Functional and structural characterization of a serine protease from Bacillus toyonensis co-expressed with short peptides from late embryogenesis abundant (LEA) proteins

The increasing demand for high-performance proteases in industrial applications necessitates strategies to enhance enzyme yield, stability, and functional robustness under extreme conditions. This study investigates a serine protease from Bacillus toyonensis , isolated from fermented kitchen waste, and its co-expression with short peptides derived from late embryogenesis abundant (LEA) proteins, LEA-I, and LEA-K. The protease gene was cloned into a pRSFDuet-1 vector and expressed in Escherichia coli BL21(DE3), followed by purification using immobilized metal affinity chromatography. Co-expression with LEA peptides significantly enhanced protease production and stability, with ProLEA-K achieving the highest purification fold (1,638.00) and specific activity (16.38 U/mg) compared to 0.01 U/mg in the crude extract. Functionally, ProLEA-K exhibited a 2.5-fold increase in activity at 60°C and retained substantial activity up to 70°C, whereas the protease expressed alone (ProWL) declined sharply at elevated temperatures and maintained high activity across a broad pH range spanning acidic to alkaline conditions, a rare feature for acidic serine proteases. Evaluation under detergent-containing conditions revealed a differential response, whereby detergents enhanced the activity of ProWL but reduced the activity of LEA-co-expressed variants, indicating a context-dependent effect of LEA peptides on enzyme performance. In silico analysis supported these experimental findings. Multiple sequence alignment revealed strong conservation of the catalytic triad (Asp171, His203, and Ser225) and an amino acid profile consistent with secreted bacterial enzymes. Structural modeling confirmed stable folding, and molecular docking with azocasein indicated stronger substrate binding in LEA-containing variants (−6.9 kcal/mol for ProLEA-K vs −5.8 kcal/mol for ProWL). These results identify LEA peptides, particularly LEA-K, as effective molecular stabilizers, improving both the yield and functional robustness of industrial proteases.

IMPORTANCE

This work highlights a simple and effective approach to improve industrial proteases using short late embryogenesis abundant peptides as natural stabilizers. The method enhanced yield, thermal stability, and pH adaptability across acidic and alkaline conditions without genetic redesign of the protease itself, offering a broadly applicable and economical strategy for enzyme improvement.