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Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes

Nature Protocols volume 2, pages 891–903 (2007)Cite this article

Abstract

Iterative saturation mutagenesis (ISM) is a new and efficient method for the directed evolution of functional enzymes. It reduces the necessary molecular biological work and the screening effort drastically. It is based on a Cartesian view of the protein structure, performing iterative cycles of saturation mutagenesis at rationally chosen sites in an enzyme, a given site being composed of one, two or three amino acid positions. The basis for choosing these sites depends on the nature of the catalytic property to be improved, e.g., enantioselectivity, substrate acceptance or thermostability. In the case of thermostability, sites showing highest B-factors (available from X-ray data) are chosen. The pronounced increase in thermostability of the lipase from Bacillus subtilis (Lip A) as a result of applying ISM is illustrated here.

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Figure 1: Schematic illustration of iterative saturation mutagenesis involving (as an example) four randomization sites A, B, C and D: confined protein sequence space for evolutionary enzyme optimization (redundancy in some cases is expected).
Figure 2
Figure 3: Enhancing the thermostability of the lipase from Bacillus subtilis (Lip A) by iterative saturation mutagenesis.
Figure 4: Generalization of CASTing.
Figure 5: Accelrys Discovery Studio Visualizer main windows.
Figure 6
Figure 7: Introduction to CASTER.
Figure 8
Figure 9: Thermostability of purified wild-type (WT) and variants X and XI of Bacillus subtilis lipase A (Lip A) as displayed by the residual activity curves.

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Acknowledgements

We thank the Fonds der Chemischen Industrie, the German–Israeli Project Cooperation (DIP) and the Deutsche Forschungsgemeinschaft (SPP 1170, RE 359/13-1) for support and the Department of Crystallography of the Max-Planck-Institut für Kohlenforschung for collaborative efforts in developing the computer program B-FITTER.

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  1. Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, Mülheim/Ruhr, 45470, Germany

    Manfred T Reetz & José Daniel Carballeira

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Correspondence to Manfred T Reetz.

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Reetz, M., Carballeira, J. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2, 891–903 (2007). https://doi.org/10.1038/nprot.2007.72

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