Self-Assembly enhance lysine decarboxylase biocatalysis performance based on rational linker engineering

Protein self-assembly offers a powerful approach to engineer robust biocatalysts capable of withstanding industrial process extremes while enabling cost-efficient enzyme recycling. Here, we developed a linker-mediated assembly strategy for lysine decarboxylase (CadA) by fusing its C-terminus with the amphiphilic 18A peptide. Through systematic evaluation of three linker architectures-flexible (GGSGG)_n, rigid (EAAAK)_n, and protease-targeting (PT)-we identified the PT linker as optimal for coupling CadA with 18A. Among the variants, the CadA-PT-18A fusion exhibited optimal assembly, demonstrating a 4.1-fold enhancement in thermostability (t₁/₂ at 60 °C) compared to the wild-type enzyme while retaining 91.7 % of its native activity. The self-assembled system displayed expanded operational limits, including broadened pH adaptability (5.0-9.0), a shifted substrate optimum from 600 mM to 1.1 M lysine, and maintained 37 % activity through 20 reuse cycles. Structural analysis revealed that the PT linker facilitated ordered aggregation via α-helix stacking of 18A peptides, minimizing steric clashes while preserving active site accessibility. This work established linker-engineered self-assembly as a generalizable paradigm for designing industrially viable biocatalysts, particularly for substrate-inhibited enzyme systems in biotransformation processes.