Immobilized Whole-Cell Transaminase Biocatalysts for Continuous-Flow Kinetic Resolution of Amines

Immobilization of transaminases creates promising biocatalysts for production of chiral amines in batch or continuous-flow mode reactions. E. coli cells containing overexpressed transaminases of various selectivities and hollow silica microspheres as supporting agent were immobilized by an improved sol-gel process to produce immobilized transaminase biocatalysts with suitable stability and mechanical properties for continuous-flow applications. The immobilized cell-based transaminase biocatalyst proved to be durable and easy-to-use in kinetic resolution of four racemic amines 1a–d. The batch and continuous-flow mode kinetic resolutions with transaminase biocatalyst of opposite stereopreference provided access to both enantiomers of the corresponding amines. By using the most suitable immobilized transaminase biocatalysts, this study describes the first transaminase-based approach for the production of both pure enantiomers of 1-(3,4-dimethoxyphenyl)ethan-1-amine 1d.

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Lipase B from Candida antarctica Immobilized on Epoxy-functionalized Hollow Silica Microspheres: Efficient Biocatalysts for Enantiomer Selective Acylation of Alcohols and Amines

Periodica Polytechnica Chemical Engineering ◽

10.3311/ppch.12517 ◽

2018 ◽

Vol 62 (4) ◽

Cited By ~ 2

Author(s):

Márk Oláh ◽

Szandra Suba ◽

Zoltán Boros ◽

Péter Kovács ◽

Mathilde Gosselin ◽

...

Keyword(s):

Continuous Flow ◽

Kinetic Resolution ◽

Operation Time ◽

Candida Antarctica ◽

Effect Of Temperature ◽

Flow Mode ◽

Silica Microspheres ◽

Lipase B ◽

Hollow Silica ◽

Racemic Amines

Hollow silica microspheres with promising physical properties (MAT540TM) as support for enzyme immobilization and biocatalyst were investigated in this study. The amine-functionalized MAT540TM was activated by six bisepoxides inclosing different spacers and used as epoxy-functionalized carrier for immobilization of lipase B from Candida antarctica (CaLB). The novel, covalently fixed CaLB biocatalysts were compared in kinetic resolution (KR) of racemic 1-phenyethanol rac-1 and five racemic amines rac-3a-e using shaken flasks and continuous-flow packed-bed microreactors. Mechanic stability, re-usability and the effect of temperature (0–90 °C) on productivity and enantiomer selectivity of the covalently immobilized CaLB were investigated. The best performing CaLB biocatalyst showed good mechanic stability after 24 h operation time in continuous-flow mode at 60 °C and provided in KRs of racemic 1-phenyethanol rac-1 with vinyl acetate and of five racemic amines with isopropyl 2-ethoxyacetate as acylating agent the non-reacted (S)-alcohol [(S)-1] or (S)-amines [(S)-3a-e] and the forming (R)-ester [(R)-2] or (R)-amide [(R)-4a-e] in good yields with high enantiomeric excess (ee > 99 %, for all).

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Enantioselective Transamination in Continuous Flow Mode with Transaminase Immobilized in a Macrocellular Silica Monolith

10.20944/preprints201701.0053.v1 ◽

2017 ◽

Author(s):

Ludivine van den Biggelaar ◽

Patrice Soumillion ◽

Damien P. Debecker

Keyword(s):

Continuous Flow ◽

Preferential Flow ◽

Kinetic Resolution ◽

Flow Reactor ◽

Sol Gel ◽

Flow Mode ◽

Chiral Amine ◽

Silica Monoliths ◽

Covalent Grafting ◽

Heterogeneous Biocatalysts

ω-Transaminases have been immobilized on macrocellular silica monoliths and used as heterogeneous biocatalysts in a continuous flow mode enantioselective transamination reaction. The support was prepared by a sol-gel method based on emulsion-templating. The enzyme was immobilized on the structured silica monoliths both by adsorption, and by covalent grafting using amino-functionalized silica monoliths and glutaraldehyde as a coupling agent. A simple reactor set-up based on the use of a heat-shrinkable Teflon tube is presented and successfully used for the continuous flow kinetic resolution of a chiral amine, 4-bromo-α-methylbenzylamine. The porous structure of the supports ensures effective mass transfer and the reactor works in the plug flow regime without preferential flow paths. When immobilized in the monolith and used in the flow reactor, transaminases retain their activity and their enantioselectivity. The solid biocatalyst is also shown to be stable both on stream and during storage. These essential features pave the way to the successful development of an environmentally friendly process for chiral amines production.

