Design of Experiments Investigation of Sericin Acetylation Using a Quantitative FTIR Approach

Sericin, a silk-derived protein recovered as a by-product of the textile industry, is a renewable biomacromolecule with considerable potential for sustainable material development. Chemical modification represents an effective strategy for its valorization. In this study, acetylation using acetyl chloride (AcCl) was selected as a model reaction to systematically investigate the reactivity of sericin in an N,N-dimethylacetamide/lithium chloride (DMA/LiCl) solvent system and to evaluate the influence of reaction parameters on both the extent of functionalization and protein secondary structure. A Design of Experiments (DoE) strategy, comprising an initial full factorial screening followed by Box-Behnken optimization, was employed to investigate the effects of AcCl equivalents, sericin concentration, and LiCl content. A quantitative FTIR workflow based on constrained Gaussian deconvolution was developed to derive functionalization index (FI) and β-sheet index (BI) from both peak areas and peak intensities, enabling the simultaneous evaluation of chemical modification and structural organization. The exploratory screening identified AcCl as the dominant factor governing sericin functionalization. During the optimization phase, the FI models described the general response trends, whereas the BI was successfully represented by robust quadratic models (R² = 0.977-0.978; adjusted R² = 0.936-0.938), revealing significant linear, interaction, and quadratic effects, with LiCl concentration and sericin concentration playing key roles in governing structural organization. The analytical workflow was verified by reproducibility assessment and independent validation experiments. Overall, this study proposes a quantitative DoE-FTIR framework for systematically investigating sericin functionalization and its associated structural evolution, providing support for future studies aimed at sericin industrial valorization.