Engineering a recombination neutral protease I from Aspergillus oryzae to improve enzyme activity at acidic pH

Extracellular neutral proteases (NPs) in Aspergillus oryzae (A. oryzae) play a role in hydrolyzing soybean proteins into smaller peptides at pH about 7.5. The optimum pH of moromi fermentation (The second stage of soy sauce fermentation.) is 4.5-5.5. NPI is acid sensitive. To decrease the pH optimum of NPI, we got a mutant NPI-Y122FK246ID382V from the error-prone PCR library that showed optimal activity at pH 5.5. The specific activity at 40 °C of the NPI-Y122FK246ID382V mutant was 1383.50 U mg^-1, which was 2.75-fold that of wild-type (503.09 U mg^-1). The Michaelis constants of the mutant decreased from 22.13 mM (wild-type) to 19.98 mM (NPI-Y122FK246ID382V). The residues at positions 122 and 246 are important in influencing hydrolytic activity at pH 5.5 through site-directed mutagenesis. And the pH optimum of double amino acid mutants (Y122FK246I) shifted dramatically to an acidic pH compared to those of single amino acid substitution. Molecular models and structural comparisons of native and mutant provided further insight on the basis to improve catalytic efficiency at acidic pH. These results indicated that we modified the neutral protease I of Aspergillus oryzae, which can effectively improve the application of the neutral protease in industrial production, and finally lay the foundation for improving the utilization rate of raw protein.