| 1. |
- Antonopoulou, Io, 1989-
(författare)
-
Development of biocatalytic processes for selective antioxidant production
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases that under normal conditions catalyze the hydrolysis of the ester bond between hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, p-coumaric acid) and sugar residues in plant cell walls. Based on their specificity towards monoferulates and diferulates, substitutions on the phenolic ring and on their amino acid sequence identity, they have been classified into four types (A-D) while phylogenetic analysis has resulted in classification into thirteen subfamilies (SF1-13). Under low water content, these enzymes are able to catalyze the esterification of hydroxycinnamic acids or the transesterification of their esters (donor) with alcohols or sugars (acceptor) resulting in compounds with modified lipophilicity, having a great potential for use in the tailor-made modification of natural antioxidants for cosmetic, cosmeceutical and pharmaceutical industries. The work described in this thesis focused on the selection,characterization and application of FAEs for the synthesis of bioactive esters with antioxidant activity in non-conventional media. The basis of the current classification systems was investigated in relation with the enzymes’ synthetic and hydrolytic abilities while the developed processes were evaluated for their efficiency and sustainability.Paper I was dedicated to the screening and evaluation of the synthetic abilities of 28 fungal FAEs using acceptors of different lipophilicity at fixed conditions in detergentless microemulsions. It was revealed that FAEs classified in phylogenetic subfamilies related to acetyl xylan esterases (SF5 and 6) showed increased transesterification rates and selectivity. In general, FAEs showed preference on more hydrophilic alcohol acceptors and in descending order to glycerol > 1-butanol > prenol. Homology modeling and small molecule docking simulations were employed as tools for the identification of a potential relationship between the predicted surface and active site properties of selected FAEs and the transesterification selectivity.Papers II- IV focused on the characterization of eight promising FAEs and the optimization of reaction conditions for the synthesis of two bioactive esters (prenyl ferulate and L-arabinose ferulate) in detergentless microemulsions. The effect of the medium composition, the donor and acceptor concentration, the enzyme load, the pH, the temperature and the agitation on the transesterification yield and selectivity were investigated. It was observed that the acceptor concentration and enzyme load were crucial parameters for selectivity. Fae125 (Type A, SF5) iiexhibited highest prenyl ferulate yield (81.1%) and selectivity (4.685) converting 98.5% of VFA to products after optimization at 60 mM VFA, 1.5 M prenol, 0.04 mg FAE mL-1, 40oC, 24 h, 53.4:43.4:3.2 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. On the other hand, FaeA1 (Type A, SF5) showed highest L-arabinose ferulate yield (52.2 %) and selectivity (1.120) at 80 mM VFA, 55 mM L-arabinose, 0.02 mg FAE mL-1, 50oC, 8 h, 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0.In paper V, the effect of reaction media on the enzyme stability and transesterification yield and selectivity was studied in different solvents for the synthesis of two bioactive esters: prenyl ferulate and L-arabinose ferulate. The best performing enzyme (Fae125) was used in the optimization of reaction conditions in the best solvent (n-hexane) via response surface methodology. Both bioconversions were best described by a two-factor interaction model while optimal conditions were determined as the ones resulting in highest yield and selectivity.Highest prenyl ferulate yield (87.5%) and selectivity (7.616) were observed at 18.56 mM prenol mM-1VFA, 0.04 mg FAE mL-1, 24.5 oC, 24.5 h, 91.8: 8.2 v/v n-hexane: 100 mM sodium acetate pH 4.7. Highest L-arabinose ferulate yield (56.2%) and selectivity (1.284) were observed at 2.96 mM L-arabinose mM-1VFA, 0.02 mg FAE mL-1, 38.9 oC, 12 h, 90.5: 5.0: 4.5 v/v/v n-hexane: dimethyl sulfoxide: 100 mM sodium acetate pH 4.7. The enzyme could be reused for six consecutive reaction cycles maintaining 66.6% of its initial synthetic activity. The developed bioconversions showed exceptional biocatalyst productivities (> 300 g product g-1FAE) and the waste production was within the range of pharmaceutical processes.Paper VI focused on the investigation of the basis of the type A classification of a well-studied FAE from Aspergillus niger(AnFaeA) by comparing its activity towards methyl and arabinose hydroxycinnamic acid esters. For this purpose, L-arabinose ferulateand caffeate were synthesized enzymatically. kcat/Kmratios revealed that AnFaeA hydrolyzed arabinose ferulate 1600 times and arabinose caffeate 6.5 times more efficiently than methyl esters. This study demonstrated that short alkyl chain hydroxycinnamate esters which are used nowadays for FAE classification can lead to activity misclassification, while L-arabinose esters could potentially substitute synthetic esters in classification describing more adequately the enzyme specificitiesin the natural environment.
|
|
| 2. |
- Karnaouri, Anthi C, et al.
