AA10 LPMO Homologues as Scaffolds for Engineered Inclusion Bodies and Carrier-Free Biocatalysts

Abstract

Traditional enzyme immobilization strategies often rely on chemical crosslinkers or solid carriers, thereby increasing processing complexity and potentially compromising catalytic efficiency. Here, we present a carrier-free approach for generating stable biocatalytic particles by exploiting the intrinsic aggregation behavior of four phylogenetically distinct AA10 LPMO homologues (Kpapp40, Karip40, Alipp40, and Psufp40) as scaffolds for catalytically active inclusion bodies (CatIBs) in Escherichia coli. Each AA10 variant was genetically fused to either mCherry or a thermostable Bacillus α-amylase (BacAmy) and expressed in E. coli BL21(DE3), resulting in the predominant formation of insoluble protein inclusion bodies (IBs). Protein partitioning was quantified by SDS–PAGE densitometry, intracellular localization by confocal microscopy, particle size and morphology by dynamic light scattering and FESEM, and secondary structure by FTIR spectroscopy. All variants assembled into submicron, structured aggregates with hydrodynamic diameters ranging from 620 to 824 nm and were enriched in α-helical and β-sheet secondary structure, consistent with the formation of structured aggregates rather than extensive amorphous misfolding. mCherry IBs retained fluorescence and displayed polar localization in vivo, while BacAmy CatIBs exhibited maximal catalytic activity at 80 °C, maintained substantial activity up to 95 °C, and demonstrated broad pH tolerance with pronounced pH stability from a slightly acidic to a mild alkaline range. FTIR analysis showed that BacAmy CatIBs contained 47–54% α-helical structure, while mCherry IBs contained 42–45% α-helical structure, indicating the preservation of partially native protein conformations within the aggregated state. Differences among variants influenced particle size, dispersity, and aggregate morphology. These findings demonstrate the potential of AA10 LPMO domains as versatile structural modules for engineering thermostable, carrier-free biocatalysts and provide a foundation for expanding their application beyond oxidative polysaccharide cleavage toward sustainable enzyme material design.