Mutagenesis for Improvement of Activity and Stability of Prolyl Aminopeptidase from Aspergillus oryzae.

Abstract

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 3Å 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 127Umg-1, 156Umg-1, and 120Umg-1, respectively, which were 27%, 56%, and 20% higher than those of the wild type, with a value of 100Umg-1. The half-life of thermostability of the mutant 3D9 was 4.5h longer than that of the wild type at 50°C. The mutant C185V improved thermostability and had a half-life 2h 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.