Prospects for robust biocatalysis: Engineering of novel specificity in a halophilic amino acid dehydrogenase

Nayla Munawar, Paul C. Engel

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting l-methionine, l-norleucine and l-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

Original languageEnglish
Pages (from-to)43-51
Number of pages9
JournalExtremophiles
Volume17
Issue number1
DOIs
Publication statusPublished - Jan 2013
Externally publishedYes

Keywords

  • Glutamate dehydrogenase
  • Halobacterium
  • Halophilic biocatalyst
  • Homology modelling
  • Methionine/norleucine dehydrogenase
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Microbiology
  • Molecular Medicine

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