| 1. |
- Hüttner, Silvia, 1984, et al.
(författare)
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Synthesis of antioxidants with free and immobilised fungal feruloyl esterases
- 2016
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Ingår i: European Symposium on Biochemical Engineering Sciences, 11-14 Sep 2016, Dublin, Ireland.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Feruloyl esterases (FAEs, E.C. 3.1.1.73, CAZy family CE1) are enzymes that are secreted by a wide range of fungi and bacteria as part of the enzymes hydrolysing plant biomass. Under conditions of low water content, FAEs can also carry out (trans)esterification reactions. Thus, their potential use as biocatalysts for the production of antioxidants with applications in food, cosmetic and pharmaceutical industries has been investigated in recent years. We characterised the biosynthetic potential of four new FAE enzymes from a thermophilic fungus. We focused on optimizing reaction conditions for the synthesis of ferulate esters with improved hydrophobic or hydrophilic properties; prenyl ferulate and 5-O-(trans-feruloyl)-arabinofuranose, respectively. In addition to using free enzymes, we also immobilised them on the mesoporous silica material SBA-15 with pore sizes ranging from 7 to 10 nm, to improve the esterification-to-hydrolysis ratio of the enzymes. It has been shown previously that immobilisation renders enzymes more resilient to adverse conditions and increases their productive life time [1]. Furthermore, immobilisation may also result in a decrease of unwanted side reactions (hydrolysis of transesterification) [2]. In agreement with that, we achieved a higher product yield with immobilised enzymes compared to free enzymes. The immobilised biocatalysts are also more easily re-usable for several production cycles, thus lowering production costs.
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| 2. |
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| 3. |
- Antonopoulou, Io, 1989-
(författare)
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Development of biocatalytic processes for selective antioxidant production
- 2018
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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.
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| 4. |
- Zerva, Anastasia, et al.
(författare)
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Optimization of Transesterification Reactions with CLEA-Immobilized Feruloyl Esterases from Thermothelomyces thermophila and Talaromyces wortmannii
- 2018
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Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 23:9
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Tidskriftsartikel (refereegranskat)abstract
- Feruloyl esterases (FAEs, E.C. 3.1.1.73) are biotechnologically important enzymes with several applications in ferulic acid production from biomass, but also in synthesis of hydroxycinnamic acid derivatives. The use of such biocatalysts in commercial processes can become feasible by their immobilization, providing the advantages of isolation and recycling. In this work, eight feruloyl esterases, immobilized in cross-linked enzyme aggregates (CLEAs) were tested in regard to their transesterification performance, towards the production of prenyl ferulate (PFA) and arabinose ferulate (AFA). After solvent screening, comparison with the activity of respective soluble enzymes, and operational stability tests, FAE125 was selected as the most promising biocatalyst. A central composite design revealed the optimum conditions for each transesterification product, in terms of water content, time, and substrate ratio for both products, and temperature and enzyme load additionally for prenyl ferulate. The optimum product yields obtained were 83.7% for PFA and 58.1% for AFA. FAE125 CLEAs are stable in the optimum conditions of transesterification reactions, maintaining 70% residual activity after five consecutive reactions. Overall, FAE125 CLEAs seem to be able to perform as a robust biocatalyst, offering satisfactory yields and stability, and thus showing significant potential for industrial applications.
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| 5. |
- Anasontzis, George E, 1980, et al.
(författare)
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Challenges in ethanol production with fusarium oxysporum through consolidated bioprocessing
- 2014
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Ingår i: Bioengineered Bugs. - : Informa UK Limited. - 1949-1018 .- 1949-1026 .- 2165-5979 .- 2165-5987. ; 5:6, s. 393-395
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Tidskriftsartikel (refereegranskat)abstract
- Fusarium oxysporum has been reported as being able to both produce the enzymes necessary to degrade lignocellulosic biomass to sugars and also ferment the monosaccharides to ethanol under anaerobic or microaerobic conditions. However, in order to become an economically feasible alternative to other ethanol-producing microorganisms, a better understanding of its physiology, metabolic pathways, and bottlenecks is required, together with an improvement in its efficiency and robustness. In this report, we describe the challenges for the future and give additional justification for our recent publication.
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| 6. |
- Matsakas, Leonidas, et al.
(författare)
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Biological Production of 3-Hydroxypropionic Acid : An Update on the Current Status
- 2018
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Ingår i: Fermentation. - : MDPI. - 2311-5637. ; 4:1
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Tidskriftsartikel (refereegranskat)abstract
- The production of high added-value chemicals from renewable resources is a necessity inour attempts to switch to a more sustainable society. 3-Hydroxypropionic acid (3HP) is a promisingmolecule that can be used for the production of an important array of high added-value chemicals,such as 1,3-propanediol, acrylic acid, acrylamide, and bioplastics. Biological production of 3HP hasbeen studied extensively, mainly from glycerol and glucose, which are both renewable resources.To enable conversion of these carbon sources to 3HP, extensive work has been performed to identifyappropriate biochemical pathways and the enzymes that are involved in them. Novel enzymeshave also been identified and expressed in host microorganisms to improve the production yieldsof 3HP. Various process configurations have also been proposed, resulting in improved conversionyields. The intense research efforts have resulted in the production of as much as 83.8 g/L 3HP fromrenewable carbon resources, and a system whereby 3-hydroxypropionitrile was converted to 3HPthrough whole-cell catalysis which resulted in 184.7 g/L 3HP. Although there are still challengesand difficulties that need to be addressed, the research results from the past four years have been animportant step towards biological production of 3HP at the industrial level.
