| 1. |
- Börner, Tim, et al.
(author)
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A Process Concept for High-Purity Production of Amines by Transaminase-Catalyzed Asymmetric Synthesis: Combining Enzyme Cascade and Membrane-Assisted ISPR
- 2015
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In: Organic Process Research & Development. - : American Chemical Society (ACS). - 1083-6160 .- 1520-586X. ; 19:7, s. 793-799
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Journal article (peer-reviewed)abstract
- For the amine transaminase (ATA)-catalyzed synthesis of chiral amines, the choice of donor substrate is of high importance for reaction and process design. Alanine was investigated as an amine donor for the reductive amination of a poorly water-soluble ketone (4-phenyl-2-butanone) in a combined in situ product removal (ISPR) approach using liquid-membrane extraction together with an enzyme cascade. This ISPR strategy facilitates very high (>98%) product purity with an integrated enrichment step and eliminates product as well as coproduct inhibition. In the presented proof-of-concept alanine shows the following advantages over the other frequently employed amine donor isopropyl amine: (i) nonextractability of alanine affords high product purity without any additional downstream step and no losses via coextraction, (ii) higher maximum reaction rates, and (iii) broader acceptance among ATAs.
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| 2. |
- Börner, Tim
(author)
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Amine transaminase: Process development aspects for chiral amine synthesis
- 2016
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Doctoral thesis (other academic/artistic)abstract
- Many modern drugs contain chiral amine moieties and the need for more efficient processes to synthesize these amines in high optical purity and concentration are greatly demanded by the fine chemical and pharma industry. Amine transaminases (ATA) allow for the direct asymmetric synthesis of optically pure amines from ketone substrates. Although academia and industry have been researching ATAs extensively since more than 20 years, the development of an efficient ATA-based process for chiral amine synthesis remains challenging.In this doctoral work, process development aspects for the ATA-catalyzed synthesis of chiral amines was investigated. The work can be divided into four parts: i) method development in order to obtain quickly detailed kinetic and stability data, ii) identify the inactivation mechanism that renders ATAs inefficient under process conditions, iii) utilize these biochemical information to stabilize ATAs through enzyme and process engineering, and iv) development of a process concept that employs two in situ product removal (ISPR) methods to overcome the thermodynamic and inhibition barrier of ATA-catalyzed reactions.The characterization of ATAs was accelerated by developing an automated high performance liquid chromatography (HPLC) platform. Equipped with a size exclusion column, this HPLC platform constitutes a substrate-independent assay that allows quantification of enzyme-bound and free cofactor species in transamination reactions. The combination of kinetic and stability studies (e.g. via melting point determination) revealed that ATAs suffer from an amine donor-induced inactivation mechanism. Besides cofactor concentration and substrate ratio, the quaternary structure of ATAs was found to affect the operational stability and the tendency to lose the aminated cofactor intermediate (pyridoxamine 5’-phosphate, PMP). The dimeric ATAs from Chromobacterium violaceum and Vibrio fluvialis exhibited lower stability than a novel tetrameric ATA. The tetramer structure stabilizes a key element for cofactor binding and reduces dissociation of PMP during catalysis.Targeted enzyme engineering yielded a mutant with improved stability towards high substrate concentrations, solvent and reaction temperature. The mutant contained two mutations in the cofactor-ring binding motif, which revealed it as a ‘hot spot’ for ATA engineering. Moreover, medium engineering strategies are demonstrated that improve ATA stability and productivity, e.g. employing optimal substrate ratios.A novel process concept was developed to address the unfavorable reaction equilibrium, product and coproduct inhibition as well as amine product purity. This integrated process concept uses alanine as amine donor in combination with two ISPR methods, i.e. a three-phase extraction system to recover the inhibiting amine product (1-methyl-3-phenylpropylamine) and an enzymatic cascade reaction to remove the inhibiting coproduct (pyruvate). The double ISPR concept yields the amine product in high purity (≥98%) without any additional purification step. Advantageously, even when poorly water-soluble ketone substrates (here: benzylacetone) are used, the amine product can be enriched in situ to high concentrations, which will ease the downstream process.
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| 3. |
- Börner, Tim, et al.
(author)
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Explaining Operational Instability of Amine Transaminases : Substrate-Induced Inactivation Mechanism and Influence of Quaternary Structure on Enzyme-Cofactor Intermediate Stability
- 2017
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In: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 7:2, s. 1259-1269
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Journal article (peer-reviewed)abstract
- The insufficient operational stability of amine transaminases (ATA) constitutes a limiting factor for high productivity in chiral amine synthesis. In this work, we investigated the operational stability of a tetrameric ATA with 92% sequence identity to a Pseudomonas sp. transaminase and compared it to the two commonly used dimeric ATAs from Chromobacterium violaceum and Vibrio fluvialis. In the presence of substrate, all three ATAs featured reduced stability in comparison to their resting stability, but the tetramer showed slower inactivation rates than the dimeric ATAs. Kinetic and thermodynamic analysis revealed an amine donor induced inactivation mechanism involving accumulation of the less stable aminated enzyme-cofactor intermediate. Dissociation of the enzyme-PMP complex forms the unstable apoenzyme, which can rapidly unfold. Crystal structure analysis shed light on the structure-function relationship suggesting that the cofactor-ring binding element is stabilized in the quaternary structure conferring higher operational stability by minimizing PMP leakage and apoenzyme formation. In contrast to the common practice, increasing the amine acceptor content improved the stability and substrate turnover of dimeric ATAs. An extra supply of the pyridoxal cofactor (PLP) enhanced the stability of dimeric and tetrameric ATAs but reduced the transamination activity. The ATA inactivation mechanism described here provides valuable aspects for both process development and protein engineering.
