Page 224 - Advances in Textile Biotechnology

Functionalisation of wool and silk fi bres using enzymes 205

−1
estimated to be about 70 μmol g (∼0.5 mol%) based on the known amino

acid sequence of the three fibroin polypeptides. Thus, the Gln content in
silk is much lower than that of wool. The content of Lys is also low, account-
−1
ing for about 40 μmol g (∼0.3 mol%). To verify whether the Gln residues

of silk fibroin could be used by TGase for transamidation reactions, an

aqueous silk fibroin solution was incubated with microbial TGase from
S. mobaraensis and then films were cast. When dissolved in aqueous solu-

tion, silk fibroin adopts a disordered random coil conformation with the

more hydrophilic polypeptide sequences exposed to the aqueous phase
(Canetti et al., 1989). This is considered the most favourable condition for
the accessibility of charged amino acid residues like Gln and Lys. However,
no detectable differences were observed in the chemical, physical, and
mechanical properties of silk fi broin films upon incubation with TGase with

respect to the control films cast without enzyme (Freddi et al., unpublished
results). Noteworthy, another enzyme, i.e. the mushroom tyrosinase from
Agaricus bisporus, under the same reaction conditions, had signifi cant
effects on silk fibroin properties (see section 9.4.2, Applications of tyrosin-

ases: advantages and limitations). These results seem to indicate that the
Gln residues of silk fibroin are hardly accessible to TGase, even under

homogeneous reaction conditions. Based on these findings it is possible to

assume that silk fibroin in the soluble state and, all the more so, in the form
of intact fibres, might not be a good substrate for TGase-catalysed modifi ca-

tion and/or functionalisation.
9.4 Tyrosinases
9.4.1 Biological properties and biotechnological utility
of tyrosinases

Tyrosinases (monophenol, o-diphenol: oxygen oxidoreductase, EC 1.14.18.1)
belong to the type 3 copper proteins having a diamagnetic spin-coupled Cu
pair in the active centre (Rosenzweig and Sazinsky, 2006; Solomon et al.,
1996). Tyrosinases, which are often referred to as polyphenol oxidases
(PPO), can catalyse both the hydroxylation of tyrosine (Tyr) into o-diphe-
nols and the oxidation of o-diphenols into o-quinones (Fig. 9.2). In the fi rst
reaction, often referred to as the monooxygenase or cresolase activity, which
is known to be the rate-determining step, a hydroxyl group is introduced
into the ortho-position of the aromatic ring. In the second reaction, often
referred to as the diphenolase or catecholase activity, the o-dihydroxy com-
pound produced in the first step is oxidised to an o-quinone. Both reactions

involve molecular oxygen as an electron acceptor which is reduced to water.
Quinones are molecular species highly susceptible to non-enzymatic reac-
tions leading to formation of brown or black pigments such as melanins.

© Woodhead Publishing Limited, 2010