Page 218 - Advances in Textile Biotechnology

Functionalisation of wool and silk fi bres using enzymes 199

The enzyme technology was exploited by Chiono et al. (2008) to produce
melt-extruded guides for peripheral nerve regeneration by blending poly-
(ε-caprolactone) (PCL) and gelatine. In one approach, gelatine was cross-
linked with microbial TGase and then blended with PCL by solution mixing
for the production of nerve guidance channels. In another approach, micro-
bial TGase was used to graft poly-l-Lys, a biomimetic molecule able to
confer to the channel guide a specifi c signalling for nerve cells attachment,
proliferation and migration. Binding of poly-l-Lys to the exposed gelatine
sequences in the inner surface of the nerve channel was proved by confocal
microscopy, whereas in vitro tests with neuroblastoma cells showed that the
nerve guides produced by melt extrusion were biocompatible and that the
grafted signalling peptide greatly improved cell response. The same research
group has recently reported the production of three-dimensional porous
hydroxyapatite/collagen composite scaffolds for bone tissue regeneration
characterised by enhanced stability and controllable swelling ratio by cross-
linking via microbial TGase-catalysed reaction (Ciardelli et al., 2010).
An alternative approach in the use of TGase for tissue engineering is to
modify the surface of biomaterials by coating with the enzyme in order to
exploit its ability to stimulate cell adhesion processes. Ball et al. (2009) used
TGase in conjunction with fibronectin as a coating for tricalcium phosphate

scaffolds and studied their effect on metabolic activity and other physiolog-
ical parameters, including the rate and extent of mineralisation of osteoblast
cells seeded on the scaffold surface. They found that fi bronectin supported
early cell differentiation, whereas increased matrix formation or stabilisa-
tion was induced by TGase, probably through nucleation of mineralisation
promoted by the TGase crosslinked extracellular matrix.

Site-specifi c modification of proteins

Enzymatic approaches to site-specifi c modification of proteins have
attracted a great deal of attention in biological research because this mod-

ification strategy enables protein manipulation without significant loss of
function, it is highly selective and works under mild conditions compared
with conventional chemical methods. Microbial TGase has been widely

applied for site-specifi c modification, crosslinking, and conjugation of pro-
teins with other biological molecules (e.g. DNA, carbohydrates). If protein
substrates are not intrinsically reactive to microbial TGase because of
reduced accessibility of target amino acid residues, the incorporation of

specific peptide tags for enzyme recognition allows enhancing substrate
specificity and reaction yield. By using an amino-derivative of poly(ethylene

glycol) (PEG) as substrate for the enzymatic reaction with TGase, it
has been possible to covalently bind the polymer to proteins of pharm-
aceutical interest thus making these protein drugs more water soluble,

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