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25 pages, 2698 KiB

Open AccessFeature PaperArticle

Modelling Approach for the Continuous Biocatalytic Synthesis of N-Acetylneuraminic Acid in Packed Bed Reactors

by Kristin Hölting, Miriam Aßmann, Paul Bubenheim, Andreas Liese and Jürgen Kuballa

Processes 2024, 12(10), 2191; https://doi.org/10.3390/pr12102191 - 9 Oct 2024

Viewed by 717

Abstract

Continuous flow technologies have become increasingly important for biocatalytic processes. In this study, we present the application and modelling of covalently immobilised N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid lyase in packed bed reactors for the synthesis of N-acetylneuraminic acid. The immobilised [...] Read more.

Continuous flow technologies have become increasingly important for biocatalytic processes. In this study, we present the application and modelling of covalently immobilised N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid lyase in packed bed reactors for the synthesis of N-acetylneuraminic acid. The immobilised enzymes were stable under continuous flow process conditions with half-life times of >28 d (epimerase immobilised on hexamethylamino methacrylate HA403/M) or 58 d (lyase immobilised on dimenthylamino methacrylate ECR8309M), suitable for continuous flow applications. Kinetic studies revealed Michaelis–Menten kinetic behaviour for both enzymes. The kinetic parameters and the inhibitions were analysed under continuous flow conditions and were integrated into a process model using Python. The model was validated by varying flow rates, the mass of immobilised enzymes and the reactor dimensions and shows a low error compared to the measured data. An error accuracy of 6% (epimerase) or 9% (lyase) was achieved. The product concentrations of the enzyme cascade at the end of the packed bed reactor can be predicted with an accuracy of 9% for the calculation of a large column (84.5 mL) or of 24% if several small columns (2.5 mL, 0.8 mL) are connected in series. The developed model has proved to be valid and will be used to optimise the process with respect to substrate concentrations, reactor dimensions and flow rate. Full article

(This article belongs to the Special Issue Development, Modelling and Simulation of Biocatalytic Processes)

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14 pages, 2681 KiB

Open AccessFeature PaperArticle

Efficient Bio-Oxidation of Cellobiose with Engineered Gluconobacter oxydans to Provide Highly Concentrated Cellobionic Acid

by Emmeran Bieringer, Lisa Pütthoff, Arne Zimmermann, Mariana de Souza Góes, Uraz Yilmaz, Armin Ehrenreich, Wolfgang Liebl and Dirk Weuster-Botz

Processes 2024, 12(7), 1464; https://doi.org/10.3390/pr12071464 - 13 Jul 2024

Viewed by 1119

Abstract

Cellobionic acid (CBA) can be obtained through the oxidation of cellobiose, the monomer of cellulose. CBA serves as a plant-based alternative to its stereoisomer lactobionic acid, which is used in the pharmaceutical, cosmetic, and food industries. Gluconobacter oxydans is a well-established whole-cell biocatalyst [...] Read more.

Cellobionic acid (CBA) can be obtained through the oxidation of cellobiose, the monomer of cellulose. CBA serves as a plant-based alternative to its stereoisomer lactobionic acid, which is used in the pharmaceutical, cosmetic, and food industries. Gluconobacter oxydans is a well-established whole-cell biocatalyst with membrane-bound dehydrogenases (mDH) for regio-specific oxidations. As G. oxydans wildtype cells show low cellobiose oxidation activities, the glucose mDH from Pseudomonas taetrolens was overexpressed in G. oxydans BP9, a multi mDH deletion strain. Whole-cell biotransformation studies were performed with resting cells of the engineered G. oxydans in stirred tank bioreactors. Initial biomass specific cellobionate formation rates increased with increasing cellobiose concentrations up to 190 g L⁻¹, and were constant until the solubility limit. The maximal volumetric CBA formation rates and the oxygen uptake rates increased linearly with the concentration of engineered G. oxydans. This enables the estimation of the maximum biocatalyst concentration limited by the maximum oxygen transfer rate of any bioreactor. Thus, 5.2 g L⁻¹ G. oxydans was sufficient to produce 502 g L⁻¹ CBA with >99% yield in a simple aerobic batch process. The highly concentrated CBA will reduce downstream processing costs considerably after cell separation. Full article

(This article belongs to the Special Issue Development, Modelling and Simulation of Biocatalytic Processes)

