58
C.B. Elias and J.B. Joshi
Trichoderma reesei cellulase in a bioreactor which combined the wet milling step
with the hydrolysis. This reactor they termed as an attrition bioreactor (ABR).
Two types of impellers were used, a marine type propeller and a curved blade
turbine. Stainless steel balls of 4.69 mm diameter were used as milling media.
The effect of an air-liquid interface was seen by sealing the reactor after
completely filling it. It was shown that the enzyme solution was 50% deactivated
in the presence of air whereas only 5% deactivation took place after 20 h in its
absence at an agitation speed of 700 rpm. Under normal operating conditions
a speed of 200 rpm was used at which there was no significant deactivation
irrespective of the presence of air.
The above experiments deal with the effects of shear on cellulase enzyme
during its use as a catalyst of cellulose hydrolysis. The enzyme however, is also
exposed to shearing conditions during its production in reactors as it is secreted
extracellularly. The production of cellulase by immobilized Trichoderma reesei
has been studied in two types of fermenters, the conventional stirred tank
reactor representative of high shear rates and a low shear stress rotating disk
fermenter [46]. The enzymes produced from the two types of reactors were
compared by subjecting them to conditions of shear by stirring at room
temperature for varying time periods and estimating the activity of the component enzymes of the complex viz. cellobiohydrolase I (CHB I) and endoglucanase
using standard assay methods for the respective enzymes. Their results showed
that the productivity of enzyme per se was reduced in the low shear stress
rotating disk fermenter. The important result that they obtained however, was
that the enzyme produced under conditions of low shear stress exhibited a
higher specific microcrystalline cellulose (MCC) hydrolyzing activity. This difference in the activity of the enzymes was not distinguishable in early stages of
cellulose hydrolysis but was seen in the later stages when the action of enzymes
is on the more crystalline domains of cellulose.
Furthermore, it was observed that not only was the enzymatic activity of the
cellulase altered by shear forces but also the ability of the enzyme molecules to
adsorb onto crystalline cellulose was affected. This may be a result of structural
changes in the enzyme molecule. These effects are discussed in a separate section
below.
Microbial lipases have widespread industrial applications as catalysts for
hydrolysis, synthesis and transesterification of triglycerols and optical resolution. Very few studies exist on the production and stability of enzymes. Lee and
Choo [47] described the deactivation of lipase due to shear forces. However, in
a later study Gordillo et al. [48], examined the effects of shear on lipase from
Candida rugosa. They subjected the enzyme to shear stress by stirring 500 ml
culture broth free from biomass in a stirred bioreactor 11 at speeds of 250, 500
and 750 rpm at 30 ~ The activity was greater than 90% after 25 h of stirring. In
another set of experiments they studied the effect of surface forces generated due
to gas-liquid interface by bubbling helium and air through a cell free culture
broth in 250 ml flasks at different flow rates. At all the flow rates studied the loss
in activity was less than 20%. A lipase containing broth when subjected to
C.B. Elias and J.B. Joshi
Trichoderma reesei cellulase in a bioreactor which combined the wet milling step
with the hydrolysis. This reactor they termed as an attrition bioreactor (ABR).
Two types of impellers were used, a marine type propeller and a curved blade
turbine. Stainless steel balls of 4.69 mm diameter were used as milling media.
The effect of an air-liquid interface was seen by sealing the reactor after
completely filling it. It was shown that the enzyme solution was 50% deactivated
in the presence of air whereas only 5% deactivation took place after 20 h in its
absence at an agitation speed of 700 rpm. Under normal operating conditions
a speed of 200 rpm was used at which there was no significant deactivation
irrespective of the presence of air.
The above experiments deal with the effects of shear on cellulase enzyme
during its use as a catalyst of cellulose hydrolysis. The enzyme however, is also
exposed to shearing conditions during its production in reactors as it is secreted
extracellularly. The production of cellulase by immobilized Trichoderma reesei
has been studied in two types of fermenters, the conventional stirred tank
reactor representative of high shear rates and a low shear stress rotating disk
fermenter [46]. The enzymes produced from the two types of reactors were
compared by subjecting them to conditions of shear by stirring at room
temperature for varying time periods and estimating the activity of the component enzymes of the complex viz. cellobiohydrolase I (CHB I) and endoglucanase
using standard assay methods for the respective enzymes. Their results showed
that the productivity of enzyme per se was reduced in the low shear stress
rotating disk fermenter. The important result that they obtained however, was
that the enzyme produced under conditions of low shear stress exhibited a
higher specific microcrystalline cellulose (MCC) hydrolyzing activity. This difference in the activity of the enzymes was not distinguishable in early stages of
cellulose hydrolysis but was seen in the later stages when the action of enzymes
is on the more crystalline domains of cellulose.
Furthermore, it was observed that not only was the enzymatic activity of the
cellulase altered by shear forces but also the ability of the enzyme molecules to
adsorb onto crystalline cellulose was affected. This may be a result of structural
changes in the enzyme molecule. These effects are discussed in a separate section
below.
Microbial lipases have widespread industrial applications as catalysts for
hydrolysis, synthesis and transesterification of triglycerols and optical resolution. Very few studies exist on the production and stability of enzymes. Lee and
Choo [47] described the deactivation of lipase due to shear forces. However, in
a later study Gordillo et al. [48], examined the effects of shear on lipase from
Candida rugosa. They subjected the enzyme to shear stress by stirring 500 ml
culture broth free from biomass in a stirred bioreactor 11 at speeds of 250, 500
and 750 rpm at 30 ~ The activity was greater than 90% after 25 h of stirring. In
another set of experiments they studied the effect of surface forces generated due
to gas-liquid interface by bubbling helium and air through a cell free culture
broth in 250 ml flasks at different flow rates. At all the flow rates studied the loss
in activity was less than 20%. A lipase containing broth when subjected to