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10.4 Reactor Options 237

product [24]. Avoiding extraction of such compounds results in considerable process
improvement, including the option of recovery and recycle of the biocatalyst [25].

10.3.2
Compartmentalization

A second development has been the move toward cell-free extracts by the deliberate
lysis of cells to overcome simultaneously transport as well as compartmentalization
limitations [18]. However, the resultant catalyst ‘‘soup’’ is rich in other enzymes,
necessitating the addition of inhibitors (or alternatively using genetic engineering
to massively overexpress the desired enzymes over other proteins). Alternatively
heat treatment can be used in cases where thermostable isoenzymes are available.
Likewise, cell debris is present in the reaction medium, necessitating the combi-
nation of microfiltration as well as ultrafiltration for recovery (and potential reuse).
A further complication remains in the control of enzyme activities which to a
large extent is dependent upon expression during the fermentation. One potential
solution is to add supplementary enzymes to the cell-free extract. Such a precedent
has already been set by a few reported cases where whole cells were mixed with
the isolated enzyme for ex vivo cofactor recycle. Despite these problems, there
is no doubt that as genetic engineering for expression of the desired enzyme is
improved, more systems will be tested in the cell-free environment [26]. At the very
least it is clear that cell-free extracts, combined with network topology analysis can
provide an excellent basis for effective analysis and targeting of the network so as
to insulate the desired pathway from undesired enzymatic reactions [27].

10.4
Reactor Options

In an analogous way to the increased number of biocatalyst types and combinations
that become possible when considering multienzyme systems, so too the number
of reactor options is increased. The basic choice is between packed bed, stirred
tank, or combinations thereof (with or without possibilities for enzyme retention
by membranes) (Figure 10.2). The packed bed reactor can only handle immobilized
enzyme(s) and operates in plug-flow mode (mixing only in the radial rather than
axial direction). For many enzymes their kinetics are such that plug flow is the
favored operating mode and therefore, in cases where mixing is required (e.g., for
pH control via addition of a neutralizing acid or alkali, or addition of an inhibitory
substrate), multiple continuous-stirred tank reactors (CSTRs) can be used [28].
For multiple enzymes, combinations of reactor configuration and/or operation
are also possible, dependent upon the kinetic characteristics of each enzyme and
the cost contribution of each enzyme, relative to the other components [6]. Such
combinations of reactor configuration and operation can also bring extra flexibility
to cope with the different characteristics of each enzyme. For example, each
reactor can operate with different enzymes, and/or different reactor hydrodynamics