TY - JOUR
T1 - Thermostable transketolase from Geobacillus stearothermophilus
T2 - Characterization and catalytic properties
AU - Abdoul-Zabar, Juliane
AU - Sorel, Isabelle
AU - Hélaine, Virgil
AU - Charmantray, Franck
AU - Devamani, Titu
AU - Yi, Dong
AU - De Berardinis, Véronique
AU - Louis, Dominique
AU - Marlière, Philippe
AU - Fessner, Wolf Dieter
AU - Hecquet, Laurence
PY - 2013/1/14
Y1 - 2013/1/14
N2 - 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.
AB - 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.
KW - C-C bond formation
KW - biocatalysis
KW - monosaccharides
KW - thermostability
KW - transketolases
UR - http://www.scopus.com/inward/record.url?scp=84872200433&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84872200433&partnerID=8YFLogxK
U2 - 10.1002/adsc.201200590
DO - 10.1002/adsc.201200590
M3 - Article
AN - SCOPUS:84872200433
VL - 355
SP - 116
EP - 128
JO - Advanced Synthesis and Catalysis
JF - Advanced Synthesis and Catalysis
SN - 1615-4150
IS - 1
ER -