The product can be spread on trays and dried in a flow hood [196]. However,
note that drying operations can readily be carried out in the bioreactor if
packed beds are used. All that is necessary is to change the conditions of the
inlet air [195]. For the production of Beauvaria bassiana on clay microgranules,
which are well suited to direct harvesting and drying without further formulation, Desgranges et al. [197] increased the air flow rate twofold and decreased
the relative humidity of the inlet air to 17%.
8.3
Leaching of Enzymes
Leaching involves contacting either the fresh fermented solid, or dried fermented solid, with a leaching solution. The difference in concentration of the
product between the liquid phase held within the solid and the extraparticle
leaching solution drives the transfer of the product from the solid phase into
the leaching solution.
For the production of extracellular fungal enzymes, it is best not to disrupt
cell walls by using overly vigorous blending or agitation during the leaching
process, because this would release intracellular enzymes and make the enzyme
purification step more difficult [198]. This is not such an important issue with
unicellular microorganisms, which are much more difficult to disrupt.
It is common that, in addition to the product, substrate components such as
polysaccharides are extracted from the fermented substrate [199]. These can
make the extract quite gummy and interfere with further purification steps. In
general polysaccharides can be precipitated by adding calcium chloride to a
final concentration of 2% (w/v), but this can only be done when the calcium
chloride does not precipitate the product as well [200].
Various issues associated with leaching are discussed below.
8.3.1
Properties of the Leaching Solution
Enzyme solubility depends on pH and ionic strength. As a general principle the
pH of the leaching solution should be away from the isoelectric point of the
enzyme so that the enzyme is charged, and the leaching solution should contain
salts in order to increase the solubility of the charged enzyme. Higher enzyme
concentrations in the leachate were demonstrated using salt (NaCl) in comparison with water, under otherwise identical extraction procedures, for the
recovery of protease from rice bran [201]. Likewise, recovery of Mucor bacilliformis acid protease was higher with aqueous solutions of NaCl in comparison
with water and with solutions of non-ionic detergents such as Triton X-100 or
Tween 80 [202]. However, in some cases the presence of salt can increase the
extraction of undesirable co-solutes. For the extraction of enzymes from
fermented wood, distilled water or dilute buffers are preferable, because adding
salts, organic solvents, or mild wetting agents, although sometimes extracting
more activity, results in increased extraction of polyphenols and decreased
enzyme recovery in subsequent purification steps [198].
126
D.A. Mitchell et al.
note that drying operations can readily be carried out in the bioreactor if
packed beds are used. All that is necessary is to change the conditions of the
inlet air [195]. For the production of Beauvaria bassiana on clay microgranules,
which are well suited to direct harvesting and drying without further formulation, Desgranges et al. [197] increased the air flow rate twofold and decreased
the relative humidity of the inlet air to 17%.
8.3
Leaching of Enzymes
Leaching involves contacting either the fresh fermented solid, or dried fermented solid, with a leaching solution. The difference in concentration of the
product between the liquid phase held within the solid and the extraparticle
leaching solution drives the transfer of the product from the solid phase into
the leaching solution.
For the production of extracellular fungal enzymes, it is best not to disrupt
cell walls by using overly vigorous blending or agitation during the leaching
process, because this would release intracellular enzymes and make the enzyme
purification step more difficult [198]. This is not such an important issue with
unicellular microorganisms, which are much more difficult to disrupt.
It is common that, in addition to the product, substrate components such as
polysaccharides are extracted from the fermented substrate [199]. These can
make the extract quite gummy and interfere with further purification steps. In
general polysaccharides can be precipitated by adding calcium chloride to a
final concentration of 2% (w/v), but this can only be done when the calcium
chloride does not precipitate the product as well [200].
Various issues associated with leaching are discussed below.
8.3.1
Properties of the Leaching Solution
Enzyme solubility depends on pH and ionic strength. As a general principle the
pH of the leaching solution should be away from the isoelectric point of the
enzyme so that the enzyme is charged, and the leaching solution should contain
salts in order to increase the solubility of the charged enzyme. Higher enzyme
concentrations in the leachate were demonstrated using salt (NaCl) in comparison with water, under otherwise identical extraction procedures, for the
recovery of protease from rice bran [201]. Likewise, recovery of Mucor bacilliformis acid protease was higher with aqueous solutions of NaCl in comparison
with water and with solutions of non-ionic detergents such as Triton X-100 or
Tween 80 [202]. However, in some cases the presence of salt can increase the
extraction of undesirable co-solutes. For the extraction of enzymes from
fermented wood, distilled water or dilute buffers are preferable, because adding
salts, organic solvents, or mild wetting agents, although sometimes extracting
more activity, results in increased extraction of polyphenols and decreased
enzyme recovery in subsequent purification steps [198].
126
D.A. Mitchell et al.