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    • Keywords

       

      Bioinformatics design; disulphide bridge; organophosphorus chemicals; organophosphorus hydrolase; thermostability

    • Abstract

       

      The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the mostfrequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has beenshown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineeringof de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rationalmethod of protein engineering. In this study, Disulphide by Design software, homology modelling and moleculardynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge toimprove protein thermostability. The thermostability of the wild-type and three selected mutant enzymes wereevaluated by half-life, ΔG inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Dataanalysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min,respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124Cmutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results werecorrelated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsicfluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi alsoincreased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results weresubmitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing adisulphide bridge after bioinformatics design, even though this design would not be always successful.

    • Author Affiliations

       

      GHOLAMREZA FARNOOSH1 KHOSRO KHAJEH2 ALI MOHAMMAD LATIFI1 HOSSEIN AGHAMOLLAEI1

      1. Applied Biotechnology Research Centre, Baqiyatallah University of Medical Sciences, Tehran, Iran
      2. Department of Biochemistry, Faculty of Biologic Science, Tarbiat Modares University, Tehran, Iran
    • Dates

       
  • Journal of Biosciences | News

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