Page 24 - Advances in Textile Biotechnology

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Design and engineering of novel enzymes for
textile applications

R. ARAÚJO, M. CASAL and A. CAVACO-PAULO,
University of Minho, Portugal

Abstract: The principles of recombinant DNA molecular cloning and
transformation of host cells are outlined, and applications for protein
engineering in the textile industry are described. High production yields
of enzymes can be achieved at competitive costs and enzymes can be
redesigned with novel properties adapted to suit industrial conditions.
Protein engineering techniques, such as site-directed mutagenesis and
directed evolution, are described in detail. Despite their complexity
and disadvantages, these techniques for enzyme design have been
successfully applied at the industrial level and examples of applications
are discussed in this chapter.
Key words: enzyme engineering, molecular genetics, site-directed
mutagenesis, directed evolution.

1.1 Basic principles of recombinant deoxyribonucleic
acid (DNA) molecular cloning

The manipulation of genetic information involves the identifi cation and

purification of a particular coding sequence of interest, i.e. DNA or ribo-
nucleic acid (RNA), which can be isolated from a biological specimen or
obtained by chemical synthesis. After the target nucleic acid molecule is

obtained in purified form, the further steps involve the construction of a
recombinant DNA molecule by covalent ligation to a well characterized
replicative system and further cloning and amplification into a desirable cell

host.
The construction of a recombinant DNA molecule makes use of restric-
tion endonucleases to create a linear double strand of DNA fragments of
both the target gene and the vector, in such way that they can recombine
in vitro becoming covalently linked. The enzyme ligase promotes the phos-
phodiester ligation of the ends of any two DNA fragments that display
blunt ends or complementary sticky ends. The resulting recombinant
DNA molecule is then transferred into a host cell in a process called
transformation.

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