(författare)
-
Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates
- 2017
-
Ingår i: Biotechnology for Biofuels. - : BioMed Central (BMC). - 1754-6834. ; 10:1
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundFilamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates.ResultsIn the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced MtEG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed.ConclusionsThe endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported.
|
|
| 3. |
- Muraleedharan, Madhu Nair, et al.
(författare)
-
Effect of lignin fractions isolated from different biomass sources on cellulose oxidation by fungal lytic polysaccharide monooxygenases
- 2018
-
Ingår i: Biotechnology for Biofuels. - London : BMC. - 1754-6834. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundLytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant lignocellulose in the presence of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. One of the possible systems that provide electrons to the LPMOs active site and promote the polysaccharide degradation involves the mediation of phenolic agents, such as lignin, low-molecular-weight lignin-derived compounds and other plant phenols. In the present work, the interaction of the bulk insoluble lignin fraction extracted from pretreated biomass with LPMOs and the ability to provide electrons to the active site of the enzymes is studied.ResultsThe catalytic efficiency of three LPMOs, namely MtLPMO9 with C1/C4 regioselectivity, PcLPMO9D which is a C1 active LPMO and NcLPMO9C which is a C4 LPMO, was evaluated in the presence of different lignins. It was correlated with the physicochemical and structural properties of lignins, such as the molecular weight and the composition of aromatic and aliphatic hydroxyl groups. Moreover, the redox potential of lignins was determined with the use of large amplitude Fourier Transform alternating current cyclic voltammetry method and compared to the formal potential of the Cu (II) center in the active site of the LPMOs, providing more information about the lignin-LPMO interaction. The results demonstrated the existence of low-molecular weight lignin-derived compounds that are diffused in the reaction medium, which are able to reduce the enzyme active site and subsequently utilize additional electrons from the insoluble lignin fraction to promote the LPMO oxidative activity. Regarding the bulk lignin fractions, those isolated from the organosolv pretreated materials served as the best candidates in supplying electrons to the soluble compounds and, finally, to the enzymes. This difference, based on biomass pretreatment, was also demonstrated by the activity of LPMOs on natural substrates in the presence and absence of ascorbic acid as additional reducing agent.ConclusionsLignins can support the action of LPMOs and serve indirectly as electron donors through low-molecular-weight soluble compounds. This ability depends on their physicochemical and structural properties and is related to the biomass source and pretreatment method.
|
|
| 4. |
- Xiros, Charilaos, 1973, et al.
(författare)
-
Hydrolysis and fermentation for cellulosic ethanol production
- 2013
-
Ingår i: Wiley Interdisciplinary Reviews. - : Wiley. - 2041-8396 .- 2041-840X. ; 2:6, s. 633-654
-
Tidskriftsartikel (refereegranskat)abstract
- Second-generation bioethanol produced from various lignocellulosic materials, such as wood, agricultural, or forest residues, has the potential to be a valuable substitute for, or a complement to, gasoline. At least three major factors—rapidly increasing atmospheric CO2 levels, dwindling fossil fuel reserves, and their rising costs—suggest that we now need to accelerate research plans to make greater use of plant-based biomass for energy production and as a chemical feedstock as part of a sustainable energy economy. Optimizing the production of bioethanol to be competitive with petrochemical fuels is the main challenge for the underlying process development. The exhaustive research on enzyme technology during the latest years, resulting in significant advances in the field, show the importance of the enzymatic hydrolysis for a profitable ethanol production process. On the other hand, the persisting challenges in biomass pretreatment, which are the initial steps in most process designs, show the remarkable recalcitrance of the lignocellulosic materials to biological degradation. The recent scientific trends show toward an integrated overall bioconversion process in which fermentation technology and genetic engineering of ethanologenic microorganisms aim not only at maximizing yields and productivities but also at widening the range of fermentation products and applications.
|
|
| 5. |
- Antonopoulou, Io, et al.
(författare)
-
Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application
- 2016
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 100:15, s. 6519-6543
-
Tidskriftsartikel (refereegranskat)abstract
- Cosmeceuticals are cosmetic products containing biologically active ingredients purporting to offer a pharmaceutical therapeutic benefit. The active ingredients can be extracted and purified from natural sources (botanicals, herbal extracts, or animals) but can also be obtained biotechnologically by fermentation and cell cultures or by enzymatic synthesis and modification of natural compounds. A cosmeceutical ingredient should possess an attractive property such as anti-oxidant, anti-inflammatory, skin whitening, anti-aging, anti-wrinkling, or photoprotective activity, among others. During the past years, there has been an increased interest on the enzymatic synthesis of bioactive esters and glycosides based on (trans)esterification, (trans)glycosylation, or oxidation reactions. Natural bioactive compounds with exceptional theurapeutic properties and low toxicity may offer a new insight into the design and development of potent and beneficial cosmetics. This review gives an overview of the enzymatic modifications which are performed currently for the synthesis of products with attractive properties for the cosmeceutical industry
|
|
| 6. |
- Chalima, Angelina, et al.
