Over the last decade or so, enzyme engineers have developed strategies for
creating variant tailored enzymes that are collectively referred to as directed
evolution (Arnold, 1998). These combinatorial methods used to alter specific
properties of enzymes have resulted in remarkable improvements in enzyme
activity for specific substrates (Stemmer, 1994b; Whittle et al.
, 2001), reversal of
enantioselectivity (Reetz et al.
, 1997), as well as changes in global properties such
as solvent (You and Arnold, 1996) and heat (Zhao and Arnold, 1999) tolerance (see
also several excellent reviews Farinas et al.
, 2001; Powell et al.
There are four key steps to engineering a desired enzyme activity successfully:
(1) identification of parental enzymes to be modified, (2) introducing variation
into the gene(s), (3) choice of host system to express the enzyme, and (4) method
for identifying improvements in property of interest. See Fig. 2.4 for a generic
scheme for altering the properties of an enzyme.
|FIGURE 2.4 Generic scheme for directed evolution of an enzyme.