Role of Hydrodynamic Shear on Activity and Structure of Proteins
67
stress at the capillary wall ranged from 9-31 N/m 2 for Reynolds number
< 2300 for the different modules tested and 34 and 60 N/m 2 were used for
Reynolds number > 2300. The above results indicate that the globular proteins
are not necessarily damaged by shear forces during ultrafiltration.
5 Role of Hydrodynamic Shear on the Structure of Proteins
The effect of fluid shear stress on the catalytic activity of proteins has been
described in several literature reports. Although the loss of activity in proteins is
related to changes in structure there are very few studies where this has been
directly reported. This may be due to the difficulties in detecting and monitoring
minor changes in protein structure whereas catalytic activity is relatively easy to
monitor. It is important however, to examine the changes in the structure of
proteins when subjected to shear especially in the case of therapeutic proteins
where the small changes in the tertiary structure may result in changes in
bioactivity. These effects also play an important role when the protein under
consideration is a multienzyme complex where the different enzyme molecules
associated in the complex are differentially affected by the shear forces and
hence the overall reaction process needs to be suitably controlled. Minor
changes in protein structure also result in altered sensitivity to shear forces. This
has been demonstrated for sickle cell haemoglobin (Hb S) [77]. The oxy-form of
Hb S is extremely susceptible to fluid shear forces and results in turbidity and
precipitation when exposed to stirring. The normal heomoglobin does not show
similar shear sensitivity. This feature has clinical importance as the shear
denaturation of Hb S results in increased red cell rigidity and change in red cell
shape.
Reese and Mandels [78] have studied the shear deactivation of the cellulase
enzyme complex. The stability of this enzyme is an important aspect of cellulose
saccharisation processes. Agitation is needed during enzymatic digestion to keep
the solid substrate suspended and for ensuring homogeneity in the reaction
vessel. The cellulase enzyme is a complex of three components endo-fl-l,4glucanase (Cx), CBH and fl-glucosidase (cellobiase). Cx activity can be measured
using carboxy methyl cellulose (CMC) as the substrate. CBH activity cannot be
directly measured as there is no substrate which is not also a substrate for Cx
activity. However, microcrystalline cellulose (Avicel) which due to its crystalline
nature is a poor substrate for either pure CBH or Cx is synergistically acted
upon by both the enzyme components. After acid pretreatment, Avicel makes
a good substrate for CBH. An enzyme mixture which shows a good Cx activity
but no activity against Avicel may be considered to be inactive due to loss of
CBH activity. Deeble and Lee [79] studied the enzymatic hydrolysis of newsprint in an attrition bioreactor and found that the CBH component was
67
stress at the capillary wall ranged from 9-31 N/m 2 for Reynolds number
< 2300 for the different modules tested and 34 and 60 N/m 2 were used for
Reynolds number > 2300. The above results indicate that the globular proteins
are not necessarily damaged by shear forces during ultrafiltration.
5 Role of Hydrodynamic Shear on the Structure of Proteins
The effect of fluid shear stress on the catalytic activity of proteins has been
described in several literature reports. Although the loss of activity in proteins is
related to changes in structure there are very few studies where this has been
directly reported. This may be due to the difficulties in detecting and monitoring
minor changes in protein structure whereas catalytic activity is relatively easy to
monitor. It is important however, to examine the changes in the structure of
proteins when subjected to shear especially in the case of therapeutic proteins
where the small changes in the tertiary structure may result in changes in
bioactivity. These effects also play an important role when the protein under
consideration is a multienzyme complex where the different enzyme molecules
associated in the complex are differentially affected by the shear forces and
hence the overall reaction process needs to be suitably controlled. Minor
changes in protein structure also result in altered sensitivity to shear forces. This
has been demonstrated for sickle cell haemoglobin (Hb S) [77]. The oxy-form of
Hb S is extremely susceptible to fluid shear forces and results in turbidity and
precipitation when exposed to stirring. The normal heomoglobin does not show
similar shear sensitivity. This feature has clinical importance as the shear
denaturation of Hb S results in increased red cell rigidity and change in red cell
shape.
Reese and Mandels [78] have studied the shear deactivation of the cellulase
enzyme complex. The stability of this enzyme is an important aspect of cellulose
saccharisation processes. Agitation is needed during enzymatic digestion to keep
the solid substrate suspended and for ensuring homogeneity in the reaction
vessel. The cellulase enzyme is a complex of three components endo-fl-l,4glucanase (Cx), CBH and fl-glucosidase (cellobiase). Cx activity can be measured
using carboxy methyl cellulose (CMC) as the substrate. CBH activity cannot be
directly measured as there is no substrate which is not also a substrate for Cx
activity. However, microcrystalline cellulose (Avicel) which due to its crystalline
nature is a poor substrate for either pure CBH or Cx is synergistically acted
upon by both the enzyme components. After acid pretreatment, Avicel makes
a good substrate for CBH. An enzyme mixture which shows a good Cx activity
but no activity against Avicel may be considered to be inactive due to loss of
CBH activity. Deeble and Lee [79] studied the enzymatic hydrolysis of newsprint in an attrition bioreactor and found that the CBH component was