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)

Research output: Contribution to journalArticleScientificpeer-review

89 Citations (Scopus)

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
Number of pages13
JournalJournal of Biotechnology
Volume116
Issue number1
DOIs
Publication statusPublished - 2005
MoE publication typeA1 Journal article-refereed

Fingerprint

Fibroins
Bombyx
Silk
Chitosan
Monophenol Monooxygenase
Agaricales
Kinetics
Quinones
Tyrosine
Infrared spectroscopy
Spectrum Analysis
Schiff Bases
Polysaccharides
Grafts
Solubility
Amino acids
High Pressure Liquid Chromatography
Transplants
Amino Acids
Oxidation

Keywords

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

Cite this

Sampaio, Sandra ; Taddei, Paola ; Monti, Patrizia ; Buchert, Johanna ; Freddi, Giuliano. / Enzymatic grafting of chitosan onto Bombyx mori silk fibroin : Kinetic and IR vibrational studies. In: Journal of Biotechnology. 2005 ; Vol. 116, No. 1. pp. 21 - 33.
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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.",
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Enzymatic grafting of chitosan onto Bombyx mori silk fibroin : Kinetic and IR vibrational studies. / Sampaio, Sandra; Taddei, Paola; Monti, Patrizia; Buchert, Johanna; Freddi, Giuliano (Corresponding Author).

In: Journal of Biotechnology, Vol. 116, No. 1, 2005, p. 21 - 33.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Enzymatic grafting of chitosan onto Bombyx mori silk fibroin

T2 - Kinetic and IR vibrational studies

AU - Sampaio, Sandra

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AU - Freddi, Giuliano

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AB - 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.

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