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13 pages, 2605 KiB

Open AccessArticle

Fluent Integration of Laboratory Data into Biocatalytic Process Simulation Using EnzymeML, DWSIM, and Ontologies

by Alexander S. Behr, Julia Surkamp, Elnaz Abbaspour, Max Häußler, Stephan Lütz, Jürgen Pleiss, Norbert Kockmann and Katrin Rosenthal

Processes 2024, 12(3), 597; https://doi.org/10.3390/pr12030597 - 16 Mar 2024

Cited by 3 | Viewed by 1672

Abstract

The importance of biocatalysis for ecologically sustainable syntheses in the chemical industry and for applications in everyday life is increasing. To design efficient applications, it is important to know the related enzyme kinetics; however, the measurement is laborious and error-prone. Flow reactors are [...] Read more.

The importance of biocatalysis for ecologically sustainable syntheses in the chemical industry and for applications in everyday life is increasing. To design efficient applications, it is important to know the related enzyme kinetics; however, the measurement is laborious and error-prone. Flow reactors are suitable for rapid reaction parameter screening; here, a novel workflow is proposed including digital image processing (DIP) for the quantification of product concentrations, and the use of structured data acquisition with EnzymeML spreadsheets combined with ontology-based semantic information, leading to rapid and smooth data integration into a simulation tool for kinetics evaluation. One of the major findings is that a flexibly adaptive ontology is essential for FAIR (findability, accessibility, interoperability, reusability) data handling. Further, Python interfaces enable consistent data transfer. Full article

(This article belongs to the Special Issue Development, Modelling and Simulation of Biocatalytic Processes)

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13 pages, 689 KiB

Open AccessArticle

Immobilized Lipases—A Versatile Industrial Tool for Catalyzing Transesterification of Phytosterols Solubilized in Plant Oils to Produce Their Fatty Acid Esters

by Sobhi Basheer and Ramez Masri

Processes 2024, 12(2), 307; https://doi.org/10.3390/pr12020307 - 1 Feb 2024

Viewed by 1010

Abstract

The conjugation of phytosterols (PSs) with fatty acids results in producing phytosterol esters (PSEs) characterized by enhanced lipophilicity and improved functional properties of major interest in food and nutraceutical applications. The use of immobilized lipases to catalyze direct transesterification reactions between PSs and [...] Read more.

The conjugation of phytosterols (PSs) with fatty acids results in producing phytosterol esters (PSEs) characterized by enhanced lipophilicity and improved functional properties of major interest in food and nutraceutical applications. The use of immobilized lipases to catalyze direct transesterification reactions between PSs and plant oils to form PSEs as a green alternative to conventional chemical production methods has attracted interest during the last two decades. The low solubility of PSs in common plant oil triglycerides, typically below 3% at ambient temperatures, remains the main challenge for bringing lipase-catalyzed direct transesterification reactions of PSs and oil triglycerides to commercial scales. This study focuses on the enzymatic synthesis of PSEs starting from solubilized PSs at concentrations of up to 30% wt./wt. of oil mixtures comprising fatty acid ethyl esters (FAEEs), monoglycerides (MGs), diglycerides (DGs), and triglycerides (TGs) as a homogeneous medium for the direct transesterification reaction. The results of this study show for the first time that the addition of FAEEs into the reaction medium results in an alteration of the substrate preference of the enzyme, making MGs the favorite fatty acyl group donors for PSs amongst all other fatty acyl donors present in the reaction system. The proposed new enzymatic route allows starting with high concentrations of solubilized PSs, making the direct transesterification of oil glycerides attractive for the production of PSEs at industrial scales. Full article

(This article belongs to the Special Issue Development, Modelling and Simulation of Biocatalytic Processes)

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16 pages, 2867 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Synthesis of 2,6-Dihydroxybenzoic Acid by Decarboxylase-Catalyzed Carboxylation Using CO₂ and In Situ Product Removal

by Daniel Ohde, Benjamin Thomas, Paul Bubenheim and Andreas Liese

Processes 2024, 12(1), 10; https://doi.org/10.3390/pr12010010 - 19 Dec 2023

Viewed by 1317

Abstract

For the enzymatic carboxylation of resorcinol to 2,6-dihydroxybenzoic acid (2,6-DHBA) using gaseous CO₂ in an aqueous triethanolamine phase, an adsorption-based in situ product removal was demonstrated. The aim is to improve the reaction yield, which is limited by an unfavourable thermodynamic equilibrium. [...] Read more.

