Applied Microbiology and Biotechnology

, Volume 94, Issue 1, pp 131–140 | Cite as

Engineering pH-tolerant mutants of a cyanide dihydratase

  • Lan Wang
  • Jean M. Watermeyer
  • Andani E. Mulelu
  • B. Trevor Sewell
  • Michael J. Benedik
Biotechnologically Relevant Enzymes and Proteins

Abstract

Cyanide dihydratase is an enzyme in the nitrilase family capable of transforming cyanide to formate and ammonia. This reaction has been exploited for the bioremediation of cyanide in wastewater streams, but extending the pH operating range of the enzyme would improve its utility. In this work, we describe mutants of Bacillus pumilus C1 cyanide dihydratase (CynDpum) with improved activity at higher pH. Error-prone PCR was used to construct a library of CynDpum mutants, and a high-throughput screening system was developed to screen the library for improved activity at pH 10. Two mutant alleles were identified that allowed cells to degrade cyanide in solutions at pH 10, whereas the wild-type was inactive above pH 9. The mutant alleles each encoded three different amino acid substitutions, but for one of those, a single change, E327G, accounted for the phenotype. The purified proteins containing multiple mutations were five times more active than the wild-type enzyme at pH 9, but all purified enzymes lost activity at pH 10. The mutation Q86R resulted in the formation of significantly longer fibers at low pH, and both E327G and Q86R contributed to the persistence of active oligomeric assemblies at pH 9. In addition, the mutant enzymes proved to be more thermostable than the wild type, suggesting improved physical stability rather than any change in chemistry accounts for their increased pH tolerance.

Keywords

Nitrilase Cyanide Bioremediation Cyanide dihydratase pH tolerance Protein stability 

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Lan Wang
    • 1
  • Jean M. Watermeyer
    • 2
  • Andani E. Mulelu
    • 2
  • B. Trevor Sewell
    • 2
  • Michael J. Benedik
    • 1
  1. 1.Department of BiologyTexas A&M UniversityCollege StationUSA
  2. 2.Division of Medical Biochemistry, Institute of Infectious Diseases and Molecular MedicineUniversity of Cape TownRondeboschSouth Africa

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