Thermostable transketolase from Geobacillus stearothermophilus: Characterization and catalytic properties

Juliane Abdoul-Zabar, Isabelle Sorel, Virgil Hélaine, Franck Charmantray, Titu Devamani, Dong Yi, Véronique De Berardinis, Dominique Louis, Philippe Marlière, Wolf Dieter Fessner, Laurence Hecquet

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

Abstract

Here we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTK gst) retained 100% activity for one week at 50 °C and for 3 days at 65 °C, and has an optimum temperature range around 60-70 °C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTKgst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65 °C for 45min, directly yielding 132mg of pure mTK gst from 1L of culture. The reaction rate of mTKgst with glycolaldehyde was 14 times higher at 70 °C than at 20 °C, and 4 times higher at 50 °C when compared to E. coli TK under identical conditions. When tested at 50 °C with other aldehydes as acceptors, mTKgst activity was approximately 3 times higher than those obtained at 20 °C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors-glycolaldehyde, D-glyceraldehyde and butyraldehyde-from which the corresponding products L-erythrulose 1, D-xylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTKgst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media.

Original languageEnglish (US)
Pages (from-to)116-128
Number of pages13
JournalAdvanced Synthesis and Catalysis
Volume355
Issue number1
DOIs
StatePublished - Jan 14 2013

Keywords

  • C-C bond formation
  • biocatalysis
  • monosaccharides
  • thermostability
  • transketolases

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