Effects Of Grafting Of Polypeptide On The Enzyme-catalyzed Modification Of Silk Fibroin

As a kind of natural polymer, silk fibroin has a wide application in the field of biomedical materials owing to its satisfactory biocompatibility. Silk fibroin(SF) contains about 10% of tyrosine residues, however, some of them are huddled in the apolar micro-environments formed from the folded hydrophobic chains, which leads to the low restricted accessibility of tyrosine residues on the fibroin chains, and also affects the efficiency of enzymatic modification of silk fibroins. In the present work, grafting of a tyrosine-containing polypeptide was carried out using a natural crosslinking agent of genipin(GP), aiming at promoting the tyrosinase-catalyzed grafting efficiency of silk fibroins.The feasibility of cross-linking of amino compounds with GP was investigated, by means of SDS-PAGE and determination of primary amine groups. Meanwhile, the tyrosine-containing polypeptide was designed and used for fibroin modification, with the sequence of GKGYGGYGK. The changes in the molecular structure of polypeptide before and after GP treatment were explored by means of UV-Vis, fluorescence spectrum, GPC and MALDI-TOF-MS. Furthermore, the grafting efficacy of the polypeptide on the surface of silk fibroin was evaluated. After incubating silk fibroin with polypeptide and GP, the fibroin membranes were prepared. The molecular structure, thermal stability and mechanical properties for the fibroin membranes were investigated, respectively, by means of FTIR and TG-DTG. The obtained biocompatibility for the fibroin material was also concerned. After grafting of the polypeptide onto fibroin with GP, the combined treatment of fibroin membrane with tyrosinase and ?-Poly-L-Lysine(?-PL) was carried out. The grafting efficiency of ?-PL, amino acid composition and antibacterial property for the modified fibroin material were examined, the enzymatic reactivity of the silk fibroin were eventually evaluated.The results indicated that GP could promote the self-crosslinking of amine compounds, resulting in the increase of molecular weight and decline of the amount of free amino groups. GP treatment led to the intramolecular or intermolecular crosslinking of polypeptide, which produced various crosslinking products with gardenia blue appearance. When the concentration of GP was 30%(m/m), the covalent reaction between silk fibroin and polypeptide occurred efficiently at 37?, which induced the increase in the molecular weight of fibroin and polypeptide complex. This evidently verified the grafting of polypeptides on the fibroin surface. Thermal stability and mechanical properties of silk fibroin membrane increased after crosslinking with polypeptide. Based on the above work, the reactivity of silk membrane containing polypeptide was further investigated, via tyrosinase-catalyzed grafting of ?-PL. The results showed that the content of ?-PL for the polypeptide-grafted fibroin membrane was noticeably higher than that of the control during the enzymatic reaction. Meanwhile, the antibacterial effect for the modified fibroin membrane exhibited higher abilities against Escherichia coli(gram-negative bacteria) and Staphylococcus aureus(gram-positive bacteria), the biocompatibility was also satisfactory, and implying grafting of tyrosine-containing polypeptide on the fibroin surface can remarkably improve the enzymatic reactivity of silk fibroins.