Enhancing leucine aminopeptidase activity in Aspergillus oryzae LL1 through multi-round UV irradiation mutagenesis with potential application in debittering.

To enhance the applicability of Aspergillus oryzae LL1 in the food industry, the strain underwent ultraviolet irradiation through random mutagenesis to enhance the enzyme activity of extracellular leucine aminopeptidase (LAP). A. oryzae LL1 was subjected to three rounds of ultraviolet mutagenesis, and a number of 210, 280, and 290 mutant clones were selected based on LAP activity and stability analysis. The mutant clone with the highest LAP activity was selected for each mutagenesis, and then proceeded to the next round of mutagenesis. The activity of the three selected mutant strains showed significantly increased LAP activity compared to the wild-type strain. The LAP specific activity of A. oryzae LL1 was 319.9 mU/mg. The LAP specific activities of the mutant strains U01, U02, and U03 were 386.3, 449.9, and 556.5 mU/mg, respectively. After three rounds of mutagenesis, the U03 strain exhibited 1.74-fold greater specific activity of LAP than the LL1 strain. A further study evaluated the debittering effect of crude enzyme of the mutant strain U03 on isolated soy protein through hydrolysis and a decrease in the content of hydrophobic amino acids. The mutant strain U03 demonstrated a significantly higher efficiency in hydrolyzing soy protein compared to the LL1 strain, as evidenced by the degradation into small peptide fragments observed in SDS-PAGE analysis, the significant increase in peptide peaks in reverse-phase HPLC analysis, and the higher total free amino acid contents. These results suggest that the U03 strain exhibits more effective hydrolytic activity and holds potential for applications in the food industry, particularly in reducing bitterness in protein hydrolysates.