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10.3 Biocatalyst Options 233

believe this chapter will serve as a useful complement to the other chapters and
provide the basis for rules to implement new processes.

10.2
Multienzyme System Classification
There are a several ways to classify multienzyme systems [5, 11], but perhaps one of
the most helpful is to divide the possibilities according to the action of the enzymes
in the reaction. There are two main groups:
• Primary enzymes: this first group refers to those enzymes that are directly involved
in the synthesis of the desired product.
• Secondary enzymes: this second group refers to those enzymes that are added
to the system in order to improve the characteristics of the main reaction (e.g.,
solutions exist where enzymes are used to recycle cofactors, supply toxic or
inhibitory substrates, and in some cases to remove toxic or inhibitory products).
Interestingly, such enzymes must have higher activity, preferably use cheaper
secondary substrates rather than the main enzymes, and must not be directly
involved in the synthesis of the desired product.
For a reaction network to be classified as multienzyme, two or more enzymes
must be involved. Hence, schemes can be envisaged with
• one primary together with one (or more) secondary enzymes, or
• two (or more) primary enzymes, (with or without secondary enzymes).

The options are shown schematically in Figure 10.1.
An excellent example of the use of secondary enzymes concerns the application
of the secondary enzyme catalase to remove the by-product hydrogen peroxide,
which is otherwise toxic at very low concentrations to both enzymes and cells
alike. Catalase converts hydrogen peroxide to water and oxygen, which are not only
harmless reagents but can also serve as a co-substrate in the case of oxidation-
based primary enzymes, thereby reducing the overall demand for oxygen. Such
a scheme has been reported to enhance primary oxidative enzymes such as
amino acid oxidase (AAO) [12] and also recently aldehyde-deformylating oxygenase
(ADO, formerly known as aldehyde decarbonylase) [13]. In the latter case, the
catalase was fused with the alkane-producing enzyme to enhance enzymatic
activity fivefold.

10.3
Biocatalyst Options

Selecting the correct type of the biocatalyst is one of the more interesting
challenges raised by the use of multiple enzymes in a single reaction scheme
and the last few years have seen several new developments. From a conceptual