface
causes a marked increase in enzyme denaturation due to shear. However, the
work with the progesterone 11 ~ hydroxylase complex which is membrane
associated makes it clear that such enzymes are not as stable as soluble enzymes
when exposed to shear forces. This should be kept in mind when carrying out
cell disruption for release of these products.
4.3 Precipitation
Precipitation is an important unit operation used for the primary separation of
proteins from culture fluids. It is practised widely for the purification of various
proteins from blood [59], recovery of soya proteins [60], casein from milk and
in the production of several microbial enzymes [61, 62].
Precipitation may be employed to reduce processing volumes and to solidify
products for finishing. Differential precipitation is also carried out to fractionate
protein mixtures [63]. Combining affinity ligands with precipitation provides
a very versatile protein fractionation method [64].
Subsequent to precipitation, centrifugation is generally carried out to
achieve separation of the precipitated protein. The efficient recovery of these
protein aggregates is controlled by the size and mechanical strength of precipitates. These characteristics of precipitates are dictated by the mixing conditions
in the reactor. Thus it is necessary to characterize the effect of hydrodynamics on
the various aspects of protein precipitation.
Protein isolation is commonly done using isoelectric precipitation with acid
to obtain a suitable pH. This method is commonly used in the precipitation of
soya protein as well as in casein precipitation from milk for cheese manufacture.
The advantage of this method is that the precipitated protein can be easily
reconstituted by simple pH adjustment. The other commonly used methods for
precipitation include salting out using ammonium salts or solvents such as
acetone and ethanol.
61
at a shear rate of 5020 s- 1 resulted in less activity than at 4300 s- 1. Further it
was observed that at all the shear rates studied there was a reduction in the
activity even after 3 min.
Experiments carried out using the cell free extract of the enzyme complex
showed that treating the enzyme complex to shear rates between 720 and
2870 s- 1 for a short time of 4 min led to an increase in the enzyme activity. After
that there was a decline in the activity over a further exposure period of 40 min.
Glycerol was added to the medium to increase the shear stress at a given shear
rate, and showed that the enzymatic activity decreased with an increase in the
shear stress. In these studies experiments were also carried out under conditions
where air was excluded from the viscometer by sealing. The comparison of
activity results in the systems wherein air was present and absent showed
distinctly that air enhanced the subsequent decline of enzyme activity after the
initial increase in activity seen after 4 min. These results are similar to those
obtained with soluble enzymes wherein the presence of an air-liquid interface
causes a marked increase in enzyme denaturation due to shear. However, the
work with the progesterone 11 ~ hydroxylase complex which is membrane
associated makes it clear that such enzymes are not as stable as soluble enzymes
when exposed to shear forces. This should be kept in mind when carrying out
cell disruption for release of these products.
4.3 Precipitation
Precipitation is an important unit operation used for the primary separation of
proteins from culture fluids. It is practised widely for the purification of various
proteins from blood [59], recovery of soya proteins [60], casein from milk and
in the production of several microbial enzymes [61, 62].
Precipitation may be employed to reduce processing volumes and to solidify
products for finishing. Differential precipitation is also carried out to fractionate
protein mixtures [63]. Combining affinity ligands with precipitation provides
a very versatile protein fractionation method [64].
Subsequent to precipitation, centrifugation is generally carried out to
achieve separation of the precipitated protein. The efficient recovery of these
protein aggregates is controlled by the size and mechanical strength of precipitates. These characteristics of precipitates are dictated by the mixing conditions
in the reactor. Thus it is necessary to characterize the effect of hydrodynamics on
the various aspects of protein precipitation.
Protein isolation is commonly done using isoelectric precipitation with acid
to obtain a suitable pH. This method is commonly used in the precipitation of
soya protein as well as in casein precipitation from milk for cheese manufacture.
The advantage of this method is that the precipitated protein can be easily
reconstituted by simple pH adjustment. The other commonly used methods for
precipitation include salting out using ammonium salts or solvents such as
acetone and ethanol.