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16 pages, 3385 KiB

Open AccessArticle

Mutagenesis and Adaptation of the Psychrotrophic Fungus Chrysosporium pannorum A-1 as a Method for Improving β-pinene Bioconversion

by Mateusz Kutyła, Jan Fiedurek, Anna Gromada, Krzysztof Jędrzejewski and Mariusz Trytek

Molecules 2020, 25(11), 2589; https://doi.org/10.3390/molecules25112589 - 02 Jun 2020

Cited by 4 | Viewed by 2506

Abstract

Mutagenesis and adaptation of the psychrotrophic fungus Chrysosporium pannorum A-1 to the toxic substrate β-pinene were used to obtain a biocatalyst with increased resistance to this terpene and improved bioconversion properties. Mutants of the parental strain were induced with UV light and N [...] Read more.

Mutagenesis and adaptation of the psychrotrophic fungus Chrysosporium pannorum A-1 to the toxic substrate β-pinene were used to obtain a biocatalyst with increased resistance to this terpene and improved bioconversion properties. Mutants of the parental strain were induced with UV light and N-methyl-N′-nitro-N-nitrosoguanidine. Mutants resistant to β-pinene were isolated using agar plates with a linear gradient of substrate concentrations. Active mutants were selected based on their general metabolic activity (GMA) expressed as oxygen consumption rate. Compared to the parental strain, the most active mutant showed an enhanced biotransformation ability to convert β-pinene to trans-pinocarveol (315 mg per g of dry mycelium), a 4.3-fold greater biocatalytic activity, and a higher resistance to H₂O₂-induced oxidative stress. Biotransformation using adapted mutants yielded twice as much trans-pinocarveol as the reaction catalyzed by non-adapted mutants. The results indicate that mutagenesis and adaptation of C. pannorum A-1 is an effective method of enhancing β-bioconversion of terpenes. Full article

(This article belongs to the Special Issue Biocatalytic Synthesis of Bioactive Compounds)

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11 pages, 697 KiB

Open AccessArticle

Conversion of Oleic Acid into Azelaic and Pelargonic Acid by a Chemo-Enzymatic Route

by Elisabetta Brenna, Danilo Colombo, Giuseppe Di Lecce, Francesco G. Gatti, Maria Chiara Ghezzi, Francesca Tentori, Davide Tessaro and Mariacristina Viola

Molecules 2020, 25(8), 1882; https://doi.org/10.3390/molecules25081882 - 18 Apr 2020

Cited by 22 | Viewed by 5727

Abstract

A chemo-enzymatic approach for the conversion of oleic acid into azelaic and pelargonic acid is herein described. It represents a sustainable alternative to ozonolysis, currently employed at the industrial scale to perform the reaction. Azelaic acid is produced in high chemical purity in [...] Read more.

A chemo-enzymatic approach for the conversion of oleic acid into azelaic and pelargonic acid is herein described. It represents a sustainable alternative to ozonolysis, currently employed at the industrial scale to perform the reaction. Azelaic acid is produced in high chemical purity in 44% isolation yield after three steps, avoiding column chromatography purifications. In the first step, the lipase-mediated generation of peroleic acid in the presence of 35% H₂O₂ is employed for the self-epoxidation of the unsaturated acid to the corresponding oxirane derivative. This intermediate is submitted to in situ acid-catalyzed opening, to afford 9,10-dihydroxystearic acid, which readily crystallizes from the reaction medium. The chemical oxidation of the diol derivative, using atmospheric oxygen as a stoichiometric oxidant with catalytic quantities of Fe(NO₃)₃∙9∙H₂O, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), and NaCl, affords 9,10-dioxostearic acid which is cleaved by the action of 35% H₂O₂ in mild conditions, without requiring any catalyst, to give pelargonic and azelaic acid. Full article

(This article belongs to the Special Issue Biocatalytic Synthesis of Bioactive Compounds)

