Optimisation and evaluation of a generic microplate-based HPLC screen for transketolase activity

Abstract

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.

Structural stability of E. coli transketolase to temperature and pH denaturation

“…Recombinant transketolase was expressed with an N-terminal His 6 tag from E. coli XL10-Gold (Stratagene, La Jolla, CA) containing the engineered plasmid pQR791 to give a mixture containing 92% apo-TK (Miller et al, 2007), then purified as described previously (Martinez-Torres et al, 2007), dialysed for 24 h against 25 mM Tris-HCl, pH 7.5 to obtain only the apo-TK form, at 4 • C then stored at 4 • C for a maximum of two weeks without loss of activity, and with no precipitation visible. This preparation was completely inactive prior to the addition of cofactors, confirming it was in the apo-TK form.…”

Droplet millifluidics for kinetic study of transketolase

“…The results are given in Table III. Several authors have contributed to the determination of the substrate specificity of TK enzymes from different sources (E. coli, S. cerevisiae, G. stearothermophilus) using different assay methods towards various phosphorylated and unphosphorylated aldoses or aldehydes. 4,[14][15][16][17][18][19][20] The activity profiles of TKs are very similar, owing to the strong homology of active sites. Our system yields the same results as those obtained with TK gst using other methods.…”

“…Measurement of chiral product formation by an optical method 16,17 is highly dependent on the substrate structure, and so not of generic utility. Determination of HPA depletion by near-UV spectroscopic monitoring 18 or HPLC (High Performance Liquid Chromatography) analysis [14][15][16][17][18][19][20] is hampered by low sensitivity or low throughput. Colorimetric determination of ketose formation with tetrazolium red-based oxidation is restricted to non-hydroxylated aldehyde acceptors such as propanal.…”

α, α -Dihydroxy Ketones and 2-Amino-1,3-diols: Synthetic and Process Strategies Using Biocatalysts

“…As described above a one-pot TK biomimetic reaction has been discovered [13] and this was used to prepare dihydroxy ketones for use in assay development. TK activity has been monitored using a range of procedures including a spectrophotometric method which monitored the HPA present using an NADH dependent enzyme assay [20,23] and product formation by HPLC [29], which has been adapted to a rapid microplate method [30]. In vitro fluorescence based assays have also been developed with substrates containing coumarin moieties [31,32], and the synthesis of small molecule probes has recently been described to detect TK activity in vivo [33].…”