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Directed Evolution of a Subtilisin with Calcium-Independent Stability

Bio/Technology volume 13, pages 669–673 (1995)Cite this article

Abstract

Extracellular proteases of the subtilisin-class depend upon calcium for stability. Calcium binding stabilizes these proteins in natural extracellular environments, but is an Achilles' heel in industrial environments which contain high concentrations of metal chelators. Here we direct the evolution of calcium-independent stability hi subtilisin BPN′. By deleting the calcium binding loop from subtilisin, we initially destabilize the protein but create the potential to use new structural solutions for stabilization. Analysis of the structure and stability of the loop-deleted prototype followed by directed mutagenesis and selection for increased stability resulted in a subtilisin mutant with native-like proteolytic activity but 1000-times greater stability in strongly chelating conditions.

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Authors and Affiliations

  1. Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD, 20850

    Susan L. Strausberg, Patrick A. Alexander & Philip N. Bryan

  2. The National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, MD, 20850

    D.Travis Gallagher & Gary L Gilliland

  3. The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, Ohio, 45239

    B.L. Barnett

Authors
  1. Susan L. Strausberg
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  2. Patrick A. Alexander
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  3. D.Travis Gallagher
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  4. Gary L Gilliland
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  5. B.L. Barnett
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  6. Philip N. Bryan
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Corresponding author

Correspondence to Philip N. Bryan.

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Strausberg, S., Alexander, P., Gallagher, D. et al. Directed Evolution of a Subtilisin with Calcium-Independent Stability. Nat Biotechnol 13, 669–673 (1995). https://doi.org/10.1038/nbt0795-669

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  • DOI: https://doi.org/10.1038/nbt0795-669

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