Surface Modification of Cellulose Acetate with Cutinase and ...

Subchapter 2.3
aliphatic polyester. This similarity and the diversified substrate recognition of cutinase
makes it able to modify the polyamide surface, showing however a slow enzymatic
kinetics (Silva et al., 2004).
The high crystallinity of the polyamide structure and the low affinity of the enzyme to
the non-natural substrate are the main factors responsible for that. A more detailed study
is needed in a way to know which process variables are the most significant in
determining the efficiency of enzymatic hydrolysis. These variables may include
different genetic modifications as well as the more usual operating conditions such as
enzyme concentration, liquor ratio, treatment time, temperature, pH and mechanical
agitation (Azevedo et al., 2000).
Site direct mutagenesis is a way of developing the cutinase in order to obtain a higher
specific activity to insoluble substrates like polyamide fiber. The modeling studies are
based on the substitution of the specific amino acid residues close to the active site of
cutinase, resulting in a modified enzyme with different properties and bigger binding
sites. A previous work was performed in order to increase cutinase activity towards
polyamide substrates (Araújo et al., 2007). Site-directed mutagenesis of cutinase was
performed and five genetically modified enzymes were obtained by changing specific
amino acids residues around the active site by alanine (L81A, N84A, L182A, V184A
and L189A). The L182A mutant form was the most efficient in the catalysis of the
amide linkages (Araújo et al., 2007).
The process of enzyme adsorption is also of main importance to the enzymatic
hydrolysis of polyamide. Different studies reveal that the adsorption of proteins follows
different steps and that the mechanical agitation plays an important role in all of them
(Cavaco-Paulo et al., 1996; Maldonado-Valderrama et al., 2005). Earlier investigation
with cellulases proved that higher mechanical agitations increased greatly the enzyme
performance at the fibers surface, although it can lead to an increase of the weight loss
(Azevedo et al., 2000; Araújo et al., 2007; Cavaco-Paulo and Almeida, 1994;
Maldonado-Valderrama et al., 2005; Cavaco-Paulo, 1998; Cavaco-Paulo and Almeida,
1994).
Different levels of mechanical agitation might lead to different levels of protein
adsorption and enzymatic hydrolysis. The surface properties have an enormous effect
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