Flow synthesis of phenylserine using threonine aldolase immobilized on Eupergit support

Tibhe, J.; Fu, Huili; Noël, Timothy; Wang, Q Qi; Meuldijk, J.; Hessel, Volker

doi:10.3762/bjoc.9.254

Cited by 23 publications

(19 citation statements)

References 49 publications

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“…Mass transfer effects were assessed through the Damköhler number (Da), which establishes the ratio of the maximum reaction rate to the maximum rate of diffusion [61]:…”

Section: Mass Transfer Effectsmentioning

confidence: 99%

Sucrose Hydrolysis in a Bespoke Capillary Wall-Coated Microreactor

2017

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Abstract:Microscale technology has been increasingly used in chemical synthesis up to production scale, but in biocatalysis the implementation has been proceeding at a slower pace. In this work, the design of a low cost and versatile continuous flow enzyme microreactor is described that illustrates the potential of microfluidic reactors for both the development and characterization of biocatalytic processes. The core structure of the developed reactor consists of an array of capillaries with 450 µm of inner diameter with their inner surface functionalized with (3-aminopropyl)triethoxysilane (APTES) and glutaraldehyde where Saccharomyces cerevisiae invertase was covalently bound. The production of invert sugar syrup through enzymatic sucrose hydrolysis was used as model system. Once the microreactor assembly reproducibility and the immobilized enzyme behavior were established, the evaluation of the immobilized enzyme kinetic parameters was carried out at flow rates ranging from 20.8 to 219.0 µL·min −1 and substrate concentrations within 2.0%-10.0% (w/v). Despite the impact of immobilization on the kinetic parameters, viz. K m(app) was increased two fold and K cat showed a 14-fold decrease when compared to solution phase invertase, the immobilization proved highly robust. For a mean residence time of 48.8 min, full conversion of 5.0% (w/v) sucrose was observed over 20 days.

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“…Mass transfer effects were assessed through the Damköhler number (Da), which establishes the ratio of the maximum reaction rate to the maximum rate of diffusion [61]:…”

Section: Mass Transfer Effectsmentioning

confidence: 99%

Sucrose Hydrolysis in a Bespoke Capillary Wall-Coated Microreactor

2017

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“…Determination of DL-threo-β-phenylserine concentration was carried out as described by Tibhe et al (2013) by HPLC using a chiral column (Chirex 3126 (D)-penicillamine Column 250× 4.6 mm) under the following conditions: 75 % 2 mM CuSO 4 solution + 25 % methanol at a flow rate of 1 mL/min. Detection of products was achieved at a wavelength of 254 nm.…”

Section: Determination Of Dl-threo-β-phenylserine Via Hplc Analyticsmentioning

confidence: 99%

Development of a growth-dependent selection system for identification of l-threonine aldolases

Bulut

Gröger

Hummel

2015

Appl Microbiol Biotechnol

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Threonine aldolases (TAs) are useful enzymes for the synthesis of β-hydroxy-α-amino acids due to their capability to catalyze asymmetric aldol reactions. Starting from two prochiral compounds, an aldehyde and glycine, two chiral stereocenters were formed in a single step via C-C bond formation. Owing to poor diastereoselectivity and low activity, the enzymatic synthesis of β-hydroxy-α-amino acids by TAs is still a challenge. For identification of new TAs, a growth-dependent selection system in Pseudomonas putida KT2440 has been developed. This bacterium is able to use aromatic compounds such as benzaldehyde, which is the cleavage product of the TA-mediated retro-aldol reaction of phenylserine, as sole carbon source via the β-ketoadipate pathway. With DL-threo-β-phenylserine as sole carbon source, this strain showed only slight growth in minimal medium. This growth deficiency can be restored by introducing and expressing genes encoding TAs. In order to develop a highly efficient selection system, the gene taPp of P. putida KT2440 encoding a TA was successfully deleted by replacement with an antibiotic resistance cassette. Different growth studies were carried out to prove the operability of the selection system. Genes encoding for L- and D-specific TAs (L-TA genes of Escherichia coli (ltaE) and Saccharomyces cerevisiae (gly1) and D-TA gene of Achromobacter xylosoxidans (dtaAX)) were introduced into the selection strain P. putida KT2440ΔtaPp, followed by cultivation on minimal medium supplemented with DL-threo-β-phenylserine. The results demonstrate that only the selection strains with plasmid-encoded L-TAs were able to grow on this racemic amino acid, whereas the corresponding strain harboring the gene coding for a D-specific TA showed no growth. In summary, it can be stated that a powerful screening tool was developed to identify easily by growth new L-specific threonine aldolases or other enzymes from genomic or metagenomic libraries liberating benzaldehyde.

