Page 40 - Advances in Textile Biotechnology

Design and engineering of novel enzymes for textile applications 19

calcium-independent variants. Although the Asp357Lys, Asp357Asn, and
Asp357Ala variants did not bind calcium, at elevated temperatures these
calcium-independent mutants showed a reduced activity. Over the whole
temperature range the activities of the Asp354Lys and Asp354Asn variants

are significantly lower than the wild-type enzyme in the presence of calcium.
The lipase from Humicola lanuginosa has also been the target of surface
engineering in order to overcome calcium dependence. The variant
Asp96Leu is less dependent of calcium presence and has an increased
stability in the presence of non-ionic and anionic surfactants (Svendsen
et al., 1997).
The proteolytic stability of P. glumae lipase in the presence of proteases
for detergent use has also been improved by protein engineering. Two dif-
ferent strategies, the replacement of susceptible amino acid residues at the
cleavage site with residues that are not accepted as substrate and the
replacement of amino acid residues with proline have been employed
(Frenken et al., 1993). Proline is the only proteinogenic amino acid and is
not accepted by most of the proteases at the potential cleavage site (Bromme
et al., 1986). In both instances, a considerably increased stability towards
proteolytic degradation was observed.
Cutinase has also been exploited for the surface modification of synthetic

fibres. Despite the potential of cutinase from Fusarium solani to hydrolyse
and improve synthetic fibres properties, these fibres are non-natural sub-

strates of cutinase and, consequently, turnover rates are quite low. By the
use of site-directed mutagenesis recombinant cutinases, with higher specifi c
activity to large and insoluble substrates such as polyethylene terephthalate
(PET) and polyamide (PA), were developed (Araújo et al., 2007). The muta-
tions Leu81Ala, Asn84Ala, Leu182Ala, Val184Ala and Leu189Ala were

used to enlarge the active site in order to better fit a larger polymer chain
(Fig. 1.3).
The new cutinase, Leu181Ala mutant, was the most effective in the catal-
ysis of amide linkages of PA and displayed a remarkable hydrolytic activity
towards PET fabrics (more than five-fold compared with native enzyme)

(Araújo et al., 2007). This recombinant enzyme was further used to study

the influence of mechanical agitation on the hydrolytic efficiency of cutinase
on PET and PA in order to design a process for successful application of
enzymes to synthetic fi bres (Silva et al., 2007; O’Neill et al., 2007). The use
of cutinase opens up new opportunities for targeted enzymatic surface
functionalisation of PET and PA, polymers formerly considered as being
resistant to biodegradation.

1.5 Advantages and limitations
The tools of enzyme engineering, well developed over the last few years
by many laboratories, are now being applied for the optimization of

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