High‐Throughput Solid‐Phase Assay for Substrate Profiling and Directed Evolution of Transketolase

Abstract

Thiamine diphosphate‐dependent enzymes, and specifically transketolases, form one of the most important families of biocatalytic tools for enantioselective carbon‐carbon bond formation yielding various hydroxyketones of biological interest. To enable substrate profiling of transketolases for acceptance of different donors and acceptors, a simple, direct colorimetric assay based on pH reaction variation was developed to establish a high‐throughput solid‐phase assay. This assay reduces the screening effort in the directed evolution of transketolases, as only active variants are selected for further analysis. Transketolase activity is detected as bicarbonate anions released from the α‐ketoacid donor substrate, which causes the pH to rise. A pH indicator, bromothymol blue, which changes color from yellow to blue in alkaline conditions, was used to directly detect, with the naked eye, clones expressing active transketolase variants, obviating enzyme extraction.

Hydroxamate Assays for High‐Throughput Screening of Transketolase Libraries Against Arylated Substrates

“…Thus, colonies do not have to be picked immediately, facilitating the simultaneous treatment of multiple membranes, and the images can be processed electronically for improved colony discrimination. It should be pointed out that during the process no dialysis step was required to remove unspecific background noise, which renders the ferric chloride assay significantly faster than related solid‐phase assays [17,26] …”

“…Therefore, solid‐phase supported assays on a colony level are an alluring alternative, because they are rapid, efficient, economic and easy to handle. However, the pH‐shift method recently reported by the Hecquet group [17] is unreliable for the target of this study because the high volatility of nitrosobenzene and the oxygen sensitivity of benzaldehyde both compromise the readings. The unique product detection strategy using the colorimetric iron (III) assay was therefore highly attractive to be transferred to a solid‐phase support, allowing screening assays directly on agar plates and thereby avoiding the extraction of enzymes and the need for specialized equipment.…”

“…It should be pointed out that during the process no dialysis step was required to remove unspecific background noise, which renders the ferric chloride assay significantly faster than related solid-phase assays. [17,26] Overall, our novel colorimetric agar plate screening method has the potential to accelerate the throughput for hit identification by at least one order of magnitude in comparison to liquid-phase methods, while simultaneously reducing both handling effort and assay costs. The same assay principle thus allows rapid hit identification on solid support and subsequent characterization in liquid-phase by quantitative microtiter plate assay (Scheme 6).…”

“…Our high‐throughput pH‐shift assay developed for TK analysis is universal, being independent of substrate structure for both aldehyde acceptor and oxo acid donor moieties [6,9,14a] . Recently, an adaption of this assay has been reported for the screening of TK variants directly on agar plate [17] . However, the pH‐shift principle only accounts for the first step in the TK catalytic cycle, i. e ., the substrate consumption by decarboxylation, whereas potential hydrolytic decomposition of the cofactor‐bound intermediate may indicate false‐positive results for the desired second step without giving rise to product formation by carboligation [11d] .…”

“…[6,9,14a] Recently, an adaption of this assay has been reported for the screening of TK variants directly on agar plate. [17] However, the pH-shift principle only accounts for the first step in the TK catalytic cycle, i. e., the substrate consumption by decarboxylation, whereas potential hydrolytic decomposition of the cofactor-bound intermediate may indicate false-positive results for the desired second step without giving rise to product formation by carboligation. [11d] The pH assay may also be perturbed by air oxidation of sensitive aldehydes (such as benzaldehyde), which generates acid that is incompatible with the assay principle.…”