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14 pages, 2251 KiB

Open AccessFeature PaperArticle

Expanding the Application Range of Microbial Oxidoreductases by an Alcohol Dehydrogenase from Comamonas testosteroni with a Broad Substrate Spectrum and pH Profile

by Daniel Bakonyi, Christine Toelzer, Michael Stricker, Werner Hummel, Karsten Niefind and Harald Gröger

Catalysts 2020, 10(11), 1281; https://doi.org/10.3390/catal10111281 - 4 Nov 2020

Cited by 3 | Viewed by 3534

Abstract

Alcohol dehydrogenases catalyse the conversion of a large variety of ketone substrates to the corresponding chiral products. Due to their high regio- and stereospecificity, they are key components in a wide range of industrial applications. A novel alcohol dehydrogenase from Comamonas testosteroni (CtADH) [...] Read more.

Alcohol dehydrogenases catalyse the conversion of a large variety of ketone substrates to the corresponding chiral products. Due to their high regio- and stereospecificity, they are key components in a wide range of industrial applications. A novel alcohol dehydrogenase from Comamonas testosteroni (CtADH) was identified in silico, recombinantly expressed and purified, enzymatically and biochemically investigated as well as structurally characterized. These studies revealed a broad pH profile and an extended substrate spectrum with the highest activity for compounds containing halogens as substituents and a moderate activity for bulky–bulky ketones. Biotransformations with selected ketones—performed with a coupled regeneration system for the co-substrate NADPH—resulted in conversions of more than 99% with all tested substrates and with excellent enantioselectivity for the corresponding S-alcohol products. CtADH/NADPH/substrate complexes modelled on the basis of crystal structures of CtADH and its closest homologue suggested preliminary hints to rationalize the enzyme’s substrate preferences Full article

(This article belongs to the Special Issue Multi-Step Syntheses in Biology & Chemistry)

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13 pages, 1661 KiB

Open AccessFeature PaperArticle

A Multi-Enzyme Cascade for the Production of High-Value Aromatic Compounds

by Claudia Engelmann, Jens Johannsen, Thomas Waluga, Georg Fieg, Andreas Liese and Paul Bubenheim

Catalysts 2020, 10(10), 1216; https://doi.org/10.3390/catal10101216 - 20 Oct 2020

Cited by 16 | Viewed by 4495

Abstract

Cascade reactions are the basis of life in nature and are adapted to research and industry in an increasing manner. The focus of this study is the production of the high-value aromatic ester cinnamyl cinnamate, which can be applied in flavors and fragrances. [...] Read more.

Cascade reactions are the basis of life in nature and are adapted to research and industry in an increasing manner. The focus of this study is the production of the high-value aromatic ester cinnamyl cinnamate, which can be applied in flavors and fragrances. A three-enzyme cascade was established to realize the synthesis, starting from the corresponding aldehyde with in situ cofactor regeneration in a two-phase system. After characterization of the enzymes, a screening with different organic solvents was carried out, whereby xylene was found to be the most suitable solvent for the second phase. The reaction stability of the formate dehydrogenase (FDH) from Candida boidinii is the limiting step during cofactor regeneration. However, the applied enzyme cascade showed an overall yield of 54%. After successful application on lab scale, the limitation by the FDH was overcome by immobilization of the enzymes and an optimized downstream process, transferring the cascade into a miniplant. The upscaling resulted in an increased yield for the esterification, as well as overall yields of 37%. Full article

(This article belongs to the Special Issue Multi-Step Syntheses in Biology & Chemistry)

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9 pages, 2227 KiB

Open AccessCommunication

Catalytic Asymmetric Chlorination of β-Ketoesters Using N-PFB-PyBidine-Zn(OAc)₂

by Junma Ma, Takumi Suzuki, Satoru Kuwano and Takayoshi Arai

Catalysts 2020, 10(10), 1177; https://doi.org/10.3390/catal10101177 - 13 Oct 2020

Cited by 2 | Viewed by 2739

Abstract

A PyBidine-Zn(OAc)₂ complex catalyzed asymmetric chlorination of β-ketoesters. With assistance of NaHCO₃, a newly developed N-pentafluorobenzyl-PyBidine (N-PFB-PyBidine)-Zn(OAc)₂ catalyst promoted the reaction of α-benzyl-β-ketoesters with N-chlorosuccinimide (NCS) to give the chlorinated products with up to 82% [...] Read more.