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Enantioselective Transamination in Continuous Flow Mode with Transaminase Immobilized in a Macrocellular Silica Monolith

10.20944/preprints201701.0053.v2 ◽

2017 ◽

Author(s):

Ludivine van den Biggelaar ◽

Patrice Soumillion ◽

Damien P. Debecker

Keyword(s):

Continuous Flow ◽

Preferential Flow ◽

Kinetic Resolution ◽

Flow Reactor ◽

Sol Gel ◽

Flow Mode ◽

Chiral Amine ◽

Silica Monoliths ◽

Covalent Grafting ◽

Heterogeneous Biocatalysts

ω-Transaminases have been immobilized on macrocellular silica monoliths and used as heterogeneous biocatalysts in a continuous flow mode enantioselective transamination reaction. The support was prepared by a sol-gel method based on emulsion templating. The enzyme was immobilized on the structured silica monoliths both by adsorption, and by covalent grafting using amino-functionalized silica monoliths and glutaraldehyde as a coupling agent. A simple reactor set-up based on the use of a heat-shrinkable Teflon tube is presented and successfully used for the continuous flow kinetic resolution of a chiral amine, 4-bromo-α-methylbenzylamine. The porous structure of the supports ensures effective mass transfer and the reactor works in the plug flow regime without preferential flow paths. When immobilized in the monolith and used in the flow reactor, transaminases retain their activity and their enantioselectivity. The solid biocatalyst is also shown to be stable both on stream and during storage. These essential features pave the way to the successful development of an environmentally friendly process for chiral amines production.

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Tailored sol–gel immobilized lipase preparates for the enzymatic kinetic resolution of heteroaromatic alcohols in batch and continuous flow systems

RSC Advances ◽

10.1039/c7ra10157k ◽

2017 ◽

Vol 7 (83) ◽

pp. 52977-52987 ◽

Cited By ~ 1

Author(s):

Mădălina Elena Moisă ◽

Cristina Georgiana Spelmezan ◽

Cristina Paul ◽

Judith Hajnal Bartha-Vári ◽

László Csaba Bencze ◽

...

Keyword(s):

Continuous Flow ◽

Kinetic Resolution ◽

Immobilized Lipase ◽

Sol Gel ◽

Flow Mode ◽

Batch Mode ◽

Flow Reactors ◽

Immobilized Lipases ◽

Enzymatic Kinetic Resolution ◽

Continuous Flow Reactors

The EKR of some heteroaromatic secondary ethanols with tailored sol–gel immobilized lipases in batch and continuous-flow reactors was studied. The productivity in continuous-flow mode is higher than in batch mode.

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Continuous Flow Synthesis of Chiral Amines in Organic Solvents: Immobilization of E. coli Cells Containing Both ω-Transaminase and PLP

Organic Letters ◽

10.1021/ol502712v ◽

2014 ◽

Vol 16 (23) ◽

pp. 6092-6095 ◽

Cited By ~ 75

Author(s):

Leandro H. Andrade ◽

Wolfgang Kroutil ◽

Timothy F. Jamison

Keyword(s):

Organic Solvents ◽

Continuous Flow ◽

Chiral Amines ◽

Flow Synthesis ◽

E Coli

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Continuous Flow Mode Biocatalytic Transamination Using Macrocellular Silica Monoliths: Optimizing Support Functionalisation and Enzyme Grafting

10.26434/chemrxiv.7853552.v1 ◽

2019 ◽

Author(s):

Ludivine van den Biggelaar ◽

Patrice Soumillion ◽

Damien Debecker

Keyword(s):

Enzyme Immobilization ◽

Continuous Flow ◽

Sol Gel ◽

Fold Increase ◽

Covalent Attachment ◽

Flow Mode ◽

Active Materials ◽

Silica Monoliths ◽

Transamination Reaction ◽

Reported Data

<div>Transaminases are immobilized onto macrocellular silica monoliths and used for carrying a continuous flow mode transamination reaction. Monoliths were prepared via an emulsion-templated sol-gel method and functionalized by amino-moieties (APTES) in order to covalently immobilize the enzymes, using glutaraldehyde as a cross-linking agent. In order to obtain higher performance and improved reproducibility, we investigate the key parameters of APTES functionalization and of enzyme grafting. Four functionalization protocols were studied. It is shown that controlling the moisture levels in monolith and in the functionalisation solution led to a 3-fold increase in activity as compared to the previously reported data, and greatly improved the reproducibility. Additionally, we report a strong beneficial effect of running the enzyme immobilization at room temperature instead of 4°C, further enhancing the obtained activity. Finally, the popular method which consists in stabilizing the covalent attachment of the enzyme by reducing the imine bonds formed between the enzyme and the functionalized surface was investigated. We highlight a strong enzyme deactivation caused by cyanoborohydride, making this strategy irrelevant in this case. All in all, the improvements presented here for enzyme immobilization in macrocellular silica monoliths, lead to the preparation of more active materials for continuous flow mode biocatalysis.<br></div>

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