Abstract / Summary
Abstract Background Aminopeptidases specifically cleave N-terminal amino acid residues and have broad applications in flavor modification, bioactive peptide preparation, and biochemical diagnosis. Lysinibacillus sphaericus metabolizes amino acids rather than carbohydrates for energy. Amp0279 was previously identified as a leucyl aminopeptidase from this bacterium. Results The newly characterized Amp0293 originating from L. sphaericus shares a conserved M29 peptidase catalytic domain, homodimeric structure, and Co²⁺- stimulated catalytic properties with Amp0279. Both enzymes have an identical optimal pH of 7.5, with optimal temperatures of 50 °C and 40 °C and optimal Co²⁺ concentrations of 1 mM and 0.1 mM, respectively. For L-Arginine- 4 -nitroanilide (Arg-pNA) and L-leucine- 4 -nitroanilide (Leu-pNA), Amp0293 shows stronger substrate affinity and better substrate-inhibition tolerance toward Arg‑pNA, while Amp0279 possesses higher catalytic efficiency toward Arg-pNA but shows comparable catalytic performance against Leu-pNA. Under specific conditions, their Co²⁺-free specific activities toward Arg-pNA exceed those of the commercial benchmark Flavourzyme ® . Recombinant Corynebacterium glutamicum strains harboring the AI-designed promoter P Ha achieved the highest intracellular expression levels (1.1 and 1.9 µg/mg CDW) and enzyme activities (19.9 and 11.8 U/mg CDW without Co²⁺ addition). Secretory expression of the two enzymes was further achieved using CspB2 and TorA signal peptides, yielding crude enzyme activities of 5.4 U/mg CDW for Amp0279 and 3.0 U/mg CDW for Amp0293, respectively, without Co²⁺ addition. Conclusion This study characterizes two cobalt-stimulated, arginine-preferring aminopeptidases. It establishes efficient intracellular and secretory expression systems in C. glutamicum , providing a solid foundation for their industrial application.