The Protein Journal

, Volume 34, Issue 1, pp 82–89 | Cite as

Compensatory Stabilizing Role of Surface Mutations During the Directed Evolution of Dienelactone Hydrolase for Enhanced Activity

  • Joanne L. Porter
  • Charles A. Collyer
  • David L. Ollis


Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations—Q110L, Y137C and N154D—on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.


Protein stability Directed evolution Dienelactone hydrolase 310 helix Urea denaturation Protein evolvability 



Dienelactone hydrolase


Polymerase chain reaction


Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Melting temperature



JLP is supported by an Australian Postgraduate Award from the Australian government and funding from the Research School of Chemistry, ANU. We thank the staff at the ACRF-BRF for performing the DNA sequencing analysis. We also thank Anithahini Jeyasingham from the Mass Spectrometry Department in the Research School of Chemistry at the ANU for conducting the mass spectrometry.

Supplementary material

10930_2015_9600_MOESM1_ESM.pdf (1.8 mb)
Supplementary material 1 (PDF 1871 kb)


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Joanne L. Porter
    • 1
  • Charles A. Collyer
    • 2
  • David L. Ollis
    • 1
  1. 1.Research School of ChemistryAustralian National UniversityCanberraAustralia
  2. 2.School of Molecular BioscienceUniversity of SydneySydneyAustralia

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