A PyBidine-Zn(OAc)₂ complex catalyzed asymmetric chlorination of β-ketoesters. With assistance of NaHCO₃, a newly developed N-pentafluorobenzyl-PyBidine (N-PFB-PyBidine)-Zn(OAc)₂ catalyst promoted the reaction of α-benzyl-β-ketoesters with N-chlorosuccinimide (NCS) to give the chlorinated products with up to 82% ee. Results of a mechanistic study suggested that zinc-enolate of β-ketoesters was formed on the basic (N-PFB-PyBidine)-Zn(OAc)₂ catalyst. The α-chlorinated-β-ketoester was successfully transformed into the chiral epoxide through sequential asymmetric chlorination/cyano-epoxidation in a one-pot synthesis. Full article

(This article belongs to the Special Issue Multi-Step Syntheses in Biology & Chemistry)

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19 pages, 3154 KiB

Open AccessFeature PaperArticle

Immobilization of Aldoxime Dehydratases and Their Use as Biocatalysts in Aqueous Reaction Media

by Alessa Hinzmann, Michael Stricker and Harald Gröger

Catalysts 2020, 10(9), 1073; https://doi.org/10.3390/catal10091073 - 17 Sep 2020

Cited by 12 | Viewed by 4118

Abstract

Immobilization of biocatalysts is a current topic in research enabling the easy recovery of catalysts from the reaction medium after the reaction, and it is often accompanied by a stabilization of the catalysts, which enables recycling. Within our ongoing research on the utilization [...] Read more.

Immobilization of biocatalysts is a current topic in research enabling the easy recovery of catalysts from the reaction medium after the reaction, and it is often accompanied by a stabilization of the catalysts, which enables recycling. Within our ongoing research on the utilization of aldoxime dehydratases in the cyanide-free synthesis of nitriles through dehydration of readily available aldoximes, a screening of different immobilization methods for free enzymes was performed. The applied immobilization methods are based on covalent binding and hydrophobic interactions of the enzyme with the carrier material and whole-cell immobilization in calcium alginate beads with and without subsequent coating. In our study, we found that the immobilization with purified free aldoxime dehydratases from OxdRE (Rhodococcus erythropolis) and OxdB (Bacillus sp. strain OxB-1) leads to high immobilization efficiencies, but also to a strong loss of activity with a residual activity of <20%, regardless of the carrier material used. However, when using whole cells for immobilization instead of purified enzymes, we could increase the residual activity significantly. Escherichia coli BL21(DE3)-CodonPlus-RIL OxdRE and OxdB whole cells were entrapped in calcium alginate beads and coated with silica using tetraethylorthosilicate (TEOS), leading to immobilized catalysts with up to 75% residual activity and a higher stability compared to the free whole cells. Even after three rounds of recycling, which corresponds to a 3 d reaction time, the immobilized OxdB whole cells showed a residual activity of 85%. Full article

(This article belongs to the Special Issue Multi-Step Syntheses in Biology & Chemistry)

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15 pages, 1587 KiB

Open AccessArticle

Flux Enforcement for Fermentative Production of 5-Aminovalerate and Glutarate by Corynebacterium glutamicum

by Carsten Haupka, Baudoin Delépine, Marta Irla, Stephanie Heux and Volker F. Wendisch

Catalysts 2020, 10(9), 1065; https://doi.org/10.3390/catal10091065 - 16 Sep 2020

Cited by 22 | Viewed by 4811

Abstract

Bio-based plastics represent an increasing percentage of the plastics economy. The fermentative production of bioplastic monomer 5-aminovalerate (5AVA), which can be converted to polyamide 5 (PA 5), has been established in Corynebacterium glutamicum via two metabolic pathways. l-lysine can be converted to [...] Read more.