For the enzymatic carboxylation of resorcinol to 2,6-dihydroxybenzoic acid (2,6-DHBA) using gaseous CO₂ in an aqueous triethanolamine phase, an adsorption-based in situ product removal was demonstrated. The aim is to improve the reaction yield, which is limited by an unfavourable thermodynamic equilibrium. First, a screening for a high-affinity adsorber was carried out. Then, the application of a suitable adsorber was successfully demonstrated. This enabled achieving reaction yields above 80% using the adsorber for in situ product removal. The applied biotransformation was scaled up to 1.5 L at lab-scale. Furthermore, a downstream process based on the elution and purification of the product bound to the adsorber was developed to obtain 2,6-DHBA in high purity. Recycling is one of the key factors in this system, making it possible to recycle the reaction medium, the adsorber and the solvents in additional batches. Full article

(This article belongs to the Special Issue Development, Modelling and Simulation of Biocatalytic Processes)

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16 pages, 1263 KiB

Open AccessFeature PaperArticle

Impact of Deep Eutectic Solvents on Kinetics and Folding Stability of Formate Dehydrogenase

by Nicolás F. Gajardo-Parra, Gabriel Rodríguez, Andrés F. Arroyo-Avirama, Astrit Veliju, Thomas Happe, Roberto I. Canales, Gabriele Sadowski and Christoph Held

Processes 2023, 11(10), 2815; https://doi.org/10.3390/pr11102815 - 22 Sep 2023

Cited by 5 | Viewed by 1449

Abstract

Specifically designed co-solvent mixtures are an efficient way to enhance the kinetics of enzyme-catalyzed reactions without compromising enzyme stability; among them, several deep eutectic solvents have emerged as exciting co-solvent mixtures for biocatalytic reactions. DESs nature allows one to tailor the enzyme-co-solvent interactions [...] Read more.

Specifically designed co-solvent mixtures are an efficient way to enhance the kinetics of enzyme-catalyzed reactions without compromising enzyme stability; among them, several deep eutectic solvents have emerged as exciting co-solvent mixtures for biocatalytic reactions. DESs nature allows one to tailor the enzyme-co-solvent interactions by using DESs constituents of diverse functional groups. In this work, the influence of co-solvents (betaine, glycerol, and sorbitol) and two DESs (betaine:glycerol and betaine:sorbitol) on the kinetics of candida boidinii Formate dehydrogenase was evaluated. The results showed a 30% increase in catalytic efficiency by adding 15 wt.-% of betaine to the buffered aqueous reaction media. Further, cbFDH folded-state stability was evaluated using differential scanning fluorimetry to finally obtain the binding affinity, unfolding curves, and thermodynamic unfolding parameters. The addition of glycerol, sorbitol, and DESs increased cbFDH protection against thermal stress, and this effect could be improved by increasing co-solvent concentrations. Moreover, DESs showed the ability to reduce the irreversibility of the unfolding process. Betaine was the only co-solvent that had a negative stability effect, which was offset by using betaine-based DESs. The latter was a result of the additivity of certain individual co-solvent effects on thermal stability. Non-monotonous stability effects were obtained by adding sorbitol to the buffer solutions, probably because hydrogen bond dynamics between cbFDH/co-solvent/water change dramatically with the amount of water present. Finally, DESs improved NAD

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binding affinity with cbFDH interestingly without direct correlation with the results obtained for kinetics. Full article

(This article belongs to the Special Issue Development, Modelling and Simulation of Biocatalytic Processes)

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12 pages, 1219 KiB

Open AccessFeature PaperArticle

Using Adsorption Energy Distribution for Parameter Estimation of Competitive Cofactor Coupled Enzyme Reaction

by Thomas Waluga and Mirko Skiborowski

Processes 2023, 11(9), 2686; https://doi.org/10.3390/pr11092686 - 7 Sep 2023

Viewed by 1100

Abstract

The chemical and biotechnology industries are facing new challenges in the use of renewable resources. The complex nature of these materials requires the use of advanced techniques to understand the kinetics of reactions in this context. This study presents an interdisciplinary approach to [...] Read more.

