<i>Tenebrio molitor</i> ‐Derived Enzyme Systems Enable Solvent‐Reduced Recovery of Intracellular Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are intracellular microbial polyesters whose commercial deployment is strongly influenced by downstream processing costs and environmental burden. Here, we demonstrate a nearly solvent-free, mild aqueous strategy for the recovery of PHAs directly from wet bacterial biomass using crude enzyme systems derived from Tenebrio molitor. Under optimized conditions (0.6 wt% crude protein, pH 7.6, 40°C), near-quantitative (≥ 95%) recovery of PHB and PHB/HV and up to 60% recovery of mcl-PHA were achieved without prior biomass drying. Proteomic analysis identified abundant digestive hydrolases, including α-amylase and cathepsin and recombinant validation confirmed their contribution to polymer release, demonstrating that targeted enzyme combinations can substantially enhance mcl-PHA recovery (up to 94%-98%). Gel permeation chromatography, NMR and thermal analysis demonstrated preservation of polymer molecular integrity and crystallinity comparable to chloroform extraction. Life cycle assessment revealed a three- to seven-fold reduction in carbon footprint relative to conventional solvent and classical enzymatic methods, primarily due to elimination of drying and solvent use. These findings establish biologically driven biomass hydrolysis as a scalable downstream strategy and highlight insect-derived enzyme systems as promising tools for integrated microbial biopolymer processing.