The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases
| Autor: | Jeremy H. Lakey, Edward J. Taylor, Simon R. Andrews, Valérie M.-A. Ducros, Harry J. Gilbert, Gavin Pell, Gideon J. Davies, Florence Vincent |
|---|---|
| Přispěvatelé: | Laboratory of Lymphocyte Signaling and Development, Centre des Matériaux (CDM), Mines Paris - PSL (École nationale supérieure des mines de Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Newcastle University [Newcastle], Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of York [York, UK], University of Newcastle [Callaghan, Australia] (UoN), Centre des Matériaux (MAT), MINES ParisTech - École nationale supérieure des mines de Paris, University of Newcastle [Australia] (UoN) |
| Jazyk: | angličtina |
| Rok vydání: | 2004 |
| Předmět: |
0106 biological sciences
Mutant chemistry.chemical_element Calcium 01 natural sciences Biochemistry 03 medical and health sciences 010608 biotechnology Hydrolase [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Binding site Molecular Biology ComputingMilieux_MISCELLANEOUS 030304 developmental biology Thermostability Cellvibrio japonicus chemistry.chemical_classification 0303 health sciences biology [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Structural Biology [q-bio.BM] [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry Molecular Biology/Molecular biology Cell Biology biology.organism_classification Amino acid [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biophysics chemistry Protein folding |
| Zdroj: | Journal of Biological Chemistry Journal of Biological Chemistry, 2004, 279 (52), pp.54369-54379. ⟨10.1074/jbc.M409044200⟩ Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2004, 279 (52), pp.54369-54379. ⟨10.1074/jbc.M409044200⟩ |
| ISSN: | 0021-9258 1083-351X |
| DOI: | 10.1074/jbc.M409044200⟩ |
| Popis: | Metal ions such as calcium often play a key role in protein thermostability. The inclusion of metal ions in industrial processes is, however, problematic. Thus, the evolution of enzymes that display enhanced stability, which is not reliant on divalent metals, is an important biotechnological goal. Here we have used forced protein evolution to interrogate whether the stabilizing effect of calcium in an industrially relevant enzyme can be replaced with amino acid substitutions. Our study has focused on the GH10 xylanase CjXyn10A from Cellvibrio japonicus, which contains an extended calcium binding loop that confers proteinase resistance and thermostability. Three rounds of error-prone PCR and selection identified a treble mutant, D262N/A80T/R347C, which in the absence of calcium is more thermostable than wild type CjXyn10A bound to the divalent metal. D262N influences the properties of the calcium binding site, A80T fills a cavity in the enzyme, increasing the number of hydrogen bonds and van der Waals interactions, and the R347C mutation introduces a disulfide bond that decreases the free energy of the unfolded enzyme. A derivative of CjXyn10A (CfCjXyn10A) in which the calcium binding loop has been replaced with a much shorter loop from Cellulomonas fimi CfXyn10A was also subjected to forced protein evolution to select for thermostablizing mutations. Two amino acid substitutions within the introduced loop and the A80T mutation increased the thermostability of the enzyme. This study demonstrates how forced protein evolution can be used to introduce enhanced stability into industrially relevant enzymes while removing calcium as a major stability determinant. |
| Databáze: | OpenAIRE |
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