Microfluidic multi-input reactor for biocatalytic synthesis using transketolase

Lawrence, James; O’Sullivan, Brian; Lye, Gary J.; Wohlgemuth, Roland; Szita, Nicolas

doi:10.1016/j.molcatb.2013.05.016

Cited by 34 publications

(31 citation statements)

References 40 publications

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“…The devices were connected as described in section 2.4.4 to form a microfluidic flow path for the transketolase‐transaminase reaction cascade using the transaminase coil reactor to provide sufficiently long residence times. The fluidic connector bars were fabricated according to previously reported designs (Gruber, Marques, Sulzer et al, 2017; Lawrence, O'Sullivan, Lye, Wohlgemuth, & Szita, 2013). …”

Section: Resultsmentioning

confidence: 99%

“…A number of routes could be pursued to further improve ABT production; by using purified enzyme the volumetric activity per unit volume could be increased; by using time‐controlled additions of MBA using a microfluidic side‐entry reactor (Gruber, Marques, Sulzer et al, 2017; Lawrence et al, 2013) the space‐time yield could be increased; by adding optical sensors and control strategies to maintain optimal reaction conditions (Gruber, Marques, Szita, & Mayr, 2017); by increasing the ERY to MBA ratio the equilibrium could be shifted further toward the product side (Scheme 1) (though at the expense of recovery efforts); or finally by implementing ISPR strategies (Gruber, Marques, O'Sullivan et al, 2017). For the latter, the use of a chromatography or a liquid‐liquid extraction unit operation could be envisaged as an ISPR step.…”

Section: Resultsmentioning

confidence: 99%

“…The transketolase and transaminase reactions are compartmentalized and the enzymes are recovered through tangential flow filtration units (Lawrence et al, 2013). ABT is separated from co‐substrate acetophenone using a liquid–liquid extraction device (Chiang, Dimov, & Szita et al, 2016)…”

Section: Resultsmentioning

confidence: 99%

“…These improvements can either relate to reactor engineering, for example by redesigning reactors to allow the additions of substrates at different points in the reaction in order to avoid enzyme deactivation due to substrate inhibition (Gruber, Marques, Sulzer et al, 2017; Lawrence et al, 2013), or relate to process monitoring and control strategies (Gruber, Marques, Szita et al, 2017), or relate to the implementation of in situ product removal strategies to drive the reaction equilibrium toward the formation of ABT (Gruber, Marques, O'Sullivan et al, 2017; Halim et al, 2014). …”

Section: Discussionmentioning

confidence: 99%

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Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Gruber

Carvalho

Marques

et al. 2017

Biotech & Bioengineering

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Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino‐alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)‐2‐amino‐1,3,4‐butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non‐chiral starting materials, by coupling a transketolase‐ and a transaminase‐catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor‐based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous‐flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase‐catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml−1. Following optimization of the transaminase‐catalyzed reaction, a volumetric activity of 10.8 U ml−1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous‐flow microreactors can be applied for the design and optimization of biocatalytic processes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Gruber

Carvalho

Marques

et al. 2017

Biotech & Bioengineering

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“…1). The millifluidic technology applied to TK-catalysed carbon-carbon bond formation reactions is of fundamental utility [13][14][15] in optimizing the reaction conditions. Several assay formats have been developed to probe the substrate tolerance of TK by measuring the remaining substrate or product formed.…”

Section: Introductionmentioning

confidence: 99%

Droplet millifluidics for kinetic study of transketolase

et al. 2016

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We present a continuous-flow reactor at the millifluidic scale coupled with an online, non-intrusive spectroscopic monitoring method for determining the kinetic parameters of an enzyme, transketolase (TK) used in biocatalysis for the synthesis of polyols by carboligation. The millifluidic system used is based on droplet flow, a well-established method for kinetic chemical data acquisition. The TK assay is based on the direct quantitative measurement of bicarbonate ions released during the transketolase-catalysed reaction in the presence of hydroxypyruvic acid as the donor, thanks to an irreversible reaction: bicarbonate ions react with phosphoenolpyruvate (PEP) in the presence of PEP carboxylase as the first auxiliary enzyme. The oxaloacetate formed is reduced to malate by NADH in the reaction catalysed by malate dehydrogenase as the second auxiliary enzyme. The extent of oxidation of NADH was measured by spectrophotometry at 340 nm. This system gives a direct, quantitative, generic method to evaluate the TK activity versus different substrates. We demonstrate the accuracy of this strategy to determine the enzymatic kinetic parameters and to study the substrate specificity of a thermostable TK from thermophilic microorganism Geobacillus stearothermophilus, offering promising prospects in biocatalysis. Millifluidic systems are useful in this regard as they can be used to rapidly evaluate the TK activity towards various substrates, and also different sets of conditions, identifying the optimal operating environment while minimizing resource consumption and ensuring high control over the operating conditions. Published by AIP Publishing. [http://dx

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Flow‐Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila: Integrated Systems of Reaction Control and Product Purification

et al. 2015

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A purine nucleoside phosphorylase from Aeromonas hydrophyla (AhPNP) was covalently immobilized in a pre‐packed stainless steel column containing aminopropylsilica particles via Schiff base chemistry upon glutaraldehyde activation. The resulting AhPNP‐IMER (Immobilized Enzyme Reactor, immobilization yield ≈50%) was coupled on‐line through a 6‐way switching valve to an HPLC apparatus containing an analytical or a semi‐preparative chromatographic column. The synthesis of five 6‐modified purine ribonucleosides was carried out by continuously pumping the reaction mixture through the AhPNP‐IMER until the highest conversion was reached, and then directing the reaction mixture to chromatographic separation. The conditions of the AhPNP‐catalyzed transglycosylations (2:1 ratio sugar donor:base acceptor; 10 mM phosphate buffer; pH 7.5; temperature 37 °C, flow rate 0.5 mL min−1) were optimized by a fractional factorial experimental design. Coupling the bioconversion step with the product purification in such an integrated platform resulted in a fast and efficient synthetic process (yield=52–89%; <10 mg) where sample handling was minimized. To date, AhPNP‐IMER has retained completely its activity upon 50 reactions in 10 months.magnified image

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Microfluidic multi-input reactor for biocatalytic synthesis using transketolase

Abstract: Graphical abstract

Cited by 34 publications

References 40 publications

Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Enzymatic synthesis of chiral amino‐alcohols by coupling transketolase and transaminase‐catalyzed reactions in a cascading continuous‐flow microreactor system

Droplet millifluidics for kinetic study of transketolase

Flow‐Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila: Integrated Systems of Reaction Control and Product Purification

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