Enzymatic Halogenation of Silk Fibroin from Bombyx mori

Silk fibroin from the silkworm, Bombyx
mori, is a unique biomaterial that has been extensively
studied for a
variety of applications that utilize robust mechanical properties,
biological compatibility, and controlled self-assembly properties.
This study tested carbon–halogen (C–X) bond halogenation
to alter the chemical composition of silk fibroin with the intention
to generate novel functional materials. In brief, silk fibroin side-chain
modification used halogen salts (NaX, X = Cl, Br, and I), hydrogen
peroxide (H2O2), and the vanadium-dependent
haloperoxidase from Curvularia inaequalis to produce primarily halogenated tyrosine residues along the amorphous
regions of the silk fibroin protein. Halogenation was confirmed with
various methods, including 1D 1H NMR, X-ray photoelectron
spectroscopy, and analysis of chymotrypsin peptide digests by Q-TOF
Liquid Chromatography–Mass Spectrometry. Secondary structure
analysis by FTIR-ATR, circular dichroism, and Raman spectroscopy revealed
increase in helical conformation of solubilized halogenated silk fibroin,
while dried film functionality demonstrated higher abundance of β-sheet
structures by maintenance of random coil content. Evaluation by contact
angle measurement demonstrated increased hydrophilicity on silk fibroin
films following addition of halogens by supporting the formation of
water insoluble hydrogels after treatment with various organic and
salt solvents. This study is the first to characterize the effects
of enzymatic halogenations on the properties of silk fibroin, and
this post-translational modification will be useful for the addition
of non-natural small molecules or ligands, introducing new material
types afforded by the silk fibroin structure.