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| 4. |
- Börner, Tim, et al.
(author)
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Generic HPLC platform for automated enzyme reaction monitoring : Advancing the assay toolbox for transaminases and other PLP-dependent enzymes
- 2016
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In: Biotechnology Journal. - : Wiley. - 1860-6768. ; 11:8, s. 1025-1036
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Journal article (peer-reviewed)abstract
- Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.
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| 5. |
- Börner, Tim, et al.
(author)
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Hydrophobic Complexation Promotes Enzymatic Surfactant Synthesis from Alkyl Glucoside/Cyclodextrin Mixtures
- 2014
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In: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 4:8, s. 2623-2634
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Journal article (peer-reviewed)abstract
- The unique ability of cyclodextrin glycosyltransferase to form and utilize the cyclic maltooligosaccharide cyclodextrin (CD) makes this enzyme an attractive catalyst for the synthesis of alkyl glycosides. Here, we characterize the sugar headgroup elongation of alkyl glucosides (acceptor) via two transglycosylation reactions from either a linear (maltohexose) or a cyclic (CD) glycosyl donor. Inclusion complex formation overcomes both poor substrate solubility and aggregation. We have used pure alkyl glucosides and alpha CD as model compounds. The complex between CD and alkyl glucoside was efficiently used as a substrate. Kinetic and thermodynamic measurements allow the prediction of the optimal synthesis conditions. This optimum corresponds to the transition between a donor-limiting and an acceptor-limiting regime. The resulting rational design should lead to the practical development of a cost-efficient industrial synthesis. Our findings with respect to the importance of complexation should also readily apply to other enzymatic systems.
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| 6. |
- Börner, Tim, et al.
(author)
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Three in One : Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase
- 2017
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In: ChemBioChem. - : Wiley. - 1439-4227. ; 18:15, s. 1482-1486
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Journal article (peer-reviewed)abstract
- Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5′-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.
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| 7. |
- Conroy, Melanie, et al.
(author)
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Uncertainty in humanities network visualization
- 2024
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In: Frontiers in Communication. - : Frontiers Media S.A.. - 2297-900X. ; 8
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Journal article (peer-reviewed)abstract
- Network visualization is one of the most widely used tools in digital humanities research. The idea of uncertain or “fuzzy” data is also a core notion in digital humanities research. Yet network visualizations in digital humanities do not always prominently represent uncertainty. In this article, we present a mathematical and logical model of uncertainty as a range of values which can be used in network visualizations. We review some of the principles for visualizing uncertainty of different kinds, visual variables that can be used for representing uncertainty, and how these variables have been used to represent different data types in visualizations drawn from a range of non-humanities fields like climate science and bioinformatics. We then provide examples of two diagrams: one in which the variables displaying degrees of uncertainty are integrated into the graph and one in which glyphs are added to represent data certainty and uncertainty. Finally, we discuss how probabilistic data and what-if scenarios could be used to expand the representation of uncertainty in humanities network visualizations.
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| 8. |
- Heintz, Søren, et al.
(author)
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Development of in situ product removal strategies in biocatalysis applying scaled-down unit operations
- 2017
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592. ; 114:3, s. 600-609
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Journal article (peer-reviewed)abstract
- An experimental platform based on scaled-down unit operations combined in a plug-and-play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs). An on-line HPLC system was applied to avoid manual sample handling and to semi-automatically characterize ω-transaminases in a scaled-down packed-bed reactor (PBR) module, showing MPPA as a strong inhibitor. To overcome the inhibition, a two-step liquid–liquid extraction (LLE) ISPR concept was tested using scaled-down unit operations combined in a plug-and-play manner. Through the tested ISPR concept, it was possible to continuously feed the main substrate benzylacetone (BA) and extract the main product MPPA throughout the reaction, thereby overcoming the challenges of low substrate solubility and product inhibition. The tested ISPR concept achieved a product concentration of 26.5 gMPPA · L−1, a purity up to 70% gMPPA · gtot −1 and a recovery in the range of 80% mol · mol−1 of MPPA in 20 h, with the possibility to increase the concentration, purity, and recovery further. Biotechnol. Bioeng. 2017;114: 600–609.
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| 9. |
- Van Daele, Timothy, et al.
(author)
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Application of iterative robust model-based optimal experimental design for the calibration of biocatalytic models
- 2017
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In: Biotechnology Progress. - : Wiley. - 8756-7938. ; 33:5, s. 1278-1293
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Journal article (peer-reviewed)abstract
- The aim of model calibration is to estimate unique parameter values from available experimental data, here applied to a biocatalytic process. The traditional approach of first gathering data followed by performing a model calibration is inefficient, since the information gathered during experimentation is not actively used to optimize the experimental design. By applying an iterative robust model-based optimal experimental design, the limited amount of data collected is used to design additional informative experiments. The algorithm is used here to calibrate the initial reaction rate of an ω-transaminase catalyzed reaction in a more accurate way. The parameter confidence region estimated from the Fisher Information Matrix is compared with the likelihood confidence region, which is not only more accurate but also a computationally more expensive method. As a result, an important deviation between both approaches is found, confirming that linearization methods should be applied with care for nonlinear models.
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