A pH‐Based High‐Throughput Assay for Transketolase: Fingerprinting of Substrate Tolerance and Quantitative Kinetics

Abstract

A pH-based high-throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active-site-modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3-hydroxypropanal and 4-hydroxybutanal for preparative synthesis of chiral deoxyketose-type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH-based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution.

Real‐time pH monitoring of industrially relevant enzymatic reactions in a microfluidic side‐entry reactor (μSER) shows potential for pH control

“…Despite this apparent correlation, the pH change is strongly dependent on the pH value and salinity of the reaction buffer and any linearity in the increase is coincidental. Whilst the increase in pH was expected, and was in fact previously monitored using a colorimetric method with nitrophenol [35], the initial drop in pH was not reported before. Indeed, Yi et al [35] did not monitor such an initial decrease of pH, though they only performed the reaction with an initial substrate concentration of 50 mM (for which our results also only showed a very small decrease, < 0.1 pH units).…”

“…Whilst the increase in pH was expected, and was in fact previously monitored using a colorimetric method with nitrophenol [35], the initial drop in pH was not reported before. Indeed, Yi et al [35] did not monitor such an initial decrease of pH, though they only performed the reaction with an initial substrate concentration of 50 mM (for which our results also only showed a very small decrease, < 0.1 pH units). A possible explanation for the drop in pH might be the hydrolysis of the glycolaldehyde dimer [36].…”

Droplet millifluidics for kinetic study of transketolase

“…It was obtained by a protocol described elsewhere. 21 Each solution was prepared fresh in glycylglycine buffer solution (100 mM, pH 7.5) except for HPA (Sigma Aldrich) stock solution, which was prepared in glycylglycine buffer solution (100 mM, pH 7).…”

“…We have recently described an assay, independent of the substrate and the product, and based on direct monitoring of pH changes with phenol red as pH indicator. 21 In the TK catalytic reaction with HPA as the donor, one equivalent of bicarbonate is produced during HPA decarboxylation, raising the pH. Phenol red exposed to alkali changes colour from yellow to bright red.…”

“…21 From the industrial point of view, enzymatic processes at elevated temperatures offer many advantages, such as higher reaction rates, and better tolerance of organic solvents. We showed that TK gst offers several advantages: (i) TK gst is thermostable and retained 100% activity for one week at 50 C, (ii) the relative velocity of TK gst catalysis at 50 C was 8 times higher than at 20 C, 21 and (iii) the high stability of TK gst in the presence of co-solvents such as organic solvents, but also ionic liquids, can be used to improve the solubility of acceptor substrates. We showed that TK gst activity was stable for 16 h in the aqueous solution containing 30%-50% [BMIm][Cl] enhancing TK gst activity towards pentoses, particularly D-ribose.…”