Page 29 - Advances in Textile Biotechnology

8 Advances in textile biotechnology

Furthermore, the existence of efficient methods to integrate several copies
of the expression cassette carrying the recombinant DNA into the genome,
eliminating the problems associated with expression from plasmids, make
this yeast the micro-organism of choice for an increasing number of bio-
technologists (Cereghino and Cregg, 2000; Hollenberg and Gellissen, 1997).
Once fermentation is completed, the micro-organisms are destroyed, and
the enzymes are isolated and further processed for commercial use.

1.3 Enzyme engineering
In broad terms, enzyme engineering refers to the creation of mutant pro-
teins with predetermined enzymatic characteristics. The three main con-
cerns of enzyme optimization as industrial biocatalysts are the volumetric
productivity or activity of the enzyme, the stability of the enzyme under
process conditions, and the availability or cost of producing the enzyme
(Marrs et al., 1999). The activity and stability of enzymes can be modifi ed

by chemical modification, immobilization, or by the use of solvents
(DeSantis and Jones, 1999; Bull et al., 1999). Recently, enzyme engineering
by molecular modeling, site-directed mutagenesis and directed evolution

has become remarkably efficient and may generally be applied in combina-
tion to develop desired mutants.

1.3.1 Site-directed mutagenesis

Site-directed mutagenesis represents a powerful technique that allows
selective engineering of gene sequences and has led to rapid advances in
the understanding of gene expression and function. In site-directed muta-
genesis, nucleotide alterations are introduced into a target sequence by
incorporating DNA base changes within an oligonucleotide utilized in the
DNA synthesis step (primer). For example, deletions, insertions and point
mutations can be easily introduced using suitable primers containing the
desired mutations.
Several protocols of site-directed mutagenesis based on polymerase
chain reaction (PCR) methods have been developed and optimized in order
to achieve efficient mutagenesis of a target DNA sequence (Hemsley et al.,

1989; Ho et al., 1989; Ito et al., 1991) Among the PCR-based protocols, the
‘megaprimer’ method, first introduced by Kammann and collaborators

(1989), is simple and cost-effective. This method involves two rounds of
PCR with two ‘flanking’ primers and one internal mutagenic primer con-

taining the desired mutation. The first PCR is performed using the muta-

genic internal primer and the fi rst flanking primer. After purifi cation, this

first PCR product, the ‘megaprimer’, is used, together with the second fl ank-

ing primer, for a second PCR round from which the final PCR product,

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