(författare)
-
Integration of a dark fermentation effluent in a microalgal-based biorefinery for the production of high-added value omega-3 fatty acids
- 2019
-
Ingår i: Applied Energy. - : Elsevier. - 0306-2619 .- 1872-9118. ; 241, s. 130-138
-
Tidskriftsartikel (refereegranskat)abstract
- Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen- for generation of energy- that however releases high amounts of polluting volatile fatty acids, such as acetic acid, in the environment. In order for this biohydrogen production process to become more competitive, the volatile fatty acids stream can be utilized through conversion to high added-value metabolites, such as omega-3 fatty acids. The docosahexaenoic acid is one of the two most known omega-3 fatty acids and has been found to be necessary for a healthy brain and proper cardiovascular function. The main source is currently fish, which obtain the fatty acid from the primary producers, microalgae, through the food chain. Crypthecodinium cohnii, a heterotrophic marine microalga, is known for accumulating high amounts of docosahexaenoic acid, while offering the advantage of assimilating various carbon sources, such as glucose, ethanol, glycerol and acetic acid. The purpose of this work was to examine the ability of a C. cohnii strain to grow on different volatile fatty acids, as well as, on a pretreated dark fermentation effluent and accumulate omega-3. The strain was found to grow well on relatively high concentrations of acetic, butyric or propionic acid as main carbon source in a fed-batch pH-auxostat. Most importantly, C. cohnii totally depleted the organic acid content of an ultra-filtrated dark fermentation effluent after 60 h of fed-batch cultivation, therefore offering a bioprocess not only able to mitigate environmental pollutants, but also to provide a solution for a sustainable energy production process. The accumulated docosahexaenoic acid content was as high as 29.8% (w/w) of total fatty acids.
|
|
| 7. |
- Chalima, Angelina, et al.
(författare)
-
Optimization of the production of docosahexaenoic fatty acid by the heterotrophic microalga Crypthecodinium cohnii utilizing a dark fermentation effluent
- 2020
-
Ingår i: Renewable energy. - : Elsevier. - 0960-1481 .- 1879-0682. ; 152, s. 102-109
-
Tidskriftsartikel (refereegranskat)abstract
- Dark fermentation is an anaerobic digestion process of biowaste, used to produce hydrogen as a fuel, which however releases high amounts of polluting volatile fatty acids in the environment. In order for the process to become more competitive, the acids stream can be utilized through conversion to high added-value docosahexaenoic acid by the microalga Crypthecodinium cohnii. Docosahexaenoic acid is one of the two main omega-3 fatty acids, necessary for human nutrition. The purpose of this work was to optimize the production of omega-3 fatty acids by the cells, utilizing the organic content of a dark fermentation effluent. For that purpose, the effect of different fermentation conditions was examined, such as incubation temperature, nitrogen source and concentration, the addition of chemical modulators, as well as the feeding composition. The volatile fatty acid content of the effluent was totally depleted in a fed-batch culture of the microalga, while the cells accumulated DHA in a percentage of 35.6% of total lipids, when fed with yeast extract or 34.2% when fed with ammonium sulfate. Taking into consideration the economic feasibility of the culture conditions proposed it was concluded that the use of yeast extract could be substituted by the much economic ammonium sulfate.
|
|
| 8. |
- Chalima, Angelina, et al.
(författare)
-
Utilization of volatile fatty acids from microalgae for the production of high added value compounds
- 2017
-
Ingår i: Fermentation. - : MDPI. - 2311-5637. ; 3:4
-
Forskningsöversikt (refereegranskat)abstract
- Volatile Fatty Acids (VFA) are small organic compounds that have attracted much attention lately, due to their use as a carbon source for microorganisms involved in the production of bioactive compounds, biodegradable materials and energy. Low cost production of VFA from different types of waste streams can occur via dark fermentation, offering a promising approach for the production of biofuels and biochemicals with simultaneous reduction of waste volume. VFA can be subsequently utilized in fermentation processes and efficiently transformed into bioactive compounds that can be used in the food and nutraceutical industry for the development of functional foods with scientifically sustained claims. Microalgae are oleaginous microorganisms that are able to grow in heterotrophic cultures supported by VFA as a carbon source and accumulate high amounts of valuable products, such as omega-3 fatty acids and exopolysaccharides. This article reviews the different types of waste streams in concert with their potential to produce VFA, the possible factors that affect the VFA production process and the utilization of the resulting VFA in microalgae fermentation processes. The biology of VFA utilization, the potential products and the downstream processes are discussed in detail.
|
|
| 9. |
- Charavgi, Maria-Despoina, et al.
(författare)
-
The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst
- 2013
-
Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69:1, s. 63-73
-
Tidskriftsartikel (refereegranskat)abstract
- The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-[beta]-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an [alpha]/[beta]-hydrolase fold with a three-layer [alpha][beta][alpha]-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.
|
|
| 10. |
|
|
