Page 30 - Advances in Textile Biotechnology

Design and engineering of novel enzymes for textile applications 9

containing the desired mutation, is obtained (Kammann et al., 1989). This

method was later modified to increase yield and optimize time consumption
(Sarkar and Sommer, 1990; Barik and Gahnski, 1991; Barik, 1995; Ke and
Madison, 1997).
Later, a new method was described combining both PCR technology and

homologous recombination for introducing site-specific alterations in any
DNA sequence cloned into a plasmidic expression vector. This approach is

based on PCR amplification of the entire plasmid DNA by mutagenic
primers divergently oriented but overlapping at their 5′ ends (Martin et al.,
1995). This approach was optimized to overcome length limitation, owing
to the plasmid size, to increase mutagenesis efficiency and to minimize the

rate of undesired mutations (Ansaldi et al., 1996; Rabhi et al., 2004).
Several commercial site-directed mutagenesis kits have now been devel-
oped. Muta-Gene M13 in vitro Mutagenesis Kit from Bio-Rad and the
QuikChange kit from Stratagene guarantees mutation frequencies higher
than 50% (Siemers et al., 1996).
Enzyme stability and activity can be optimized by site-directed mutagen-
esis but this technique is dependent on the availability of structural and
biochemical information. First, the 3D-structure of the protein of interest

must be solved to allow the identification of important amino acids, then,
the protein variants are constructed based on predictions derived from the
analysis of the 3D-structure and, finally, these variants are biochemically

characterized.

1.3.2 Directed evolution
Directed evolution involves the application of repeated rounds of random
mutagenesis, in vitro recombination, and selection to develop enzymes
with improved properties. This strategy mimics natural evolution, as an
initial parent gene is chosen and a diverse library of offspring genes is
created through mutagenesis or recombination. A screening is applied to
the library and the mutants that exhibit the greatest improvement in the
desired properties are chosen to become the parents to the next genera-
tion (Bloom et al., 2005). Directed evolution is a more general strategy for
the isolation of catalysts as it does not necessarily require structural infor-
mation of the enzyme or its catalytic mechanism (Cherry and Fidantsef,
2003; Farinas et al., 2001; Jaeger et al., 2001; Powell et al., 2001; Tao and
Cornish, 2002) and it can be applied to most chemical reactions in aqueous
solutions (Kettling et al., 1999). However, as it results in a large number
of variants, the success of this approach is dependent on an effi cient
screening procedure, so that variants with improved and desired properties

can be identified. Directed evolution has been used to alter a diverse range

of enzyme performance properties including modification of stability and

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