8 Enzyme Engineering and Technology 207Although site-directed mutagenesis is widely
used, it is not always feasible due to the limited
knowledge of the protein structure-function relation-
ship and the approximate nature of computer-graphic
modeling. In addition, rational design approaches
can fail due to unexpected influences exerted by the
substitution of one or more amino acid residues.
Irrational approaches can therefore be preferable
alternatives for engineering enzymes with highly
specialized traits.
DIRECTEDENZYMEEVOLUTION
Directed evolution by DNA recombination can be
described as a mature technology for accelerating
protein evolution. Evolution is a powerful algorithm
with proven ability to alter enzyme function and
especially to “tune” enzyme properties. The methods
of directed evolution use the process of natural selec-
tion, but in a directed way (Altreuter and Clark 1999).
The major step in a typical directed enzyme evolution
experiment is first to make a set of mutants and then
to find the best variants through a high-throughput
selection or screening procedure. The process can be
iterative, so that a “generation” of molecules can be
created in a few weeks or even in a few days, with
large numbers of progeny subjected to selective pres-
sures not encountered in nature (Arnold 2001).
There are many methods to create combinatorial
libraries using directed evolution. Some of these
are random mutagenesis using mainly error-prone
PCR (Ke and Madison 1997), DNA shuffling (Stem-
mer 1994, Crameri et al. 1998), StEP (staggered
extension process; Zhao et al. 1998), RPR (random-
priming in vitro recombination; Shao et al. 1998),
and incremental truncation for the creation of hybrid
enzymes (ITCHY; Lutz et al. 2001). The most fre-
quently used methods for DNA shuffling are shown
in Figure 8.19.Figure 8.19.A schematic representation of the most frequently used methods for DNA shuffling.