| 1. |
- Enman, Josefine
(författare)
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Fungal production and solid state chemistry of eritadenine : an integrated approach to development of an active pharmaceutical ingredient
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The present thesis demonstrates an integrated approach to the development of a potential active pharmaceutical ingredient, eritadenine, a cholesterol reducing compound originating from the shiitake mushroom (Lentinus edodes). The main areas covered in the thesis are a method for quantification of eritadenine, production of eritadenine by submerged cultivation of fungal mycelia and investigation of the influence of process parameters on mycelial growth and production, and finally solid state characterizations of eritadenine. The usage of the fungus as a source of eritadenine requires an analytical tool for quantification of the compound. An HPLC method was hence developed for identification and quantification of eritadenine, using chemically synthesized eritadenine as a reference. The amount of eritadenine in fruit bodies of selected strains of shiitake was determined and with the method developed in this study, eritadenine concentrations up to ten times higher than previously reported were detected. Since both fruit bodies and mycelia of shiitake have been shown to contain eritadenine submerged cultivation of shiitake mycelia was investigated as an alternative source for this compound. The mycelia were cultivated in various submerged conditions, both in shake flasks and in bioreactors. It was found that both the mycelia and the culture media contained eritadenine, of which the major part was detected in the culture media. While the biomass concentrations were higher in shake flasks, the eritadenine concentrations were considerably higher in the bioreactors, which were assigned to morphological variations. In an attempt to improve the mycelial growth and eritadenine production, a growth promotive substance in the form of a water extract of DDGS, a by-product from drygrind ethanol facilities, was added to the culture media. It was demonstrated that an amendment of the cultivation media with this extract caused a considerable growth promotive effect on shiitake mycelia in bioreactor cultivations, along with enhanced eritadenine production. If eritadenine will be used as a pharmaceutical agent, understanding about the solid state chemistry of the compound is required. Raman spectroscopy is a valuable technique for investigation of structural properties; hence, a Raman reference spectrum with line assignments for the solid state of synthetic eritadenine was established. To further investigate the solid state chemistry of eritadenine, its synthetic analogue was slowly crystallized from water and different ethanol concentrations, at different temperatures. Solids formed from slow cooling of either water or aqueous ethanol showed crystallinity. No polymorphism was detected, irrespective of solvent system or temperature. However, dissimilar thermal behaviours were observed, deducing crystals derived from water as dihydrates and crystals derived from aqueous ethanol as 2.5 hydrates.
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| 2. |
- Enman, Josefine, et al.
(författare)
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Growth promotive conditions for enhanced eritadenine production during submerged cultivation of Lentinus edodes
- 2012
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Ingår i: Journal of chemical technology and biotechnology (1986). - : Wiley. - 0268-2575 .- 1097-4660. ; 87:7, s. 903-907
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Tidskriftsartikel (refereegranskat)abstract
- Background: Mycelium of the medicinal mushroom shiitake, Lentinus edodes, is a potential source for production of the blood cholesterol reducing compound eritadenine. To increase the mycelial biomass and in turn the production of eritadenine, a potential growth promoting substance in the form of a water extract of distillers dried grains with solubles (DDGS) was added to the culture media.Results: The hot water extract of DDGS was shown to considerably increase the growth of shiitake mycelia in bioreactor cultivations; the mycelial yield was 2-3 times higher than in the control, and the highest final biomass concentration obtained was 3.4 g L -1. Further, by using shake flask cultures as inoculums the bioreactor cultivation time could be reduced by 1 week for some of the experiments. The highest final titer of eritadenine in the present study was 25.1 mg L -1, which was about 2 times higher than in the control, and was also obtained when a water extract of DDGS was added to the culture medium.Conclusion: It was demonstrated that a water extract of DDGS promoted the growth of shiitake mycelia in bioreactor cultivations, along with enhanced eritadenine production.
