Page 232 - Advances in Textile Biotechnology

Functionalisation of wool and silk fi bres using enzymes 213

lysates. In a first enzymatic step, the penta-Tyr tagged green fl uorescent
protein was bound to the stimuli-responsive polysaccharide chitosan via
tyrosinase-catalysed conjugation, thus enabling multiple washing steps for

protein purification. Then, the protein of interest could be released by
another enzymatic step involving either an enterokinase or a chitosanase.
More interestingly, Lewandowski et al. (2008) further developed the concept
of enzymatic coupling of proteins onto micropatterned surfaces upon tyros-
inase activation of Tyr-tagged proteins, leading to the production of bio-
functional microfabricated devices (see section 9.2.2).
Binding proteins without loss of biological activity (e.g. growth factors or
enzymes) onto the surface of drug delivery devices or tissue engineering

scaffolds is still a field of intensive research. Some authors have recently

developed a novel approach based on a highly specific coiled-coil capture/
release system to functionalise the surface of chitosan scaffolds with teth-
ered epidermal growth factor (EGF) (Demolliens et al., 2008). In this
approach, one of the coil partners (Kcoil) is coupled to chitosan via tyros-
inase-catalysed oxidation of a Tyr residue of the peptide sequence. Then the
EGF coil-tagged (Ecoil) protein is captured onto the surface of the chitosan
scaffold by formation of a stable K/E coiled-coil structure. This versatile
strategy for capturing proteins onto the surface of biomaterials, which
exploits the chemoselectivity of tyrosinase in one of the key steps of the
reaction protocol, seems very promising for the preparation of multifunc-
tional devices applicable in regenerative medicine.

9.5 Functionalisation of protein fi bres
using tyrosinases

The quinone-mediated crosslinking of biopolymers is the basis of many
biological processes dealing with the production of strong composite mate-
rials such as the exoskeleton of insects and the byssus threads of marine
mussels. Their astonishing chemical, physical, and mechanical properties
have stimulated researchers to exploit the quinone chemistry for the pro-
duction of biomimetic materials such as adhesives (Yamada et al., 2000 and
2008) and novel hybrid fi bres (Kuboe et al., 2004; Tonegawa et al., 2004).
Chitosan and poly-l-Lys N-substituted with the tetrapeptides ‘Lys-Gly-Tyr-
Gly’ were synthesised and blended with the anionic polysaccharide gellan,

and then fibres were spun with addition of tyrosinase to the spinning dope.
In this way, the polycation–polyanion hybrid fibres formed by polyionic

complexation could be further reinforced by enzymatic-assisted crosslink-
ing, as confirmed by the results of tensile measurements.

Mushroom tyrosinase from A. bisporus has been widely used to activate/

functionalise/crosslink protein fibres, e.g. wool and silk, as well as protein
fractions extracted from them (keratin hydrolysate, fibroin, and sericin

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