Page 32 - Advances in Textile Biotechnology

Design and engineering of novel enzymes for textile applications 11

sequence were designed: the substitution Lys269Ala and the deletion of
Arg176 and Gly177. Both mutations have drastically increased thermosta-
bility of BAA. The Arg176-Gly177 deletion was further applied to LAMY
α-amylase derived from alkaliphilic Bacillus species with similar effects on
the thermostability (Igarashi et al., 1998). The same group also reported for

the first time the improvement of LAMY stability by proline substitution
using site-directed mutagenesis (Igarashi, 1999).
One of the most important criteria for the use of amylases in detergents
is to maintain optimal activity under the very oxidizing washing environ-
ment (Ito et al., 1998). Oxidative stability of α-amylases was achieved by
site-directed mutagenesis, replacing oxidation-sensitive aminoacids such as
cysteines and methionines (Brosnan et al., 1992) with non-oxidizable resi-
dues as shown in the case of BLA (Brzozowski et al., 2000) and AmyK,
α-amylase from an alkaliphilic Bacillus strain KSM-1378 (Igarashi et al.,
2003). The commercial tradenames of the two currently available oxida-
®
tively stable α-amylases are Purastar OxAm from Genencor Intl and
®
Duramyl from Novozymes.
Another drawback related to the use of amylase in liquid detergents is
the presence of calcium-containing chelating agents, known as builders. The
primary function of these additives is to soften the wash water by binding
calcium ions. Amylases in direct contact with these builders lose the calcium
ions that support the enzyme molecule thus affecting long-term storage
stability. Several groups have attempted to produce calcium-independent
and acid-stable α-amylases by protein engineering. The x-ray structure of
the α-amylase from Aspergillus oryzae was used as a template to study the
importance of the region vicinal to the structural calcium-binding site. This

region was modified by amino acid substitution so that the negative poten-
tial around the site was increased and made more attractive for cations such
as calcium. The result was an amylase that bound calcium more tightly and
was more thermostable than the wild-type parent enzyme from B. licheni-
TM
formis (Van der Laan and May, 1995). Termamyl LC , which was produced
by site-directed mutagenesis, also showed high calcium independence
(Hashida and Bisgaard-Frantzen, 2000). The amino acids that lead to the
tightening of calcium binding between the A domain and the B domain of
the enzyme were substituted reducing, in this way, the destabilizing electro-
static interactions at low pH (Fig. 1.2).
Ben Ali et al. (2001) reported the cloning and characterization of an
atypically thermostable α-amylase from Geobacillus stearothermophilus
US100 strain (AmyUS100), and also used site-directed mutagenesis to
create an AmyUS100ΔIG mutant, which exhibited improved thermostabil-
ity and very low calcium requirement, by the deletion of residues Ile214
and Gly215. (Ben Ali et al., 2006). This improved enzyme was also engi-
neered with the aim of enhancing its resistance towards chemical oxidation

© Woodhead Publishing Limited, 2010