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| 3. |
- Enman, Josefine
(författare)
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Production and quantification of eritadenine, a cholesterol reducing compound in shiitake (Lentinus edodes)
- 2007
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cardiovascular diseases are among the main causes of death in our society and there is a strong correlation between enhanced blood cholesterol levels and the development of such diseases. The popular edible fungus, shiitake mushroom (Lentinus edodes), has been shown to produce a blood cholesterol lowering compound designated eritadenine, and the hypocholesterolemic action of this compound has been quite extensively examined in rats. Eritadenine is suggested to accelerate the removal of blood cholesterol either by stimulating tissue uptake or by inhibiting tissue release; there are no indications of this compound inhibiting the biosynthesis of cholesterol. If shiitake mushrooms are to be used as a source for a potential cholesterol reducing product, it is of great importance to determine the content of eritadenine in the mushrooms as accurately as possible. Hence, in paper I methanol extraction was used to recover as much as possible of the hypocholesterolemic agent from the fungal cells. In order to analyse the target compound, a reliable and reproducible HPLC method for separation, identification and quantification of eritadenine was developed. The amounts of eritadenine in fruit bodies of four commercially cultivated shiitake mushrooms were determined, and the mushrooms under investigation exhibited up to ten times higher levels of eritadenine (3.17-6.33 mg/g dry mushrooms) than previously reported. Not only the fruit bodies of shiitake, but also its mycelia contain eritadenine. Growing fruit bodies of shiitake is a fairly demanding and time consuming process. Hence, in search for a source of eritadenine, submerged (liquid) cultivation of shiitake mycelia could be an alternative. The reason why shiitake mushrooms synthesize eritadenine is yet not clarified; i.e. the function of this secondary metabolite and the growth conditions that favour its production are not elucidated. In addition, like other filamentous fungi, shiitake exhibits different hyphal morphologies in submerged cultures depending on cultivation conditions such as medium composition, temperature, pH, inoculum concentration, dissolved oxygen and shear. The fungal metabolism and hence production of secondary metabolites is in turn affected by the morphology, as have been shown in several studies on filamentous fungi. Submerged cultivation of shiitake mycelia offers a convenient way to change the cultivating conditions in order to improve eritadenine yield and productivity. The study in paper II focused on cultivation of mycelia at different conditions, both in shake flasks and in bioreactors, to investigate the effect of pH and stirring rate on production of eritadenine. The shiitake mycelia were found to produce eritadenine, and the compound of interest was found in both the fungal cells and the growth media. The major part (90-99%) was found in the culture medium, which offers a facilitated downstream processing if large scale production of the compound is to be conducted. The mycelial morphology in the shake flask cultures were macroscopic aggregates, pellets, and the specific productivity of eritadenine was relatively low; 6.56 mg/g dry cell weight (DCW). In the bioreactor cultivations, the mycelia grew as freely dispersed filaments, showing a higher specific productivity than in the shake flasks, ranging between 26.00- 39.58 mg/g DCW. This indicates the influence of morphology on eritadenine production. The biomass yield in shake flasks and bioreactors was in parity; 0.45 g in the shake flasks and 0.25- 0.62 g in the bioreactors. A stirring rate of 50 rpm in the bioreactors was preferable for eritadenine production, whereas for biomass production it was 250 rpm, indicating the influence of agitation on both growth and productivity. The pH did not have any major impact on growth, whereas the specific productivity in the bioreactors was higher when pH was uncontrolled than controlled at 5.7.
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| 4. |
- Enman, Josefine, et al.
(författare)
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Production of the bioactive compound eritadenine by submerged cultivation of shiitake (Lentinus edodes) mycelia
- 2008
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 56:8, s. 2609-2612
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Tidskriftsartikel (refereegranskat)abstract
- Fruit bodies and mycelia of shiitake mushroom (Lentinus edodes) have been shown to contain the cholesterol-reducing compound eritadenine, 2(R),3(R)-dihydroxy-4-(9-adenyl)butyric acid. In the search for a production method for eritadenine, shiitake mycelia were investigated in the present study. The mycelia were cultivated both in shake flasks and in bioreactors, to investigate the effects of pH, stirring rate, and reactor type on the production and distribution of eritadenine. Both the biomass and the culture broth were examined for their eritadenine content. In the shake flasks, the final concentration of eritadenine was 1.76 mg/L and eritadenine was equally distributed between the mycelia and the growth media. In the bioreactors, the shiitake mycelia were found to contain eritadenine in relatively low levels, whereas the majority, 90.6-98.9%, was detected in the growth media. Applying a stirring rate of 250 rpm during bioreactor cultivation resulted in the highest eritadenine concentrations: 10.23 mg/L when the pH was uncontrolled and 9.59 mg/L when the pH was controlled at 5.7. Reducing the stirring rate to 50 rpm resulted in a decreased eritadenine concentration, both at pH 5.7 (5.25 mg/L) and when pH was not controlled (5.50 mg/L). The mycelia in the shake flask cultures appeared as macroscopic aggregates, whereas mycelia cultivated in bioreactors grew more as freely dispersed filaments. This study demonstrates for the first time the extra- and intracellular distribution of eritadenine produced by shiitake mycelial culture and the influence of reactor conditions on the mycelial morphology and eritadenine concentrations.
