The application of reaction engineering to biocatalysis

Ringborg, Rolf Hoffmeyer; Woodley, John M.

doi:10.1039/c5re00045a

Cited by 56 publications

(53 citation statements)

References 108 publications

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“…Of these, polyphosphate kinase makes use of the cheapest and most stable substrate, polyphosphate. Polyphosphate is also a substrate for polyphosphate-AMP Indeed, in developing new biocatalytic processes the use of kinetic modelling is widely advocated, yet often not used in process development (32,33). The development of a kinetic model early on in the development process can be invaluable in cost/benefit or feasibility analysis (33).…”

Section: Cofactor Regenerationmentioning

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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Section: Cofactor Regenerationmentioning

confidence: 99%

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

Finnigan

Cutlan

Šnajdrová

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…In this way it would be possible to direct screening to focus on evolving improved enzymatic kinetic properties, which are ideal for process implementation. To realize such a scheme, it is necessary to develop an automated characterization system . Herein, we present one such system focused on collecting kinetic data for oxygen‐dependent enzymes.…”

Section: Figurementioning

confidence: 99%

Automated Determination of Oxygen‐Dependent Enzyme Kinetics in a Tube‐in‐Tube Flow Reactor

2017

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Enzyme‐mediated oxidation is of particular interest to synthetic organic chemists. However, the implementation of such systems demands knowledge of enzyme kinetics. Conventionally collecting kinetic data for biocatalytic oxidations is fraught with difficulties such as low oxygen solubility in water and limited oxygen supply. Here, we present a novel method for the collection of such kinetic data using a pressurized tube‐in‐tube reactor, operated in the low‐dispersed flow regime to generate time‐series data, with minimal material consumption. Experimental development and validation of the instrument revealed not only the high degree of accuracy of the kinetic data obtained, but also the necessity of making measurements in this way to enable the accurate evaluation of high K MO enzyme systems. For the first time, this paves the way to integrate kinetic data into the protein engineering cycle.

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“…Once identified, directed process and protein engineering strategies to address these bottlenecks may be identified and implemented . Hence, reaction characterisation is fundamental to the identification of bottlenecks during enzymatic process development . It follows that, if such information is gained at an early stage in process development, industrial implementation will proceed at a faster pace, which can alone determine the success of many processes, especially in the pharmaceutical industry .…”

Section: Introductionmentioning

confidence: 99%

“…[4] Hence, reaction characterisation is fundamentaltothe identification of bottlenecks during enzymatic process development. [5,6] It follows that, if such information is gained at an early stage in process development, industrial implementation will proceed at af aster pace, which can alone determine the successo fm any processes, especially in the pharmaceuticali ndustry. [7] Central to this characterisation is the necessity for accurate analytics.…”

Section: Introductionmentioning

confidence: 99%

Online Measurement of Oxygen‐Dependent Enzyme Reaction Kinetics

2017

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As the application of biocatalysis to complement conventional chemical and catalytic approaches continues to expand, an increasing number of reactions involve poorly water‐soluble substrates. At required industrial concentrations necessary for industrial implementation, this frequently leads to heterogeneous reaction mixtures composed of multiple phases. Such systems are challenging to sample, and therefore, it is problematic to measure representative component concentrations. Herein, an online method for following the progress of oxygen‐dependent reactions through accurate measurement of the oxygen mass balance in the gas phase of a reactor is demonstrated and validated. The method was successfully validated and demonstrated by using two model reactions: firstly, the oxidation of glucose by glucose oxidase and, secondly, the Baeyer–Villiger oxidation of macrocyclic ketones to lactones. Initial reaction rate constants and time‐course progressions calculated from the oxygen mass balance were validated against conventional online methods of dissolved oxygen tension and pH titration measurements. A feasible operating window and the sensitivity to dynamic changes of reaction rates were established by controlling oxygen transfer through the operating parameters of the reactor. Such kinetic data forms the basis for reaction characterisation, from which bottlenecks may be made evident and directed improvement strategies can be identified and implemented.

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The application of reaction engineering to biocatalysis

Abstract: Biocatalysis is a growing area of synthetic and process chemistry with the ability to deliver not only improved processes for the synthesis of existing compounds, but also new routes to new compounds.

Cited by 56 publications

References 108 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

Automated Determination of Oxygen‐Dependent Enzyme Kinetics in a Tube‐in‐Tube Flow Reactor

Online Measurement of Oxygen‐Dependent Enzyme Reaction Kinetics

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