Enzymatic grafting of chitosan onto Bombyx mori silk fibroin: Kinetic and IR vibrational studies

Sandra Sampaio, Paola Taddei, Patrizia Monti, Johanna Buchert, Giuliano Freddi (Corresponding Author)

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Abstract

The potential for using tyrosinase to graft the polysaccharide chitosan (Ch) onto Bombyx mori silk fibroin (SF) was examined. FT-IR spectroscopy coupled to HPLC amino acid analysis showed that mushroom tyrosinase (MT) catalyses the oxidation of tyrosine (Tyr) of SF to electrophilic o-quinones. Kinetic studies showed that only a fraction of the Tyr residues available on the SF chain were oxidized. This result was interpreted in the light of the structure assumed by SF in aqueous solution: Tyr aromatic side chain groups buried into the folded hydrophobic portions of the chain were probably less accessible to MT for steric reasons. Using slightly acidic conditions (pH 6), it was possible to modify SF under homogeneous conditions. FT-IR spectroscopy provided evidence that Ch was grafted onto MT-oxidized SF: the o-quinones were found to undergo a subsequent non-enzymatic reaction with nucleophilic amino groups of Ch via Schiff-base and Michael addition mechanisms. Various factors, i.e. reaction time, pH, MT/SF ratio, were found to influence the grafting yield. The highest grafting yield was achieved at pH 7, i.e. more favorable to MT activity rather than to Ch solubility, suggesting that the determining step of the grafting reaction is the formation of o-quinones. The FT-IR spectroscopy revealed that grafting induced a β-sheet → random coil conformational transition.
Original languageEnglish
Pages (from-to)21-33
JournalJournal of Biotechnology
Volume116
Issue number1
DOIs
Publication statusPublished - 2005
MoE publication typeA1 Journal article-refereed

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Keywords

  • Chitosan
  • Bombyx mori silk fibroin
  • Tyrosinase
  • Infrared spectroscopy

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