The Protein Journal

, Volume 31, Issue 7, pp 615–622 | Cite as

Additive Effect of Single Amino Acid Replacements on the Kinetic Stability of β-Glucosidase B

  • Menandro Camarillo-Cadena
  • Gerogina Garza-Ramos
  • Mariana Peimbert
  • Julio Polaina
  • Gerardo Pérez-Hernández
  • Rafael A. Zubillaga


Previously, we applied in vitro evolution to generate the thermoresistant triple mutant H62R/N223Y/M319I of β-glucosidase B (BglB) from Paenibacillus polymyxa. In order to dissect the energetic contributions to protein stabilization achieved by these mutations, we measured the kinetic constants of the heat denaturation of wild type BglB, the triple mutant and the three single mutants (H62R, N223Y, M319I) by circular dichroism at various temperatures. Our results show that all four mutants delayed the denaturation process. Based on the Transition State theory, the increase of the activation barrier for the thermal denaturation of the triple mutant (ΔΔG N→TS ) is equivalent to that produced by the sum of the contributions from the three single mutants, whose C β s are located at least 18 Å apart. This analysis provides a formal demonstration of the generally accepted idea that protein thermal stability can be increased through sequential addition of individual mutations. Each of the mutations described here contribute in part to the overall effect, which in this case affects the unfolding barrier.


Circular dichroism Enzyme thermostabilization Irreversible denaturation Protein kinetic stability 



β-Glucosidase B


Triple mutant of BglB


Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Circular dichroism




Half-denaturation temperature


Denaturing kinetic constant


Free energy difference between the Transition State and the Native sate

\( \Updelta \Updelta G_{MUT - WT}^{N \to TS} \)

Change in the activation barrier for a mutant respect to that of the wild type protein



This work was supported by the National Council of Science and Technology grant number 46166 and by a PhD scholarship to MCC (204689).


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Menandro Camarillo-Cadena
    • 1
  • Gerogina Garza-Ramos
    • 2
  • Mariana Peimbert
    • 3
  • Julio Polaina
    • 4
  • Gerardo Pérez-Hernández
    • 3
  • Rafael A. Zubillaga
    • 1
  1. 1.Departamento de QuímicaUniversidad Autónoma Metropolitana-IztapalapaMexico, D. F.Mexico
  2. 2.Departamento de BioquímicaUniversidad Nacional Autónoma de MéxicoMexico, D. F.Mexico
  3. 3.Departamento de Ciencias NaturalesUniversidad Autónoma Metropolitana-CuajimalpaMexico, D. F.Mexico
  4. 4.Departamento de Biotecnología de AlimentosIATA-CSICPaternaSpain

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