Structure modifications induced in silk fibroin by enzymatic treatments. A Raman study

Raman spectroscopy was used to investigate various enzyme-catalyzed reactions onto silk fibroin, i.e. the biodegradation of Tussah (Antheraea pernyi) silk fibroin films by a proteolytic enzyme, the oxidation of domestic (Bombyx mori) silk fibroin by mushroom tyrosinase and the subsequent grafting of chitosan onto oxidized silk. The spectra of Tussah silk fibroin films exposed to a bacterial protease for different times demonstrated that the cleavage of sensitive peptide bonds in the amorphous glycine-rich domains resulted in the loss of various amino acid residues (Tyr, Trp, Asp, etc.). The bands attributed to the crystalline alanine-rich sequences increased in intensity, and the β-sheet molecular conformation was not affected by biodegradation. Following oxidation with mushroom tyrosinase, the tyrosine bands of Bombyx mori fibroin decreased in intensity but did not disappear. The increase of the I853/I829 intensity ratio indicated that the Tyr residues not accessible to the enzyme were located in a strongly hydrophobic environment. Raman spectroscopy provided evidence that chitosan was effectively grafted onto oxidized silk, probably via the Schiff-base mechanism, as shown by the behavior of the imine band at about 1646 cm−1. Grafting chitosan onto silk fibroin resulted in a β-sheet→random coil conformational transition of the protein component in the bioconjugated product.