Effect of degumming conditions on the fluorescence intensity of fluorescent silk cocoons: A combined experimental and molecular dynamics study

Silk is a unique natural biopolymer with outstanding biocompatibility, high mechanical strength, and superior optical transparency. Due to its excellent properties, silk has been widely reported as an ideal biomaterial for several biomedical applications. Recently, fluorescent silk protein, a variant of native silk, has been reported as a biophotonic material with the potential for bioimaging and biosensing. Despite the realization of fluorescent silk, the traditional degumming process of fluorescence silk is crude and often results in fluorescence loss. The loss of fluorescent properties is attributed to the sensitivity of silk fibroin to temperature and solvent concentration during degumming. However, there is no comprehensive information on the influence of these processing parameters on fluorescence evolution and decay during fluorescent silk processing. Therefore, we conducted a spectroscopic study on fluorescence decay as a function of temperature, concentration, and duration for fluorescent silk cocoon degumming. Sodium carbonate solution was tested for degumming the fluorescent silk cocoons with different concentrations and temperatures; also, sodium carbonate solution is combined with Alcalase enzyme and triton x-100 to find optimal degumming conditions. Additionally, we conducted a molecular dynamics study to investigate the fundamental effect of temperature on the stability of the fluorescent protein. We observed degumming temperature as the prime source of fluorescent intensity reduction. From the MD study, fluorescence degradation originated from the thermal agitation of fluorescent protein Cα atoms and fluctuations of amino acid residues located in the chromophore region. Overall, degumming fluorescent silk with sodium carbonate and Alcalase enzyme solution at 25 ℃ preserved fluorescence.