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Engineering of serine protease for improved thermostability and catalytic activity using rational design.

Authors
  • Ashraf, Naeem Mahmood1
  • Krishnagopal, Akshaya2
  • Hussain, Aadil1
  • Kastner, David3
  • Sayed, Ahmed Mahmoud Mohammed2
  • Mok, Yu-Keung2
  • Swaminathan, Kunchithapadam4
  • Zeeshan, Nadia5
  • 1 Department of Biochemistry and Biotechnology, University of Gujrat, Hafiz Hayat Campus, Gujrat, Punjab 50700, Pakistan. , (Pakistan)
  • 2 Department of Biological Sciences, National University of Science, 117543, Singapore. , (Singapore)
  • 3 Department of Biophysics, Brigham Young University, Provo, UT 84602, USA.
  • 4 Department of Biological Sciences, National University of Science, 117543, Singapore. Electronic address: [email protected] , (Singapore)
  • 5 Department of Biochemistry and Biotechnology, University of Gujrat, Hafiz Hayat Campus, Gujrat, Punjab 50700, Pakistan. Electronic address: [email protected] , (Pakistan)
Type
Published Article
Journal
International journal of biological macromolecules
Publication Date
Apr 01, 2019
Volume
126
Pages
229–237
Identifiers
DOI: 10.1016/j.ijbiomac.2018.12.218
PMID: 30590144
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

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 Tm of A29G and V336I showed an increase by 5 °C and also a substantial increase in residual activity of the enzyme at elevated temperature. Moreover, the catalytic activity of A29G and V336I 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. Copyright © 2018. Published by Elsevier B.V.

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