Thermostable transketolase from Geobacillus stearothermophilus: Characterization and catalytic properties

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 mTK_gst 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 mTK_gst 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, mTK_gst 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 mTK_gst 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.