Optimized enzyme-microwave-assisted extraction and LC-MS/MS characterization of phycobiliproteins from the underutilized green alga Chaetomorpha aerea.

Phycobiliproteins (PBPs) are high-value fluorescent pigments widely used in biomedical and industrial applications, yet their efficient recovery from recalcitrant macroalgal biomass remains a challenge. This study established an optimized protocol for the extraction and purification of PBPs from the green macroalga Chaetomorpha aerea, an underutilized biomass with a recalcitrant cell wall structure. To overcome mass transfer limitations, a dual enzyme-assisted extraction (EAE) and microwave-assisted extraction (MAE) approach was employed. Using Response Surface Methodology (RSM), the extraction conditions were optimized to a 2.08% cellulase concentration (90 min incubation) followed by microwave irradiation at 270.06 W for 130.17 s. Under these conditions, the extraction yields reached 0.269 mg/mL for Allophycocyanin (APC), 0.404 mg/mL for C-Phycocyanin (C-PC), and 0.346 mg/mL for R-Phycoerythrin (R-PE). The crude extract was subsequently purified using ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography, achieving high purity indices of 2.23 ± 0.07 (APC), 4.21 ± 0.25 (C-PC), and 3.23 ± 0.11 (R-PE). LC-MS/MS analysis confirmed the identity of the purified pigments with high peptide confidence scores (≥ 98.5%). Furthermore, biological assays revealed that the purified PBPs possessed significant antioxidant potential and exerted dose-dependent cytotoxicity against MCF-7 breast cancer cells. These findings demonstrate that the integrated EAE-MAE strategy effectively enhances pigment recovery, positioning C. aerea as a sustainable source for functional foods, biotechnology, and pharmaceutical applications.