Journal of Molecular Evolution

, Volume 86, Issue 5, pp 283–292 | Cite as

Protein Evolution is Potentially Governed by Protein Stability: Directed Evolution of an Esterase from the Hyperthermophilic Archaeon Sulfolobus tokodaii

  • Ryo Kurahashi
  • Satoshi Sano
  • Kazufumi Takano
Original Article


The study of evolution is important to understand biological phenomena. During evolutionary processes, genetic changes confer amino acid substitutions in proteins, resulting in new or improved functions. Unfortunately, most mutations destabilize proteins. Thus, protein stability is a significant factor in evolution; however, its role remains unclear. Here, we simply and directly explored the association between protein activity and stability in random mutant libraries to elucidate the role of protein stability in evolutionary processes. In the first random mutation of an esterase from Sulfolobus tokodaii, approximately 20% of the variants displayed higher activity than wild-type protein (i.e., 20% evolvability). During evolutionary processes, the evolvability depended on the stability of template proteins, indicating that protein evolution is potentially governed by protein stability. Furthermore, decreased activity could be recovered during evolution by maintaining the stability of variants. The results suggest that protein sequence space for its evolution is able to expand during nearly neutral evolution where mutations are slightly deleterious for activity but rarely fatal for stability. Molecular evolution is a crucial phenomenon that has continued since the birth of life on earth, and mechanism underlying it is simple; therefore, this could be demonstrated by our simple experiments. These findings also can be applied to protein engineering.


Esterase Sulfolobus tokodaii Evolvability Sequence space Fitness landscape Nearly neutral theory of molecular evolution 



This work was supported in part by grants from the Japan Society for the Promotion of Science to KT (KAKENHI Nos. 25440194 and 17K07368), by a Research Encouragement Project from the Academic Promotion Fund of Kyoto Prefectural University to RK (2016), and by a Young Researcher Development Support Project from the Kyoto Prefectural Public University Corporation to RK (2017).

Author Contributions

KT conceived and supervised the experiments. RK performed the experiments. RK and KT wrote the paper. SS helped in interpretation of data and discussion of results. All the authors have read and approved the final manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare no competing financial interest.


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Authors and Affiliations

  1. 1.Department of Biomolecular ChemistryKyoto Prefectural UniversityKyotoJapan

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