Research on Enhancing the Activity of Streptomyces noursei Polylysine Synthetase through Rational Design Methods

Abstract

ε-Polylysine synthetase (ε-Poly-L-lysine synthetase, Pls) is a novel single-module non-ribosomal The activity of peptide synthetase (NRPS) is an important factor affecting the synthesis degree of ε-polylysine (ε-Poly-L-lysine, ε-PL). The laboratory obtained Pls from Streptomyces nourans, S. albulus NBRC 14147, but its Less active. This project is based on rational design methods and explores the purpose of locating key site mutations through structural analysis to improve its activity. The experiment uses the homology modeling tool SWISS-MODEL to construct the three-dimensional structure of the original and mutant Pls; through Pymol. The visualization software observes and analyzes the structural characteristics of the key regions of Pls to determine potential mutation sites; it uses molecular docking methods and molecular dynamics simulation methods to test the theoretical effects of mutations. Key region structure analysis found that there are many amino acid residues with larger side chains on the Pls active region channel, which has a greater impact on the entry and exit of small substrate molecules into the active region. steric hindrance. Based on this discovery, this project intends to improve its activity by reducing the steric hindrance near the reaction channel. After further analysis and screening, the four key residues Arg370, Arg401, Asp404 and Lys499 were finally determined as mutations location. Molecular docking and molecular dynamics analysis results show that in addition to the Lys499 site, the binding ability of other site mutants to substrate molecules has been improved to a certain extent, among which Asp404 mutant has the strongest binding ability to substrate, Arg401 mutant comes second, Arg370 Mutants third. The results of this paper deepen our understanding of the catalytic mechanism of ε-polylysine synthase, and provide a theory for the laboratory engineering of polylysine synthase protein. Guidance to facilitate the industrial production of ε-polylysine.