Journal
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 126, Issue -, Pages 229-237Publisher
ELSEVIER
DOI: 10.1016/j.ijbiomac.2018.12.218
Keywords
Pseudomonas aeruginosa; Serine protease; Protein engineering; Site-directed mutagenesis; Thermostability; Catalytic efficiency
Funding
- Higher Education Commission of Pakistan [NRPU-4571]
- International Research Support Initiative Program (IRSIP), HEC
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The study involves the isolation and characterization of a serine peptidase, named SP, from Pseudomonas aeruginosa. In addition to basic characterization, the protein was engineered, by site-directed mutagenesis of selected non-catalytic residues, to increase its thermal stability and catalytic activity. Among the eight-point mutations, predicted by FireProt, two mutants, A29G and V336I, yielded a positive impact. The T-m of A29G and V3361 showed an increase by 5 degrees C and also a substantial increase in residual activity of the enzyme at elevated temperature. Moreover, the catalytic activity of A29G and V3361 also showed an increase of 1.4-fold activity, compared to the wild-type (WT). Moreover, molecular docking simulations also predicted better substrate affinity of the mutants. We have also performed molecular dynamics (MD) simulations at 315 and 345 K, and the MD data at 345 K demonstrates improved thermostability for the mutants, compared to the WT. Our findings not only contribute to a better understanding of the structure-stability-activity relationship of SP but also highlights, that modification of non-catalytic residues could also promote favourable catalytic behaviour. (C) 2018 Published by Elsevier B.V.
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