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12 pages, 1294 KiB

Open AccessArticle

A Gram-Scale Limonene Production Process with Engineered Escherichia coli

by Jascha Rolf, Mattijs K. Julsing, Katrin Rosenthal and Stephan Lütz

Molecules 2020, 25(8), 1881; https://doi.org/10.3390/molecules25081881 - 18 Apr 2020

Cited by 49 | Viewed by 6045

Abstract

Monoterpenes, such as the cyclic terpene limonene, are valuable and important natural products widely used in food, cosmetics, household chemicals, and pharmaceutical applications. The biotechnological production of limonene with microorganisms may complement traditional plant extraction methods. For this purpose, the bioprocess needs to [...] Read more.

Monoterpenes, such as the cyclic terpene limonene, are valuable and important natural products widely used in food, cosmetics, household chemicals, and pharmaceutical applications. The biotechnological production of limonene with microorganisms may complement traditional plant extraction methods. For this purpose, the bioprocess needs to be stable and ought to show high titers and space-time yields. In this study, a limonene production process was developed with metabolically engineered Escherichia coli at the bioreactor scale. Therefore, fed-batch fermentations in minimal medium and in the presence of a non-toxic organic phase were carried out with E. coli BL21 (DE3) pJBEI-6410 harboring the optimized genes for the mevalonate pathway and the limonene synthase from Mentha spicata on a single plasmid. The feasibility of glycerol as the sole carbon source for cell growth and limonene synthesis was examined, and it was applied in an optimized fermentation setup. Titers on a gram-scale of up to 7.3 g·L_org⁻¹ (corresponding to 3.6 g·L⁻¹ in the aqueous production phase) were achieved with industrially viable space-time yields of 0.15 g·L⁻¹·h⁻¹. These are the highest monoterpene concentrations obtained with a microorganism to date, and these findings provide the basis for the development of an economic and industrially relevant bioprocess. Full article

(This article belongs to the Special Issue Biocatalytic Synthesis of Bioactive Compounds)

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17 pages, 3606 KiB

Open AccessArticle

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity

by Nina-Katharina Krahe, Ralf G. Berger and Franziska Ersoy

Molecules 2020, 25(7), 1536; https://doi.org/10.3390/molecules25071536 - 27 Mar 2020

Cited by 23 | Viewed by 3281

Abstract

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and [...] Read more.

Alkene cleavage is a possibility to generate aldehydes with olfactory properties for the fragrance and flavor industry. A dye-decolorizing peroxidase (DyP) of the basidiomycete Pleurotus sapidus (PsaPOX) cleaved the aryl alkene trans-anethole. The PsaPOX was semi-purified from the mycelium via FPLC, and the corresponding gene was identified. The amino acid sequence as well as the predicted tertiary structure showed typical characteristics of DyPs as well as a non-canonical Mn²⁺-oxidation site on its surface. The gene was expressed in Komagataella pfaffii GS115 yielding activities up to 142 U/L using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) as substrate. PsaPOX exhibited optima at pH 3.5 and 40 °C and showed highest peroxidase activity in the presence of 100 µM H₂O₂ and 25 mM Mn²⁺. PsaPOX lacked the typical activity of DyPs towards anthraquinone dyes, but oxidized Mn²⁺ to Mn³⁺. In addition, bleaching of β-carotene and annatto was observed. Biotransformation experiments verified the alkene cleavage activity towards the aryl alkenes (E)-methyl isoeugenol, α-methylstyrene, and trans-anethole, which was increased almost twofold in the presence of Mn²⁺. The resultant aldehydes are olfactants used in the fragrance and flavor industry. PsaPOX is the first described DyP with alkene cleavage activity towards aryl alkenes and showed potential as biocatalyst for flavor production. Full article

(This article belongs to the Special Issue Biocatalytic Synthesis of Bioactive Compounds)

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15 pages, 2241 KiB

Open AccessArticle

Enrichment of Polyglucosylated Isoflavones from Soybean Isoflavone Aglycones Using Optimized Amylosucrase Transglycosylation

by Young Sung Jung, Ye-Jin Kim, Aaron Taehwan Kim, Davin Jang, Mi-Seon Kim, Dong-Ho Seo, Tae Gyu Nam, Chan-Su Rha, Cheon-Seok Park and Dae-Ok Kim

