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A Study of Type II ɛ-PL Degrading Enzyme (pldII) in Streptomyces albulus through the CRISPRi System
- Source :
- International Journal of Molecular Sciences, Vol 23, Iss 12, p 6691 (2022)
- Publication Year :
- 2022
- Publisher :
- MDPI AG, 2022.
-
Abstract
- ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25–35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the ε-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the ε-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the ε-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the ε-PL was changed after repression. The repression strain S1 showed a particular peak with a polymerization degree of 44, and other repression strains also generated ε-PL with a polymerization degree of over 40. Furthermore, the homology modeling and substrate docking of pldII, a typical endo-type metallopeptidase, were performed to resolve the degradation mechanism of ε-PL in S. albulus. The hydrolysis of ε-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of ε-PL.
Details
- Language :
- English
- ISSN :
- 14220067 and 16616596
- Volume :
- 23
- Issue :
- 12
- Database :
- Directory of Open Access Journals
- Journal :
- International Journal of Molecular Sciences
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.7016e390a3174e2889fb6848ef38964b
- Document Type :
- article
- Full Text :
- https://doi.org/10.3390/ijms23126691