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

Area of Science:

• Biocatalysis and Protein Engineering
• Biotechnology
• Enzyme Technology

Background:

• Protein self-assembly is a key strategy for developing robust biocatalysts.
• Industrial processes often require enzymes with enhanced stability and recyclability.
• Lysine decarboxylase (CadA) is a valuable enzyme for biotransformation.

Purpose of the Study:

• To engineer a self-assembling lysine decarboxylase (CadA) using a linker-mediated strategy.
• To optimize linker architecture for enhanced enzyme stability and performance.
• To establish a generalizable method for creating industrially viable biocatalysts.

Main Methods:

• Fusion of CadA with the amphiphilic 18A peptide using three distinct linker architectures: flexible (GGSGG)n, rigid (EAAAK)n, and protease-targeting (PT).
• Systematic evaluation of linker performance through activity assays, thermostability tests, and operational stability assessments.
• Structural analysis to elucidate the mechanism of self-assembly and active site accessibility.

Main Results:

• The protease-targeting (PT) linker enabled optimal CadA-18A self-assembly, yielding a 4.1-fold increase in thermostability at 60 °C.
• The engineered enzyme retained 91.7% of its native activity and showed broadened pH adaptability (5.0-9.0).
• The self-assembled biocatalyst demonstrated a higher substrate optimum (1.1 M lysine) and maintained 37% activity over 20 reuse cycles.

Conclusions:

• Linker-engineered self-assembly is an effective strategy for creating robust and recyclable biocatalysts.
• The CadA-PT-18A system offers improved performance for industrial biotransformation, especially for substrate-inhibited enzymes.
• This approach provides a generalizable paradigm for designing advanced enzyme systems.