Molecules 2020, 25(1), 181; https://doi.org/10.3390/molecules25010181 - 01 Jan 2020

Cited by 16 | Viewed by 4521

Abstract

Isoflavones in soybeans are well-known phytoestrogens. Soy isoflavones present in conjugated forms are converted to aglycone forms during processing and storage. Isoflavone aglycones (IFAs) of soybeans in human diets have poor solubility in water, resulting in low bioavailability and bioactivity. Enzyme-mediated glycosylation is [...] Read more.

Isoflavones in soybeans are well-known phytoestrogens. Soy isoflavones present in conjugated forms are converted to aglycone forms during processing and storage. Isoflavone aglycones (IFAs) of soybeans in human diets have poor solubility in water, resulting in low bioavailability and bioactivity. Enzyme-mediated glycosylation is an efficient and environmentally friendly way to modify the physicochemical properties of soy IFAs. In this study, we determined the optimal reaction conditions for Deinococcus geothermalis amylosucrase-mediated α-1,4 glycosylation of IFA-rich soybean extract to improve the bioaccessibility of IFAs. The conversion yields of soy IFAs were in decreasing order as follows: genistein > daidzein > glycitein. An enzyme quantity of 5 U and donor:acceptor ratios of 1000:1 (glycitein) and 400:1 (daidzein and genistein) resulted in high conversion yield (average 95.7%). These optimal reaction conditions for transglycosylation can be used to obtain transglycosylated IFA-rich functional ingredients from soybeans. Full article

(This article belongs to the Special Issue Biocatalytic Synthesis of Bioactive Compounds)

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9 pages, 814 KiB

Open AccessArticle

Mapping the Biotransformation of Coumarins through Filamentous Fungi

by Jainara Santos do Nascimento, Wilson Elias Rozo Núñez, Valmore Henrique Pereira dos Santos, Josefina Aleu, Sílvio Cunha and Eliane de Oliveira Silva

Molecules 2019, 24(19), 3531; https://doi.org/10.3390/molecules24193531 - 29 Sep 2019

Cited by 10 | Viewed by 2668

Abstract

Natural coumarins are present in remarkable amounts as secondary metabolites in edible and medicinal plants, where they display interesting bioactivities. Considering the wide enzymatic arsenal of filamentous fungi, studies on the biotransformation of coumarins using these microorganisms have great importance in green chemical [...] Read more.

Natural coumarins are present in remarkable amounts as secondary metabolites in edible and medicinal plants, where they display interesting bioactivities. Considering the wide enzymatic arsenal of filamentous fungi, studies on the biotransformation of coumarins using these microorganisms have great importance in green chemical derivatization. Several reports on the biotransformation of coumarins using fungi have highlighted the achievement of chemical analogs with high selectivity by using mild and ecofriendly conditions. Prompted by the enormous pharmacological, alimentary, and chemical interest in coumarin-like compounds, this study evaluated the biotransformation of nine coumarin scaffolds using Cunninghamella elegans ATCC 10028b and Aspergillus brasiliensis ATCC 16404. The chemical reactions which were catalyzed by the microorganisms were highly selective. Among the nine studied coumarins, only two of them were biotransformed. One of the coumarins, 7-hydroxy-2,3-dihydrocyclopenta[c]chromen-4(1H)-one, was biotransformed into the new 7,9-dihydroxy-2,3-dihydrocyclopenta[c]chromen-4(1H)-one, which was generated by selective hydroxylation in an unactivated carbon. Our results highlight some chemical features of coumarin cores that are important to biotransformation using filamentous fungi. Full article

(This article belongs to the Special Issue Biocatalytic Synthesis of Bioactive Compounds)

