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10.3 Biocatalyst Options 235

Table 10.2 Limitations with use of whole-cell biocatalysts.

Reaction limitations ∙ Side reactions
∙ Difficult reaction reproducibility
Biocatalyst ∙ Transport barriers – diffusion trough the cell membrane
limitations ∙ Permeability issues
∙ Highly complex cellular system
∙ Often product formation growth-associated
∙ Measurement and stability of enzyme activity
∙ Compartmentalization in some cases
Process limitations ∙ Operation at low substrate/product concentrations
∙ Accumulation of toxic intermediates
∙ Difficult control and optimization
∙ Models often empirical or stochastic
∙ Low purity of the desired product – complex mixture of compounds
Economic limitations ∙ High cost of product recovery

Adapted from [5, 18–20].

10.3.1
Transport Limitations

In many cases, the use of nonnatural substrates for a biocatalytic reaction results
in limited transport of substrate(s) into the cell, and product(s) out of the cell.
A comprehensive review about permeability issues in whole cells suggests that
in some cases limitations may result in observed activities up to one to two
orders of magnitude lower than in cell-free extracts or with soluble enzyme [21].
Conventionally permeabilization has been carried out with solvents or detergents
[22], which bring about several limitations, including limited recycle of the cells.
A more recent approach involves the use of display technologies to overcome the
limitations inside the cell. However, such an approach is not universal. Today
permeabilization of the cells can even be enhanced by washing the low molecular
weight compounds out of the cell prior to use as a biocatalyst (see Table 10.3).
Combined with genetic engineering to solely express the desired enzymes (and
even block non productive pathways and individual enzymes), the concept of the
minimal cell allows the cell to act merely as a structural support. Nevertheless, the
recovery and recycle of such cells is yet to be proven and will necessitate effective
microfiltration techniques to avoid damage to the delicate biological structures.
Yet another approach to overcoming transport problems is to engineer transporter
proteins. For example, a recent publication reports for the first time the use
of transporters belonging to the ATP-binding cassette (ABC) protein family of
proteins for the enhancement of isoprenoid biofuel secretion [23]. Interestingly it
is suggested that this enhancement also results in lower toxic effects on the cell.
Nevertheless, the correct transporters must be identified because in some cases
biocatalysis results in considerable intracellular concentration of substrate and