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“…Enantioselective enzyme syntheses using slug flow (Koch et al, 2008a(Koch et al, , 2008b or single-phase flow (Bartha-Vari et al, 2015;Kataoka et al, 2010;Kawakami et al, 2012;Tibhe et al, 2013) arrangements have been carried out in microreactors. Almost enantiopure cyanohydrins were synthesized by hydroxynitril lyase in a slug flow microreactor (Koch et al, 2008a(Koch et al, , 2008b.…”

Section: Introductionmentioning

confidence: 99%

“…Lipase immobilized in monolith and tubular microreactors allowed for significant increase of enantioselectivity of transesterification reactions (Kataoka et al, 2010;Kawakami et al, 2012). Recently, Tibhe et al (2013) carried out synthesis of L-phenylserine from glycine and benzaldehyde catalyzed by LTA ( Fig. 1) in two types of microreactors.…”

Section: Introductionmentioning

confidence: 99%

Aldolase catalyzed L-phenylserine synthesis in a slug-flow microfluidic system – Performance and diastereoselectivity studies

Čech

Hessel

Přibyl

2017

Chemical Engineering Science

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L-phenylserine was produced by threonine aldolase in slug-flow microfluidic system. Substrate benzaldehyde was continuously supplied from the organic phase. TBME solvent provides mild conditions and high product concentration. MeTHF solvent provides high concentration of L-syn-phenylserine. Toluene provides low value of syn-anti forms ratio in short residence time. a b s t r a c tWe study synthesis of L-phenylserine catalyzed by the enzyme L-threonine aldolase in a slug-flow microfluidic system. Slug-flow arrangement allows for the continuous refilling of sparingly soluble substrate (benzaldehyde) into an aqueous reaction mixture. We identified suitable composition of an organic phase to provide stable slug-flow in a wide range of operational parameters. Solvent screening revealed that tert-butyl methyl ether (TBME) as the organic solvent provides the most friendly environment for L-phenylserine synthesis due to a low degree of enzyme deactivation and high benzaldehyde concentration in the reaction mixture. The effects of substrate concentrations, enzyme concentration, and dimethylsulfoxide (DMSO) concentration on the L-phenylserine concentration in the product stream were examined and proper reaction conditions were identified. Experimental results on the L-phenylserine diastereoselectivity demonstrate that the amount of syn-conformation of L-phenylserine increases with the reaction time. High syn-to anti-concentration ratio is achieved with 2-methyltetrahydrofuran (MeTHF) solvent in a system with long residence time and, finally, low syn-to anticoncentration ratio is provided by toluene environment and short residence time. (J. Čech), v.hessel@tue.nl (V. Hessel), pribylm@vscht.cz (M. Přibyl). Please cite this article as: Čech, J., et al., Aldolase catalyzed L-phenylserine synthesis in a slug-flow microfluidic system -Performance and diastereoselectivity studies. Chem. Eng. Sci. (2016), http://dx.

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Flow synthesis of phenylserine using threonine aldolase immobilized on Eupergit support

Cited by 23 publications

References 49 publications

Sucrose Hydrolysis in a Bespoke Capillary Wall-Coated Microreactor

Sucrose Hydrolysis in a Bespoke Capillary Wall-Coated Microreactor

Development of a growth-dependent selection system for identification of l-threonine aldolases

Aldolase catalyzed L-phenylserine synthesis in a slug-flow microfluidic system – Performance and diastereoselectivity studies

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