Page 235 - Advances in Textile Biotechnology

216 Advances in textile biotechnology

-(Gly-Ala) n - sequences (R) responsible for the formation of the more
ordered and crystalline regions in both regenerated silk fi broin materials

and fibres. It is reasonable to assume that these residues were hardly acces-
sible to A. bisporus tyrosinase owing to steric hindrance. The remaining Tyr
residues are located in the amorphous (A) and in the N- and C-terminus
sequences, which are characterised by a less regular amino acid sequence
and by a loose arrangement of the chain segments. When regenerated

fibroin materials such as gels are immersed in aqueous solution the A-rich
sequences can swell quite easily thus favouring the diffusion of the enzyme
towards the Tyr residues. About a half of these theoretically accessible
tyrosyl residues of fibroin gels were oxidised by tyrosinase.

When fi broin substrates in powder (mechanically generated from fi bres)

or fibre form were incubated with mushroom tyrosinase, the yield of oxida-

tion was very low or undetectable. In general, silk fibres display a high
degree of inertness towards enzymes owing to a combination of morpho-
logical and structural factors. In practice, after removal of sericin, the surface
of silk fibres appears very smooth, without any gap to the inside, which

makes it very difficult for the enzyme molecules to find paths for diffusing

into the fibre matrix. In addition to morphology, structural features of silk
fibres, such as the close packing of the fibroin chains and their high degree

of order, orientation, and crystallinity, also hinder penetration and diffusion
inside the fibre of relatively large molecules like enzymes. In contrast to

wool, where the scaled surface structure and the continuous, poorly cross-
linked CMC phase allow paths for enzymes to penetrate inside the fi bre
and to reach the target amino acids, only amino acid residues available at
the surface of silk fibres can be reasonably considered to be accessible to

enzymes. The behaviour of wool and silk with proteases supports this
assumption. Both fibres are susceptible to proteolytic degradation, but pro-

teases hydrolyse more readily wool than silk. Large decreases in strength

and weight are suffered by wool fibres upon exposure to proteases for
relatively short times (Shen et al., 1999), whereas it takes days to detect

extensive fibre degradation of silk (Arai et al., 2004). Accordingly, A. bispo-
rus tyrosinase was able to oxidise wool fi bres (Jus et al., 2008), but failed to
oxidise tyrosyl residues of silk fi bres (Freddi et al., 2006). A protease pre-
treatment lasting up to 14 days was slightly effective in opening the fi bre
texture and enhancing the accessibility of surface-available Tyr residues
(Freddi et al., unpublished results). Hence, these results demonstrate that
the accessibility of reactive amino acid residues plays a key role in deter-
mining the outcome of enzymatic reactions on protein fi bres.

FTIR and Raman measurements confirmed that tyrosinase oxidised the
accessible Tyr residues of silk fibroin and sericin and converted them into

reactive quinones and corresponding tautomers (Taddei et al., 2007). The
intensity of the Tyr Raman bands decreased upon oxidation. The Raman

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