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12 pages, 2095 KiB

Open AccessArticle

Potential Industrial Production of a Well-Soluble, Alkaline-Stable, and Anti-Inflammatory Isoflavone Glucoside from 8-Hydroxydaidzein Glucosylated by Recombinant Amylosucrase of Deinococcus geothermalis

by Te-Sheng Chang, Tzi-Yuan Wang, Szu-Yi Yang, Yu-Han Kao, Jiumn-Yih Wu and Chien-Min Chiang

Molecules 2019, 24(12), 2236; https://doi.org/10.3390/molecules24122236 - 15 Jun 2019

Cited by 21 | Viewed by 3169

Abstract

8-Hydroxydaidzein (8-OHDe), an ortho-hydroxylation derivative of soy isoflavone daidzein isolated from some fermented soybean foods, has been demonstrated to possess potent anti-inflammatory activity. However, the isoflavone aglycone is poorly soluble and unstable in alkaline solutions. To improve the aqueous solubility and stability [...] Read more.

8-Hydroxydaidzein (8-OHDe), an ortho-hydroxylation derivative of soy isoflavone daidzein isolated from some fermented soybean foods, has been demonstrated to possess potent anti-inflammatory activity. However, the isoflavone aglycone is poorly soluble and unstable in alkaline solutions. To improve the aqueous solubility and stability of the functional isoflavone, 8-OHDe was glucosylated with recombinant amylosucrase of Deinococcus geothermalis (DgAS) with industrial sucrose, instead of expensive uridine diphosphate-glucose (UDP-glucose). One major product was produced from the biotransformation, and identified as 8-OHDe-7-α-glucoside, based on mass and nuclear magnetic resonance spectral analyses. The aqueous solubility and stability of the isoflavone glucoside were determined, and the results showed that the isoflavone glucoside was almost 4-fold more soluble and more than six-fold higher alkaline-stable than 8-OHDe. In addition, the anti-inflammatory activity of 8-OHDe-7-α-glucoside was also determined by the inhibition of lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells. The results showed that 8-OHDe-7-α-glucoside exhibited significant and dose-dependent inhibition on the production of nitric oxide, with an IC₅₀ value of 173.2 µM, which remained 20% of the anti-inflammatory activity of 8-OHDe. In conclusion, the well-soluble and alkaline-stable 8-OHDe-7-α-glucoside produced by recombinant DgAS with a cheap substrate, sucrose, as a sugar donor retains moderate anti-inflammatory activity, and could be used in industrial applications in the future. Full article

(This article belongs to the Special Issue Biocatalytic Synthesis of Bioactive Compounds)

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34 pages, 8896 KiB

Open AccessReview

Halogenating Enzymes for Active Agent Synthesis: First Steps Are Done and Many Have to Follow

by Alexander Veljko Fejzagić, Jan Gebauer, Nikolai Huwa and Thomas Classen

Molecules 2019, 24(21), 4008; https://doi.org/10.3390/molecules24214008 - 05 Nov 2019

Cited by 25 | Viewed by 7449

Abstract

Halogens can be very important for active agents as vital parts of their binding mode, on the one hand, but are on the other hand instrumental in the synthesis of most active agents. However, the primary halogenating compound is molecular chlorine which has [...] Read more.

Halogens can be very important for active agents as vital parts of their binding mode, on the one hand, but are on the other hand instrumental in the synthesis of most active agents. However, the primary halogenating compound is molecular chlorine which has two major drawbacks, high energy consumption and hazardous handling. Nature bypassed molecular halogens and evolved at least six halogenating enzymes: Three kind of haloperoxidases, flavin-dependent halogenases as well as α-ketoglutarate and S-adenosylmethionine (SAM)-dependent halogenases. This review shows what is known today on these enzymes in terms of biocatalytic usage. The reader may understand this review as a plea for the usage of halogenating enzymes for fine chemical syntheses, but there are many steps to take until halogenating enzymes are reliable, flexible, and sustainable catalysts for halogenation. Full article

(This article belongs to the Special Issue Biocatalytic Synthesis of Bioactive Compounds)

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