The chemical and biotechnology industries are facing new challenges in the use of renewable resources. The complex nature of these materials requires the use of advanced techniques to understand the kinetics of reactions in this context. This study presents an interdisciplinary approach to analyze cofactor coupled enzymatic two-substrate kinetics and competitive two-substrate kinetics in a fast and efficient manner. By studying the adsorption energy distribution (AED), it is possible to determine the individual parameters of the reaction kinetics. In the case of a single alcohol reaction, the AED is able to identify parameters in agreement with the literature with few experimental data points compared to classical methods. In the case of a competitive reaction, AED analysis can automatically determine the number of competing substrates, whereas traditional nonlinear regression requires prior knowledge of this information for parameter identification. Full article

(This article belongs to the Special Issue Development, Modelling and Simulation of Biocatalytic Processes)

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Review

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29 pages, 8224 KiB

Open AccessReview

The Promising Role of Amine Transaminase Cascades in the Synthesis of Non-Canonical Amino Acids

by Najme Gord Noshahri and Jens Rudat

Processes 2024, 12(11), 2566; https://doi.org/10.3390/pr12112566 - 16 Nov 2024

Viewed by 562

Abstract

Amine transaminases (ATA) are critical players in producing non-canonical amino acids, essential building blocks in pharmaceuticals and fine chemicals. Significant progress has been made in discovering and engineering enzymes in this field, enhancing their use in organic synthesis. However, challenges such as co-factor [...] Read more.

Amine transaminases (ATA) are critical players in producing non-canonical amino acids, essential building blocks in pharmaceuticals and fine chemicals. Significant progress has been made in discovering and engineering enzymes in this field, enhancing their use in organic synthesis. However, challenges such as co-factor regeneration, substrate, and product inhibition remain significant limitations to widespread industrial enzyme application. (Chemo-)enzymatic cascades offer efficient and environmentally friendly pathways for synthesizing amino acids, reducing the need for multiple synthesis steps and saving the purification of intermediates. This review focuses specifically on the synthesis of non-canonical amino acids, emphasizing the use of enzymatic and chemoenzymatic cascades involving ATA. Full article

(This article belongs to the Special Issue Development, Modelling and Simulation of Biocatalytic Processes)

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25 pages, 2920 KiB

Open AccessReview

Thermostable α-Amylases and Laccases: Paving the Way for Sustainable Industrial Applications

by Nivedita Jaiswal and Pundrik Jaiswal

Processes 2024, 12(7), 1341; https://doi.org/10.3390/pr12071341 - 27 Jun 2024

Cited by 2 | Viewed by 3037

Abstract

The growing demand in industrial and biotechnological settings for more efficient enzymes with enhanced biochemical features, particularly thermostability and thermotolerance, necessitates a timely response. Renowned for their versatility, thermostable enzymes offer significant promise across a range of applications, including agricultural, medicinal, and biotechnological [...] Read more.

The growing demand in industrial and biotechnological settings for more efficient enzymes with enhanced biochemical features, particularly thermostability and thermotolerance, necessitates a timely response. Renowned for their versatility, thermostable enzymes offer significant promise across a range of applications, including agricultural, medicinal, and biotechnological domains. This comprehensive review summarizes the structural attributes, catalytic mechanisms, and connection between structural configuration and functional activity of two major classes of thermostable enzymes: α-amylases and laccases. These enzymes serve as valuable models for understanding the structural foundation behind the thermostability of proteins. By highlighting the commercial importance of thermostable enzymes and the interest these generate among researchers in further optimization and innovation, this article can greatly contribute to ongoing research on thermostable enzymes and aiding industries in optimizing production processes via immobilization, use of stabilizing additives, chemical modification, protein engineering (directed evolution and mutagenesis), and genetic engineering (through cloning and expression of thermostable genes). It also gives insights to the exploration of suitable strategies and factors for enhancing thermostability like increasing substrate affinity; introducing electrostatic, intramolecular, and intermolecular hydrophobic interactions; mitigating steric hindrance; increasing flexibility of an active site; and N- and C-terminal engineering, thus resulting in heightened multipronged stability and notable enhancements in the enzymes’ industrial applicability. Full article

(This article belongs to the Special Issue Development, Modelling and Simulation of Biocatalytic Processes)

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