Bio-based plastics represent an increasing percentage of the plastics economy. The fermentative production of bioplastic monomer 5-aminovalerate (5AVA), which can be converted to polyamide 5 (PA 5), has been established in Corynebacterium glutamicum via two metabolic pathways. l-lysine can be converted to 5AVA by either oxidative decarboxylation and subsequent oxidative deamination or by decarboxylation to cadaverine followed by transamination and oxidation. Here, a new three-step pathway was established by using the monooxygenase putrescine oxidase (Puo), which catalyzes the oxidative deamination of cadaverine, instead of cadaverine transaminase. When the conversion of 5AVA to glutarate was eliminated and oxygen supply improved, a 5AVA titer of 3.7 ± 0.4 g/L was reached in microcultivation that was lower than when cadaverine transaminase was used. The elongation of the new pathway by 5AVA transamination by GABA/5AVA aminotransferase (GabT) and oxidation by succinate/glutarate semialdehyde dehydrogenase (GabD) allowed for glutarate production. Flux enforcement by the disruption of the l-glutamic acid dehydrogenase-encoding gene gdh rendered a single transaminase (GabT) in glutarate production via the new pathway responsible for nitrogen assimilation, which increased the glutarate titer to 7.7 ± 0.7 g/L, i.e., 40% higher than with two transaminases operating in glutarate biosynthesis. Flux enforcement was more effective with one coupling site, thus highlighting requirements regarding the modularity and stoichiometry of pathway-specific flux enforcement for microbial production. Full article

(This article belongs to the Special Issue Multi-Step Syntheses in Biology & Chemistry)

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11 pages, 2091 KiB

Open AccessFeature PaperArticle

Enhancement of S-Adenosylmethionine-Dependent Methylation by Integrating Methanol Metabolism with 5-Methyl-Tetrahydrofolate Formation in Escherichia coli

by Kenji Okano, Yu Sato, Shota Inoue, Shizuka Kawakami, Shigeru Kitani and Kohsuke Honda

Catalysts 2020, 10(9), 1001; https://doi.org/10.3390/catal10091001 - 2 Sep 2020

Cited by 10 | Viewed by 5451

Abstract

S-Adenosylmethionine (SAM)-dependent methyltransferases are important tools for the biocatalytic methylation of diverse biomolecules. Methylation by a whole-cell biocatalyst allows the utilization of intrinsic SAM and its regeneration system, which consists of a cyclic and multi-step enzymatic cascade. However, low intracellular availability of [...] Read more.

S-Adenosylmethionine (SAM)-dependent methyltransferases are important tools for the biocatalytic methylation of diverse biomolecules. Methylation by a whole-cell biocatalyst allows the utilization of intrinsic SAM and its regeneration system, which consists of a cyclic and multi-step enzymatic cascade. However, low intracellular availability of 5-methyl-tetrahydrofolate (5-methyl-THF), which functions as a methyl group donor, limits SAM regeneration. Here, we integrated methanol metabolism with 5-methyl-THF formation into SAM-dependent methylation system in Escherichia coli, driven by heterologously expressed methanol dehydrogenase (MDH). The coupling of MDH-catalyzed methanol oxidation with the E. coli endogenous reactions enhances the formation of 5-methyl-THF using methanol as a source of methyl group, thereby promoting both the SAM regeneration and methylation reactions. Co-expression of the mutant MDH2 from Cupriavidus necator N-1 with the O-methyltransferase 5 from Streptomyces avermitilis MA-4680 enhanced O-methylation of esculetin 1.4-fold. Additional overexpression of the E. coli endogenous 5,10-methylene-THF reductase, which catalyzes the last step of 5-methyl-THF formation, further enhanced the methylation reaction by 1.9-fold. Together with deregulation of SAM biosynthesis, the titer of methylated compounds was increased about 20-fold (from 0.023 mM to 0.44 mM). The engineered E. coli strain with enhanced 5-methyl-THF formation is now available as a chassis strain for the production of a variety of methylated compounds. Full article

(This article belongs to the Special Issue Multi-Step Syntheses in Biology & Chemistry)

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8 pages, 6439 KiB

Open AccessCommunication

Vanadium(V) Complex-Catalyzed One-Pot Synthesis of Phenanthridines via a Pictet-Spengler-Dehydrogenative Aromatization Sequence

by Makoto Sako, Romain Losa, Tomohiro Takiishi, Giang Vo-Thanh, Shinobu Takizawa and Hiroaki Sasai

Catalysts 2020, 10(8), 860; https://doi.org/10.3390/catal10080860 - 2 Aug 2020

Cited by 8 | Viewed by 4808

Abstract

Phenanthridine and its derivatives are important structural motifs that exist in natural products, biologically active compounds, and functional materials. Here, we report a mild, one-pot synthesis of 6-arylphenanthridine derivatives by a sequential cascade Pictet-Spengler-dehydrogenative aromatization reaction mediated by oxovanadium(V) complexes under aerobic conditions. [...] Read more.

