A double mutant of highly purified Geobacillus stearothermophilus lactate dehydrogenase recognises l-mandelic acid as a substrate

Barış Binay, Richard B Sessions, Nevin Gül Karagüler, Nevin Gül Karagüler

Research output: Contribution to journalArticle (Academic Journal)peer-review

5 Citations (Scopus)


Lactate dehydrogenase from the thermophilic organism Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) (bsLDH) has a crucial role in producing chirally pure hydroxyl compounds. α-Hydroxy acids are used in many industrial situations, ranging from pharmaceutical to cosmetic dermatology products. One drawback of this enzyme is its limited substrate specificity. For instance, l-lactate dehydrogenase exhibits no detectable activity towards the large side chain of 2-hydroxy acid l-mandelic acid, an α-hydroxy acid with anti-bacterial activity. Despite many attempts to engineer bsLDH to accept α-hydroxy acid substrates, there have been no attempts to introduce the industrially important l-mandelic acid to bsLDH. Herein, we describe attempts to change the reactivity of bsLDH towards l-mandelic acid. Using the Insight II molecular modelling programme (except 'program' in computers) and protein engineering techniques, we have successfully introduced substantial mandelate dehydrogenase activity to the enzyme. Energy minimisation modelling studies suggested that two mutations, T246G and I240A, would allow the enzyme to utilise l-mandelic acid as a substrate. Genes encoding for the wild-type and mutant enzymes were constructed, and the resulting bsLDH proteins were overexpressed in Escherichia coli and purified using the TAGZyme system. Enzyme assays showed that insertion of this double mutation into highly purified bsLDH switched the substrate specificity from lactate to l-mandelic acid.

Original languageEnglish
Pages (from-to)393-9
Number of pages7
JournalEnzyme and Microbial Technology
Issue number6-7
Publication statusPublished - 10 May 2013

Bibliographical note

Copyright © 2013 Elsevier Inc. All rights reserved.


  • Binding Sites
  • Biotechnology
  • Escherichia coli
  • Geobacillus stearothermophilus
  • Kinetics
  • L-Lactate Dehydrogenase
  • Mandelic Acids
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Engineering
  • Substrate Specificity


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