Studies on the enzymatic synthesis of N-acetylneuraminic acid with continuously operated enzyme membrane reactors on a milliliter scale

Schmideder, Andreas; Schottroff, Felix; Klermund, Ludwig; Castiglione, Kathrin; Weuster‐Botz, Dirk

doi:10.1016/j.bej.2016.12.007

Cited by 8 publications

(16 citation statements)

References 34 publications

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“…Lastly, it can support the making of fundamental choices in the development of enzyme cascade transformations, in particular which reactions should be telescoped in one pot and which rather not (e.g., Klermund et al, 2017;Rexer et al, 2020). The NAL cascade provides an interesting example for it could involve synthesis of Neu5Ac (Kragl et al, 1991;Lin et al, 2013;Lv et al, 2017;Schmideder et al, 2017;Tao et al, 2011) spatiotemporally separated from the sialoside formation, or integrated with it as shown here.…”

Section: Discussionmentioning

confidence: 90%

Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis

Schelch

Eibinger

Belduma

et al. 2021

Biotech & Bioengineering

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Sialo-oligosaccharides are important products of emerging biotechnology for complex carbohydrates as nutritional ingredients. Cascade bio-catalysis is central to the development of sialo-oligosaccharide production systems, based on isolated enzymes or whole cells. Multienzyme transformations have been established for sialooligosaccharide synthesis from expedient substrates, but systematic engineering analysis for the optimization of such transformations is lacking. Here, we show a mathematical modeling-guided approach to 3ʹ-sialyllactose (3SL) synthesis from N-acetyl-D-neuraminic acid (Neu5Ac) and lactose in the presence of cytidine 5ʹ-triphosphate, via the reactions of cytidine 5ʹ-monophosphate-Neu5Ac synthetase and α2,3-sialyltransferase. The Neu5Ac was synthesized in situ from N-acetyl-Dmannosamine using the reversible reaction with pyruvate by Neu5Ac lyase or the effectively irreversible reaction with phosphoenolpyruvate by Neu5Ac synthase.We show through comprehensive time-course study by experiment and modeling that, due to kinetic rather than thermodynamic advantages of the synthase reaction, the 3SL yield was increased (up to 75%; 10.4 g/L) and the initial productivity doubled (15 g/L/h), compared with synthesis based on the lyase reaction. We further show model-based optimization to minimize the total loading of protein (saving: up to 43%) while maintaining a suitable ratio of the individual enzyme activities to achieve 3SL target yield (61%-75%; 7-10 g/L) and overall productivity (3-5 g/L/h).Collectively, our results reveal the principal factors of enzyme cascade efficiency for 3SL synthesis and highlight the important role of engineering analysis to make multienzyme-catalyzed transformations fit for oligosaccharide production.

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Section: Discussionmentioning

confidence: 90%

Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis

Schelch

Eibinger

Belduma

et al. 2021

Biotech & Bioengineering

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“…Since AGE was inhibited by pyruvate and Neu5Ac, cascades of EMRs employing both enzymes in each reactor were implemented to reduce the influence of a product inhibition. Neu5Ac was obtained as the final product at lower conversion (33 %) and yield (25 %), but better space‐time yield (199 mg Neu5Ac 1/L.h) …”

Section: In Vitro Cascades Requiring For Each Step a Biocatalystmentioning

confidence: 99%

“…With the application of enzyme membrane reactors (EMRs), a similar concept was reported for the two‐step synthesis of Neu5Ac . For continuous operation mode, the reactor was filled with 15.5 mL potassium phosphate buffer (100 mM, pH 7.5, T=30 °C) containing the substrates GlcNAc (50 mM) and pyruvate (100 mM), as well as the allosteric activator ATP (1 mM) without consuming it.…”

Section: In Vitro Cascades Requiring For Each Step a Biocatalystmentioning

confidence: 99%

Extending Designed Linear Biocatalytic Cascades for Organic Synthesis

2018

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Artificial cascade reactions involving biocatalysts have demonstrated a tremendous potential during the recent years. This review just focuses on selected examples of the last year and putting them into context to a previously published suggestion for classification. Subdividing the cascades according to the number of catalysts in the linear sequence, and classifying whether the steps are performed simultaneous or in a sequential fashion as well as whether the reaction sequence is performed in vitro or in vivo allows to organise the concepts. The last year showed, that combinations of in vivo as well as in vitro are possible. Incompatible reaction steps may be run in a sequential fashion or by compartmentalisation of the incompatible steps either by using special reactors (membrane), polymersomes or flow techniques.

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“…The EMRs allow continuous enzymatic processes to run for longer periods of time (> 85 h) under stable process conditions, with good parallel reproducibility. In this study, a mutant of Anabaena variabilis AGE enzyme, which showed an improved tolerance for pyruvate, was used along with NAL from E. coli K12 and reported an efficient production of NeuAc with space-time yield of 198.5 ± 7.8 mg L h −1 when cascades of EMRs were used to minimize enzymes inhibition by final product [68].…”

Section: Two-step Enzymatic Bio-catalysis For Neuac Productionmentioning

confidence: 99%

“…Furthermore, the retention of costly enzymes is required to accomplish a cost-effective production process, and enzyme immobilization is considered an efficient approach for enzyme retention. Enzyme membrane reactors (EMRs) are widely used for enzyme immobilization, in addition to other approaches [68]. Kragle et al [3] and Schmideder et al [68] used two enzymes immobilized on an enzyme membrane reactor (EMR) for effective NeuAc synthesis.…”

Section: Enzyme Immobilizationmentioning

confidence: 99%

Enzymatic production of N-acetylneuraminic acid: advances and perspectives

Hussain

Zhang

Basharat

et al. 2021

Syst Microbiol and Biomanuf

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N-Acetyl-d-neuraminic acid (NeuAc), a well-known and well-studied sialic acid, is found in cell surface glycolipids and glycoproteins, where it performs a variety of biological functions. The use of NeuAc as a nutraceutical for infant brain development and as an intermediate for pharmaceutical production demands its production on an industrial scale. Natural extraction, chemical synthesis, enzymatic synthesis, and biosynthesis are the methods used for NeuAc production. Among these methods, enzymatic synthesis using N-acetyl-glucosamine (GlcNAc) 2-epimerase (AGE) for epimerization and N-acetyld-neuraminic acid lyase (NAL) for aldol condensation, has been reported to produce NeuAc with high production efficiency. In this review, we discuss advances in the two-step enzymatic synthesis of NeuAc using pyruvate and GlcNAc as substrates. The major challenges in producing NeuAc with high yield are highlighted, including multiple parameter-dependent processes, undesirable reversibility, and diminished solubility of AGEs and NALs. Further, different strategies applied to overcome the limitations of the two-step enzymatic production are discussed, such as pyruvate concentration and temperature shift during the process to increase conversion yield, use of mathematical and computational simulations for process optimization, enzyme engineering to make enzymes highly efficient, and the use of tags and chaperones to increase enzyme solubility. We suggest future directions and the strategies that can be followed to improve enzymatic synthesis of NeuAc.

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Studies on the enzymatic synthesis of N-acetylneuraminic acid with continuously operated enzyme membrane reactors on a milliliter scale

Cited by 8 publications

References 34 publications

Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis

Engineering analysis of multienzyme cascade reactions for 3ʹ‐sialyllactose synthesis

Extending Designed Linear Biocatalytic Cascades for Organic Synthesis

Enzymatic production of N-acetylneuraminic acid: advances and perspectives

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