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84 Advances in textile biotechnology

using the drop method; the results were 4 s for cutinase-treated fabrics,
>180 s for untreated fabrics, and ∼120 s for cutinase-treated fabrics and
>45 min for untreated fabrics. Results from the drop test are strongly
affected by the porosity of the fabric; this makes it diffi cult to compare the
results. Brueckner et al. (2008) demonstrated using XPS that, after enzy-

matic treatment, novel superficial carboxyl and hydroxyl groups are formed,
whereas a hydroxide treatment does not result in the formation of new

superficial groups. Donelli et al. (2008, 2009) assessed the surface changes

using attenuated total reflectance Fourier transform infrared spectroscopy
(ATR-FTIR). Donelli et al. (2008, 2009) successfully demonstrated that the
free carboxylic surface groups generated by enzymatic hydrolysis of PET
can be functionalized with functional molecules or groups. Müller et al.
(2005) and Vertommen et al. (2005) demonstrated that cutinase displays
higher activity towards amorphous PET and little activity towards highly
crystalline PET. The amount and ratio of hydrolysis products formed/
released from the PET surface [viz. terephthalic acid (TPA), mono(2-
hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephtha-
late (BHET), Fig. 4.1] depends on the enzyme, the source of the enzyme,
the substrate, the enzyme–substrate ratio, the enzyme concentration and
the progress of the reaction (Brueckner et al., 2008; Hooker et al., 2003;
Vertommen et al., 2005). Despite the positive results obtained (increased

hydrophilicity, significant depilling, increased reactivity with cationic dyes,
and a small weight decrease) a better understanding of the action of cuti-
nase on PET, the interaction of cutinase with PET and protein removal is
required in order to make the potential of cutinase technology available
for industry.

Analytical techniques

To monitor the changes resulting from the cutinase treatment a large variety

of analytical techniques exist: techniques to assess the modifications of the
PET itself and to determine the hydrolysis products released. When assess-
ing surface properties, it is necessary to remove adsorbed cutinase thor-
oughly (Vertommen et al., 2005).
Brueckner et al. (2008), Vertommen et al. (2005) and Yoon et al. (2002)
used high-performance liquid chromatography (HPLC) to detect hydroly-
sis products formed (TPA, MHET and BHET), whereas O’Neil and Cavaco-
Paulo (2004) used a spectroscopic assay to determine the release of TPA.
Scanning electron microscopy (SEM) (Brueckner et al., 2008; Feuerhack
et al., 2008; Kim et al., 2006, 2008), XPS (Brueckner et al., 2008; Vertommen
et al., 2005) and ATR-FTIR (Donelli et al., 2008, 2009) have been used to
analyse surface hydrolysis and surface properties. Donelli et al. (2008, 2009)
and Brueckner et al. (2008) measured hydrophilicity using contact angle

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