Optimisation and evaluation of a generic microplate-based HPLC screen for transketolase activity.
A microplate-based HPLC assay for transketolase is described for rapidly determining substrate and product concentration suitable for optimisation of biocatalytic process conditions and screening directed evolution libraries. Transketolase catalyses the enantioselective carbon-carbon bond formation of chiral keto-diol products. The assay was used to determine dissociation constants for the two cofactors required by transketolase with 5-11% error. The preparation of samples by microplate-based fermentation, cell lysis, addition of cofactor, addition of substrates was also evaluated and optimised for increased transketolase activity. The whole process enables 3-fold improved enzyme variants to be identified from a single measurement.
Miller
Oliver J
OJ
Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.
Hibbert
Edward G
EG
Ingram
Christine U
CU
Lye
Gary J
GJ
Dalby
Paul A
PA
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology*; D005285:Fermentation; D013379:Substrate Specificity; D014174:Transketolase / Q000032:analysis* / Q000378:metabolism
D014174:Transketolase