Modelling of immobilised enzyme biocatalytic membrane reactor performance

Preez, Ryne du; Clarke, Kim Gail; Callanan, Linda H.; Burton, Stephanie G.

doi:10.1016/j.molcatb.2015.05.015

Cited by 13 publications

(10 citation statements)

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“…In contrast, the use of several chemical steps together will often have differing operational requirements . Using isolated enzymes in biotransformations offers advantages including reduced complexity and cost in downstream processing, fewer side reactions and the removal of rate limiting diffusion across cellular membranes . Most attractively, parameters such as enzyme or substrate concentration, co‐solvents, pH or temperature, can easily be manipulated .…”

Section: Introductionmentioning

confidence: 99%

“…The development of a kinetic model early on in the development process can be invaluable in cost/benefit or feasibility analysis . It permits evidence‐based decision‐making, and critically allows for the identification of bottlenecks and the quantification of process problems (e. g. feedback inhibition or inhibition by side‐products) . Mechanistic models, which seek to describe enzyme mechanisms as accurately as possible, seek to understand a system as well as to predict it.…”

Section: Introductionmentioning

confidence: 99%

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Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts

et al. 2019

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Multi‐step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi‐step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi‐step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts

et al. 2019

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“…It permits evidence-based decision-making (34), and critically allows for the identification of bottlenecks and the quantification of process problems (e.g. feedback inhibition or inhibition by side-products) (6). Mechanistic models, which seek to describe enzyme mechanisms as accurately as possible, seek to understand a system as well as to predict it.…”

Section: Cofactor Regenerationmentioning

confidence: 99%

“…In contrast, the use of several chemical steps together will often have differing operational requirements (4). Using isolated enzymes in biotransformations offers advantages (5) including reduced complexity and cost in downstream processing (6), fewer side reactions (7) and the removal of rate limiting diffusion across cellular membranes (8). Most attractively, parameters such as enzyme or substrate concentration, co-solvents, pH or temperature, can easily be manipulated (9).…”

Section: Introductionmentioning

confidence: 99%

Engineering a seven enzyme biotransformation using mathematical modelling and characterized enzyme parts

Finnigan

Cutlan

Šnajdrová

et al. 2019

Preprint

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Multi-step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi-step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi-step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized. Significance statementThis study examines the challenge of modeling and optimizing multi-enzyme cascades. We detail the development, testing and optimization of a deterministic model of a three enzyme cascade with four cofactor regeneration enzymes. Significantly, the model could be easily used to predict the optimal concentrations of each enzyme in order to get maximum flux through the cascade. This prediction was strongly validated experimentally. The success of our model demonstrates that robust models of systems of at least seven enzymes are † Present address: Manchester readily achievable. We highlight the importance of following good modeling practice to evaluate model quality and limitations. Examining deviations from expected behavior provided additional insight into the model and enzymes. This work provides a template for developing larger deterministic models of enzyme cascades. phosphotransferase (PAP), which allows the regeneration of ADP from AMP (24). The combination of PAP and PPK therefore allowed complete regeneration of ATP from AMP (25). In an alternative approach, adenylate kinase (which catalyzes the reversible phosphorylation of AMP by ATP) was used in place of PPK. Coupling of this enzyme with PAP pushes the equilibrium towards ATP regeneration ( Figure 1B) (26). The application of process modellingSynthetic biology has long promised the use of well-characterized parts for the rational design of new metabolic pathways (27)(28)(29). However the use of modelling to optimize these in vivo reactions is yet to be fully realized (30). In contrast, modelling of enzymes in vitro can be fairly robust and offers solutions for reaction engineering (31). This could allow the combination of enzymes, for which validated mathematical models exist, to be rapidly engineered and tested in silico (2).

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“…Aldolases [10], amidases [11], transaminases [12], oxidases [13,14], peroxidases [15] are some enzymes that have been successfully applied to a continuous-flow approach. Additionally, lipase-mediated esterification [16][17][18][19][20][21], interesterification [22,23], transesterification [24][25][26] and enzymatic kinetic resolution (EKR) of racemic compounds [27][28][29][30][31][32], have demonstrated the potential of combining continuous-flow systems and biocatalysis.…”

mentioning

confidence: 99%

Biocatalysis in continuous-flow mode: A case-study in the enzymatic kinetic resolution of secondary alcohols via acylation and deacylation reactions mediated by Novozym 435®

Thomas¹,

Burich²,

Bandeira³

et al. 2017

Biocatalysis

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Enzymatic kinetic resolution reactions are a well-established way to achieve optically active compounds. When enzymatic reactions are combined to continuous-flow methodologies, other benefits are added, including reproducibility, optimized energy use, minimized waste generation, among others. In this context, we herein report a case study involving lipase-mediated transesterification by acylation and deacylation reactions of secondary alcohols/esters in batch and continuous-flow modes. Acylation reactions were performed with high values of enantiomeric excess (72 up to >99%) and enantioselectivity (E > 200) for both batch and continuous-flow modes. On the other hand, for deacylation reactions using n-butanol as nucleophile, enatiomeric excess ranged between 38 to >99% and E from 6 to >200 were observed for batch mode. For deacylation reactions in continuous-flow mode, results were disappointing, as in some cases, very low or no conversion was observed. Enantiomeric excess ranged from 16 to >99% and enantioselectivity from 5 to >200 were observed. In terms of productivity, continuous-flow mode reactions were superior in both strategies (acylation: r from 1.1 up to 18.1-fold higher, deacylation: 2.8 up to 7.4- fold higher in continuous-flow than in batch mode).

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Modelling of immobilised enzyme biocatalytic membrane reactor performance

Cited by 13 publications

References 13 publications

Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts

Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts

Engineering a seven enzyme biotransformation using mathematical modelling and characterized enzyme parts

Biocatalysis in continuous-flow mode: A case-study in the enzymatic kinetic resolution of secondary alcohols via acylation and deacylation reactions mediated by Novozym 435®

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