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| 5. |
- Enman, Josefine, et al.
(författare)
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Quantification of the bioactive compound eritadenine in selected strains of shiitake mushroom (Lentinus edodes)
- 2007
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Ingår i: Journal of Agricultural and Food Chemistry. - : American Chemical Society (ACS). - 0021-8561 .- 1520-5118. ; 55:4, s. 1177-1180
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Tidskriftsartikel (refereegranskat)abstract
- Cardiovascular disease is one of the most common causes of death in the Western world, and a high level of blood cholesterol is considered a risk factor. The edible fungus, shiitake mushroom (Lentinus edodes), contains the hypocholesterolemic agent eritadenine, 2(R),3(R)-dihydroxy-4-(9-adenyl)-butyric acid. This study was conducted to quantify the amount of the cholesterol reducing agent eritadenine in shiitake mushrooms, in search of a potential natural medicine against blood cholesterol. The amounts of eritadenine in the fruit bodies of four different shiitake mushrooms, Le-1, Le-2, Le-A, and Le-B, were investigated in this study. To achieve this goal, methanol extraction was used to recover as much as possible of the hypocholesterolemic agent from the fungal cells. In addition, enzymes that degrade the fungal cell walls were also used to elucidate if the extraction could be further enhanced. To analyze the target compound, a reliable and reproducible HPLC method for separation, identification, and quantification of eritadenine was developed. The shiitake strains under investigation exhibit up to 10 times higher levels of eritadenine than previously reported for other shiitake strains. Further, pretreating the mushrooms with hydrolytic enzymes before methanol extraction resulted in an insignificant increase in the amount of eritadenine released. These results indicate the potential for delivery of therapeutic amounts of eritadenine from the ingestion of extracts or dried concentrates of shiitake mushroom strains.
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| 6. |
- Enman, Josefine, et al.
(författare)
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Raman analysis of synthetic eritadenine
- 2008
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Ingår i: Journal of Raman Spectroscopy. - : Wiley. - 0377-0486 .- 1097-4555. ; 39:10, s. 1464-1468
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Tidskriftsartikel (refereegranskat)abstract
- Eritadenine, 2(R),3(R)-dihydroxy-4-(9-adenyl)-butyric acid, is a cholesterol-reducing compound naturally occurring in the shitake mushroom (Lentinus edodes). To identify the unknown Raman spectrum of this compound, pure synthetic eritadenine was examined and the vibrational modes were assigned by following the synthesis pathway. This was accomplished by comparing the known spectra of the starting compounds adenine and D-ribose with the spectra of a synthesis intermediate, methyl 5-(6-Aminopurin-9H-9-yl)-2,3-O-isopropylidene-5-deoxy-β-D-ribofuranoside (MAIR) and eritadenine. In the Raman spectrum of eritadenine, a distinctive vibrational mode at 773 cm-1 was detected and ascribed to vibrations in the carbon chain, ν(C--C). A Raman line that arose at 1212 cm-1, both in the Raman spectrum of MAIR and eritadenine, was also assigned to ν(C--C). Additional Raman lines detected at 1526 and at 1583 cm-1 in the Raman spectrum of MAIR and eritadenine were assigned to ν(N--C) and a deformation of the purine ring structure. In these cases the vibrational modes are due to the linkage between adenine and the ribofuranoside moiety for MAIR, and between adenine and the carbon chain for eritadenine. This link is also the cause for the disappearance of adenine specific Raman lines in the spectrum of both MAIR and eritadenine. Several vibrations observed in the spectrum of D-ribose were not observed in the Raman spectrum of eritadenine due to the absence of the ribose ring structure. In the Raman spectrum of MAIR some of the D-ribose specific Raman lines disappeared due to the introduction of methyl and isopropylidene moieties to the ribose unit. With the approach presented in this study the so far unknown Raman spectrum of eritadenine could be successfully identified and is presented here for the first time.
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| 7. |
- Enman, Josefine, et al.
