Transketolases

Wohlgemuth, Roland; Smith, Mark E.; Dalby, Paul A.; Woodley, John M.

doi:10.1002/9780470054581.eib569

Cited by 7 publications

(5 citation statements)

References 53 publications

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Section: Abstract: Microreactor · Online Monitoring · Optical Sensor mentioning

confidence: 99%

“…[19, 20]. The synthetic utility of transketolasecatalyzed reactions is based on the excellent selectivity and versatility of the two-carbon chain elongation of suitable aldehydes to more complex chiral compounds in combination with the irreversibility of this reaction by the use of hydroxypyruvate as carbon donor [21][22][23][24][25].In this contribution, we demonstrate real-time monitoring of pH in a microfluidic reactor as a further step towards their use as novel process development tools for biocatalysis. To achieve this, optical pH sensor layers with dual lifetime referencing [26] were integrated in a microfluidic side-entry reactor.…”

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confidence: 99%

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

Gruber

Marques

Sulzer

et al. 2017

Biotechnology Journal

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Monitoring and control of pH is essential for the control of reaction conditions and reaction progress for any biocatalytic or biotechnological process. Microfluidic enzymatic reactors are increasingly proposed for process development, however typically lack instrumentation, such as pH monitoring. We present a microfluidic side-entry reactor (µSER) and demonstrate for the first time real-time pH monitoring of the progression of an enzymatic reaction in a microfluidic reactor as a first step towards achieving pH control. Two different types of optical pH sensors were integrated at several positions in the reactor channel which enabled pH monitoring between pH 3.5 and pH 8.5, thus a broader range than typically reported. The sensors withstood the thermal bonding temperatures typical of microfluidic device fabrication. Additionally, fluidic inputs along the reaction channel were implemented to adjust the pH of the reaction. Time-course profiles of pH were recorded for a transketolase- and a penicillin G acylase-catalyzed reaction. Without pH adjustment, the former showed a pH increase of one pH unit and the latter a pH decrease of about 2.5 pH units. With pH adjustment, the pH drop of the penicillin G acylase-catalyzed reaction was significantly attenuated, the reaction condition kept at a pH suitable for the operation of the enzyme, and the product yield increased. This contribution represents a further step towards fully instrumented and controlled microfluidic reactors for biocatalytic process development. Keywords: Microreactor · Online monitoring · Optical sensor · Penicillin G acylase · pH sensor · TransketolaseCorrespondence: Prof. Nicolas Szita, University College London, Biochemical Engineering, Bernard Katz Building, Gordon Street, London, WC1H 0AH, UK E-mail: n.szita@ucl.ac.uk Abbreviations: 6-APA, 6-amino benzyl penicillanic acid; ERY, L-erythrulose; GA, glycol aldehyde; GC, gas chromatography; HPA, hydroxyl pyruvate; HPLC, high performance liquid chromatography; ISFET, ion-sensitive field effect transistor; PG, penicillin G; PGA, penicillin G acylase; TFA, trifluoroacetic acid; TK, transketolase; µSER, microfluidic side-entry reactor Biotechnol. J. 2017, 12, 1600475 electrode becomes less practical. This integration challenge is exacerbated in microfluidic devices, where channels are normally small, narrow, and enclosed. Reactions can in principle be monitored at-line or off-line, for example with a HPLC or a GC; however, in reactions where a change in a common analyte such as oxygen or pH occurs, online monitoring is preferable, as it provides a direct, i.e. real time, measure of the progress of the reaction. On-line monitoring at the microfluidic scale thus enables the rapid analysis of enzymatic reactions with small amounts of enzymes, and -due to the fine control over the fluid flow afforded by microfluidics -with precise control over reaction conditions. Monitoring of pH can be accomplished using electrochemical sensors, such as ion-sensitive field effect transistors (ISFETs) [3]. Since their ...

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Section: Abstract: Microreactor · Online Monitoring · Optical Sensor mentioning

confidence: 99%

mentioning

confidence: 99%

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

Gruber

Marques

Sulzer

et al. 2017

Biotechnology Journal

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“…The stereospecifically controlled carbon‐carbon bond‐forming ability of TK makes it very promising as a biocatalyst in industry, for the synthesis of complex carbohydrates and other high‐value compounds . The large‐scale fermentation, downstream processing and purification of TK to give a stable lyophilised powder have been established at Sigma‐Aldrich (Buchs, Switzerland) , and TK has also been used in large‐scale biocatalytic reactions for the production of unusual sugars . However, to achieve wider industrial applications in large‐scale processes, a robust TK toolbox is desired, in which the variants should be able to accept a diverse range of substrates including both acceptors and donors.…”

Section: Introductionmentioning

confidence: 99%

Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α‐hydroxyketones

López

Steadman

et al. 2019

The FEBS Journal

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A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimisation of both substrate specificities is also required for the rapid expansion of accepted substrates. Transketolase (TK) catalyses the reversible transfer of a C 2 -ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for industrial applications. Herein, TK from Escherichia coli was engineered to accept both pyruvate, as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid (FBA). Twenty single-mutant variants were first designed and characterised experimentally. Beneficial mutations were then recombined to construct a small library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to wild-type (WT). Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% enantiomeric excess (ee) for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-FBA. The best variant was able to convert 47% of substrate into product within 24 h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor recognition, which would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high-value products.

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“…Both techniques were applied to immobilise His-tagged transketolase (TK) in a microfluidic device made of PMMA. TK was chosen due to its wide substrate tolerance and high enantio- and regio-specificity [28], which make it an attractive biocatalyst for the asymmetric synthesis of chiral metabolites [[29], [30], [31], [32], [33]] and highly relevant in the pharmaceutical industry. However, the method is applicable to a large variety of proteins that can be engineered with a His-tag [34].…”

Section: Introductionmentioning

confidence: 99%

Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

et al. 2018

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Poly(methyl methacrylate) (PMMA) microfluidic devices have become promising platforms for a wide range of applications. Here we report a simple method for immobilising histidine-tagged enzymes suitable for PMMA microfluidic devices. The 1-step-immobilisation described is based on the affinity of the His-tag/Ni-NTA interaction and does not require prior amination of the PMMA surface, unlike many existing protocols. We compared it with a 3-step immobilisation protocol involving amination of PMMA and linking NTA via a glutaraldehyde cross-linker. These methods were applied to immobilise transketolase (TK) in PMMA microfluidic devices. Binding efficiency studies showed that about 15% of the supplied TK was bound using the 1-step method and about 26% of the enzyme was bound by the 3-step method. However, the TK-catalysed reaction producing l-erythrulose performed in microfluidic devices showed that specific activity of TK in the device utilising the 1-step immobilisation method was approximately 30% higher than that of its counterpart. Reusability of the microfluidic device produced via the 1-step method was tested for three cycles of enzymatic reaction and at least 85% of the initial productivity was maintained. The device could be operated for up to 40 h in a continuous flow and on average 70% of the initial productivity was maintained. The simplified immobilisation method required fewer chemicals and less time for preparation of the immobilised microfluidic device compared to the 3-step method while achieving higher specific enzyme activity. The method represents a promising approach for the development of immobilised enzymatic microfluidic devices and could potentially be applied to combine protein purification with immobilisation.

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Transketolases

Cited by 7 publications

References 53 publications

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

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

Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α‐hydroxyketones

Simplified immobilisation method for histidine-tagged enzymes in poly(methyl methacrylate) microfluidic devices

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