Journal
ADVANCED SYNTHESIS & CATALYSIS
Volume 350, Issue 16, Pages 2631-2638Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/adsc.200800489
Keywords
biotransforniations; alpha,alpha'-dihydroxy ketones; enzymes; molecular evolution; transketolase
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Funding
- UK Engineering and Physical Sciences Research Council (EPSRC)
- Bioconversion Integrated with Chemistry and Engineering (BiCE) [GR/S62505/01]
- Engineering and Physical Sciences Research Council [GR/S62505/01, GR/S02532/01] Funding Source: researchfish
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Chiral auxiliary methodology and chiral assays have been developed to establish the enantiomeric purities of erythrulose and 1,3-dihydroxypentan-2-one generated using wild-type (WT) Escherichia coli transketolase (TK). L-Erythrulose was formed in 95% ee and (3S)-1,3-dihydroxypentan-2-one in 58% ee. Since the latter compound was formed in moderate ee, TK libraries were screened to identify higher performing mutants. A colorimetric screen and chiral assay were successfully applied to a 96-well format, and new active TK mutants were identified, which gave 1,3-dihydroxypentan-2-one in high stereoselectivities. Remarkably, active-site single-point mutants were identified that were able to both enhance and reverse the stereoselectivity of TK.
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