(författare)
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Solid state characterization of sodium eritadenate
- 2011
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Ingår i: American Journal of Analytical Chemistry. - : Scientific Research Publishing, Inc.. - 2156-8251 .- 2156-8278. ; 2:2, s. 164-173
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Tidskriftsartikel (refereegranskat)abstract
- Knowledge of the solid state is of great importance in the development of a new active pharmaceutical ingredient, since the solid form often dictates the properties and performance of the drug. In the present study, solid state characteristics of the sodium salt of the candidate cholesterol reducing compound eritadenine, 2(R), 3(R))-dihydroxy-4-(9-adenyl)-butanoic acid, were investigated. The compound was crystallized by slow cooling from water and various aqueous ethanol solutions, at different temperatures. Further, the compound solution was subjected to lyophilization and to high vacuum drying. The resulting solids were screened for polymorphism by micro Raman spectroscopy (λex = 830 nm) and the crystallinity was investigated by X-ray powder diffraction. Further, thermal analysis was applied to study possible occurrence of solvates or hydrates. Solids obtained from slow cooling showed crystallinity, whereas rapid cooling gave rise to more amorphous solids. Analysis of difference spectra of the Raman data for solids obtained from slow cooling of solution revealed subtle differences in the structures between crystals derived from pure water and crystals derived from aqueous ethanol solutions. Finally, from the thermal analysis it was deduced that crystals obtained from pure water were stoichiometrically dihydrates whereas crystals obtained from aqueous ethanol solutions were 2.5 hydrates; this formation of different hydrates were supported by the Raman difference analysis.
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| 8. |
- Faisal, Abrar, et al.
(författare)
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Recovery of l-Arginine from Model Solutions and Fermentation Broth Using Zeolite-Y Adsorbent
- 2019
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Ingår i: ACS Sustainable Chemistry and Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 7:9, s. 8900-8907
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Tidskriftsartikel (refereegranskat)abstract
- Arginine was produced via fermentation of sugars using the engineered microorganism Escherichia coli. Zeolite-Y adsorbents in the form of powder and extrudates were used to recover arginine from both a real fermentation broth and aqueous model solutions. An adsorption isotherm was determined using model solutions and zeolite-Y powder. The saturation loading was determined to be 0.2 g/g using the Sips model. Arginine adsorbed from a real fermentation broth using either zeolite-Y powder or extrudates both showed a maximum loading of 0.15 g/g at pH 11. This adsorbed loading is very close to the corresponding value obtained from the model solution showing that under the experimental conditions the presence of additional components in the broth did not have a significant effect on the adsorption of arginine. Furthermore, a breakthrough curve was determined for extrudates using a 1 wt % arginine model solution. The selectivity for arginine over ammonia and alanine from the real fermentation broth at pH 11 was 1.9 and 8.3, respectively, for powder, and 1.0, and 4.1, respectively, for extrudates. To the best of our knowledge, this is the first time recovery of arginine from real fermentation broths using any type of adsorbent has been reported.
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| 9. |
- Ginesy, Mireille, et al.
(författare)
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Metabolic engineering of Escherichia coli for enhanced arginine biosynthesis
- 2015
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Background: Arginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants. Results: Firstly, argR (encoding an arginine responsive repressor protein), speC, speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO, which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation. Conclusions: In this study, Escherichia coli was successfully engineered for enhanced arginine production. The Delta adiA, Delta speC, Delta speF, Delta argR, Delta argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of Escherichia coli as an industrial producer of arginine.
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| 10. |
- Ginesy, Mireille, et al.
(författare)
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Simultaneous Quantification of L-Arginine and Monosaccharides during Fermentation : An Advanced Chromatography Approach
- 2019
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Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 24:4
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Tidskriftsartikel (refereegranskat)abstract
- Increasing demand for L-arginine by the food and pharmaceutical industries has sparked the search for sustainable ways of producing it. Microbial fermentation offers a suitable alternative; however, monitoring of arginine production and carbon source uptake during fermentation, requires simple and reliable quantitative methods compatible with the fermentation medium. Two methods for the simultaneous quantification of arginine and glucose or xylose are described here: high-performance anion-exchange chromatography coupled to integrated pulsed amperometric detection (HPAEC-IPAD) and reversed-phase ultra-high-performance liquid chromatography combined with charged aerosol detection (RP-UHPLC-CAD). Both were thoroughly validated in a lysogeny broth, a minimal medium, and a complex medium containing corn steep liquor. HPAEC-IPAD displayed an excellent specificity, accuracy, and precision for arginine, glucose, and xylose in minimal medium and lysogeny broth, whereas specificity and accuracy for arginine were somewhat lower in medium containing corn steep liquor. RP-UHPLC-CAD exhibited high accuracy and precision, and enabled successful monitoring of arginine and glucose or xylose in all media. The present study describes the first successful application of the above chromatographic methods for the determination and monitoring of L-arginine amounts during its fermentative production by a genetically modified Escherichia coli strain cultivated in various growth media.
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