Alanine aminopeptidase from Bacillus licheniformis E7 expressed in Bacillus subtilis efficiently hydrolyzes soy protein to small peptides and free amino acids

Extensive proteolysis has become an important approach to add value to local bioresources. Here, we isolated novel strain Bacillus licheniformis E7 from protein-rich soil. This strain produces an alanine aminopeptidase, PepN, the encoding gene for which was cloned using genome walking technology. Recombinant PepN was expressed in B. subtilis 168—extra- and intracellular (secreted) forms of the enzyme were obtained (BlPepN^ex and BlPepNⁱⁿ, respectively), which showed different enzymatic properties. Their activities toward Ala-p-nitroaniline were 401.3 ± 16.9 U/mg and 113.8 ± 3.4 U/mg, respectively. The enzymes maintained >80% relative activity after incubation at 8.0–9.0 for 2 days, and >80% relative activity after incubation at 45 °C for 1 h. BlPepN^ex had an approximately fourfold higher k_cat/K_M value for Ala-p-nitroanilines than BlPepNⁱⁿ. When combined with an alkaline protease, BlPepN^ex and BlPepNⁱⁿ showed similar hydrolytic effects to those of a commercial aminopeptidase. The combinations hydrolyzed soybean protein to approximately 75% peptides of <500 Da and 45% peptides of <150 Da; >2000 mg/L free amino acids were also produced. This work supplies an effective, safe aminopeptidase for improving the degree of hydrolysis of protein-rich materials to release multitudinous peptides and amino acids.