Applied Biochemistry and Biotechnology, Vol.191, No.4, 1483-1498, 2020
Mutagenesis for Improvement of Activity and Stability of Prolyl Aminopeptidase from Aspergillus oryzae
In this study, the prokaryotic expression system of Escherichia coli was used to modify prolyl aminopeptidase derived from Aspergillus oryzae JN-412 (AoPAP) via random mutagenesis and site-directed saturation mutagenesis. A random mutant library with a capacity of approximately 3000 mutants was compiled using error-prone polymerase chain reaction, and nonconservative amino acids within 3 angstrom of the substrate L-proline-p-nitroaniline were selected as site-directed saturation mutagenesis sites via homologous simulation and molecular docking of AoPAP. Variants featuring high catalytic efficiency were screened by a high-throughput screening method. The specific activities of the variants of 3D9, C185V, and Y393W were 127 U mg(-1), 156 U mg(-1), and 120 U mg(-1), respectively, which were 27%, 56%, and 20% higher than those of the wild type, with a value of 100 U mg(-1). The half-life of thermostability of the mutant 3D9 was 4.5 h longer than that of the wild type at 50 degrees C. The mutant C185V improved thermostability and had a half-life 2 h longer than that of the wild type at a pH of 6.5. Prolyl aminopeptidase had improved stability within the acidic range and thermostability after modification, making it more suitable for a synergistic combination with various acidic and neutral endoproteases.