Surface Modification of Cellulose Acetate with Cutinase and ...

Influence of Mechanical Agitation on Cutinases and Protease Activity Toward Polyamide Substrates
disappearance of the water-mirror on the surface (in other words the time for the water
drop to lose its reflective power) was measured as the wetting time. This procedure was
applied to both untreated and treated fabrics.
2.6. Enzymatic hydrolysis of polyamide model substrate (trimmer)
2.6.1. Cutinases
In the first part of the work, a native and a mutated cutinase (L182A) were used to
incubate the polyamide trimmer. Two sets of experiments were performed where 0.01 g
of a polyamide model substrate was incubated in two different solutions. The first
solution contained 10 mL of phosphate buffer (pH 7.5) and 35 U (μmol of p-NP per
min)/mL of native cutinase and the second solution contained the same amount of
buffer and 47 U (μmol of p-NP per min)/mL of cutinase mutant (L182A). Both
experiments were performed at 35 ºC for 8 hours under continuous shaking (using an
AHIBA Spectradye, from Datacolor, with vertical agitation). At different periods of
incubation, the total protein content in the solution was determined as described in
section 2.2. After 8 hours of incubation, a protein precipitation step was performed and
the primary amino groups resulting from enzymatic hydrolysis were quantified by the
TNBS method (Silva et al., 2004).
2.6.2. Protease
On this set of experiments, 10 mL of Tris-HCl buffer (pH 7.6) containing 18 U (μmol of
Tyrosine per min)/mL of protease were incubated with 0.01 g of model substrate under
the same conditions already described for cutinases. At different periods of incubation,
the total protein content in solution was determined as described in section 2.2. After 8
hours of incubation, a protein precipitation step was performed and the primary amino
groups resulting from enzymatic hydrolysis were quantified by the TNBS method (Silva
et al., 2004).
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