Phenanthridine and its derivatives are important structural motifs that exist in natural products, biologically active compounds, and functional materials. Here, we report a mild, one-pot synthesis of 6-arylphenanthridine derivatives by a sequential cascade Pictet-Spengler-dehydrogenative aromatization reaction mediated by oxovanadium(V) complexes under aerobic conditions. The reaction of 2-(3,5-dimethoxyphenyl)aniline with a range of commercially available aryl aldehydes provided the desired phenanthridine derivatives in up to 96% yield. The ability of vanadium(V) complexes to function as efficient redox and Lewis acid catalysts enables the sequential reaction to occur under mild conditions. Full article

(This article belongs to the Special Issue Multi-Step Syntheses in Biology & Chemistry)

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11 pages, 2761 KiB

Open AccessFeature PaperArticle

Aldoxime Dehydratase Mutants as Improved Biocatalysts for a Sustainable Synthesis of Biorenewables-Based 2-Furonitrile

by Ji Eun Choi, Suguru Shinoda, Yasuhisa Asano and Harald Gröger

Catalysts 2020, 10(4), 362; https://doi.org/10.3390/catal10040362 - 26 Mar 2020

Cited by 14 | Viewed by 3305

Abstract

2-Furonitrile is an interesting nitrile product for the chemical industry due to its use as intermediate in the field of fine chemicals and pharmaceuticals or as a potential sweetener, as well as due to its access from biorenewables. As an alternative to current [...] Read more.

2-Furonitrile is an interesting nitrile product for the chemical industry due to its use as intermediate in the field of fine chemicals and pharmaceuticals or as a potential sweetener, as well as due to its access from biorenewables. As an alternative to current processes based on, e.g., the ammoxidation of furfural with ammonia as a gas phase reaction running at > 400 °C, we recently reported an enzymatic dehydration of 2-furfuryl aldoxime being obtained easily from furfural and hydroxylamine. However, improving the catalytic properties of the aldoxime dehydratase biocatalyst from Rhodococcus sp. YH3-3 (OxdYH3-3) in terms of activity and stability remained a challenge. In this contribution, the successful development of aldoxime dehydratase OxdYH3-3 mutants that were generated by directed evolution and its enhanced activity toward 2-furfuryl aldoxime is reported. The mutant OxdYH3-3 N266S showed an improved activity of up to six times higher than the wild type when utilizing a substrate concentration of 50–100 mM of 2-furfuryl aldoxime. Full article

(This article belongs to the Special Issue Multi-Step Syntheses in Biology & Chemistry)

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Review

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13 pages, 3156 KiB

Open AccessReview

Fatty Acid Hydratases: Versatile Catalysts to Access Hydroxy Fatty Acids in Efficient Syntheses of Industrial Interest

by Jana Löwe and Harald Gröger

Catalysts 2020, 10(3), 287; https://doi.org/10.3390/catal10030287 - 3 Mar 2020

Cited by 27 | Viewed by 6342

Abstract

The utilization of hydroxy fatty acids has gained more and more attention due to its applicability in many industrial building blocks that require it, for example, polymers or fragrances. Furthermore, hydroxy fatty acids are accessible from biorenewables, thus contributing to a more sustainable [...] Read more.

The utilization of hydroxy fatty acids has gained more and more attention due to its applicability in many industrial building blocks that require it, for example, polymers or fragrances. Furthermore, hydroxy fatty acids are accessible from biorenewables, thus contributing to a more sustainable raw material basis for industrial chemicals. Therefore, a range of investigations were done on fatty acid hydratases (FAHs), since these enzymes catalyze the addition of water to an unsaturated fatty acid, thus providing an elegant route towards hydroxy-substituted fatty acids. Besides the discovery and characterization of fatty acid hydratases (FAHs), the design and optimization of syntheses with these enzymes, the implementation in elaborate cascades, and the improvement of these biocatalysts, by way of mutation in terms of the substrate scope, has been investigated. This mini-review focuses on the research done on process development using fatty acid hydratases as a catalyst. It is notable that biotransformations, running at impressive substrate loadings of up to 280 g L⁻¹, have been realized. A further topic of this mini-review is the implementation of fatty acid hydratases in cascade reactions. In such cascades, fatty acid hydratases were, in particular, combined with alcohol dehydrogenases (ADH), Baeyer-Villiger monooxygenases (BVMO), transaminases (TA) and hydrolases, thus enabling access to a broad variety of molecules that are of industrial interest. Full article

(This article belongs to the Special Issue Multi-Step Syntheses in Biology & Chemistry)

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