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| 7. |
- Nair, Madhu Muraleedharan
(författare)
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Lytic Polysaccharide MonoOxygenases; their role for lignocellulose depolymerization and production of (functional) biobased compounds
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Increased environmental concerns over petroleum-based products triggered the quest to find a sustainable alternative for fuels, chemicals etc. Lignocellulose biomass, due to its abundance, is considered as one of the most promising sustainable sources for the production of fuels and chemicals, while replacing the traditional petroleum resources. In a biorefinery, by choosing a greener biochemical conversion process with cellulolytic enzymes, cellulose from biomass is depolymerized into monomeric sugars and residual fibers; which can be later converted into a spectra of value added products.Lytic polysaccharide monooxygenases (LPMOs) are one of the essential groups of enzymes in the bioconversion of lignocellulose. They are copper active enzymes that are produced by different polysaccharide degrading organisms in nature, such as lignocellulolytic fungi. In lignocellulose degradation, they are different from the traditional hydrolytic cellulolytic enzymes with their unique way of oxidative breakage of cellulose, in the presence of a co-substrate such as oxygen, and a reducing agent like lignin in the biomass. Their ability to enhance the action of traditional cellulases in cellulose depolymerization make them an integral part of today’s commercial cellulosic cocktails.Primary goals of biorefinery research include efficient liquefaction of lignocellulose in order to increase the release of monomeric sugars towards the production of various chemicals and fuels, together with the potential use of residual fibers for the production of value-added products; all by minimizing the release of undesired by-products and the environmental impact of the process. LPMOs, along with other cellulases, have been shown to be very much beneficial in this.This thesis comprises the study of LPMOs from different fungal origin, in their depolymerization ability on various substrates, including both model substrates and natural biomass samples. The evaluation was done based on their ability to release neutral and oxidized sugars, as well as their capability to promote liquefaction. Effect of various pretreatment methods of lignocellulose on the action of LPMOs was studied, together with their capability to use lignin present in the wood as a reducing agent, which gives a better understanding about their function in nature. Lastly, their role in producing value added materials such as nanocellulose, the prebiotic disaccharide cellobiose, from lignocellulose was also evaluated.
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| 8. |
- Najjarzadeh, Nasim, 1985-
(författare)
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SOUnd-DRIven BIOtechnology
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ultrasound has been vastly applied in different areas such as medical imaging and the food industry, but not much attempt has been made to investigate its influence on microorganisms and its potential to be applied in microbial biotechnology. Previous studies show the potential of acoustic waves to increase biomass yield, production of secondary metabolites and enhanced enzyme-catalyzed reactions, probably due to improved mass transfer and cell retention. The influence of audible waves, however, is not properly elucidated, and more studies are needed to understand how audible waves affect living cells. As most of the applied equipment in biological fields are originally designed for other purposes, there has been limited control of frequency, input power and pressure distribution in biological reactor system. So far, the lack of well-defined sono(bio)reactors that uniformly distribute sound waves has prevented more systematic studies on how acoustics affect biological systems. To obtain in-depth knowledge in this specific field, a sonobioreactor was designed that enables control of the pressure distribution. The viability of a model lignocellulose degrading fungus, Fusarium oxysporum, was studied at different intensities of resonance frequency. The online growth measurement showed that sonication did not adversely affect the cells up to 6 W and the best growth was recorded at 4W. In addition, SEM analysis showed that sound waves can disrupt the mycelium and the higher the applied input power, the greater the number of these breaks. Therefore, in the next parts of the thesis, the influence of sonication on production of lignocellulose degrading enzymes was studied. Prior to studying the effect of ultrasound on lignocellulose enzyme activity, a series of experiments were conducted to identify effective cellulase and xylanase inducers. In these trials, inducers such as cellooligosaccharides, xylooligosacharides, sophorose and lactose were tested and the induced enzymatic activities were measured. Besides, the influence of consumed carbon source (sucrose vs glycerol) for biomass production on cellulase activity was also investigated. The obtained result showed that the efficiency of the inducer depends on the carbon source. Specifically, when using sucrose for the production of fungal biomass, cellooligosaccharides with a higher degree of polymerization (cellotetraose) were better inducers of endoglucanase, exoglucanase, cell-bound and extracellular β-glucosidase, while when using glycerol, cellobiose resulted in an enhanced induction of endoglucanase and exoglucanase, while cellohexaose promoted both β-glucosidases. When it comes to xylanase induction, it was found that the chain size of xylooligosacharides plays an important role with the highest induction occurring with xylotetraose. Finally, transcriptomics analysis was done for both cellulases and xylanases induction, by using cellobiose and xylotetraose respectively, to understand the mechanism of induction of biomass degrading enzymes by revealing differentially expressed genes or transcription factors. The bioinformatics analysis showed that by adding each inducer, a series of carbohydrate degrading enzymes, transcription factors, transporters, as well as genes necessary for translation are differentially expressed. In the final part, the effect of ultrasound and audible sound on the induction of cellulase was studied by using cellobiose as inducer. Prior to this, the effect of different sonication parameters, specifically the time, on the enzyme induction was investigated, and it could be concluded that continuous sonication had a negative effect on enzyme induction. As such, sonication for 2, 8, and 20 hours were compared where 8 hours sonication were selected for further studies. The impact of three different frequencies, namely 40000 kHz and its resonance frequencies in audible sound and higher ultrasound frequencies, on the cellobiose induction of cellulase was studied by using 4W input power and 8-hour sonication. To acquire detailed information on the mechanism of sound influence on the microbial cell, enzyme activity results were used in combination with transcriptomics, metabolomics, proteomics, and secretomics and phosphoproteomics analyses. The OMICs analysis showed that based on the applied frequency, the cell responds differently by activating different metabolic pathways, resulting in the production of different proteins and phosphoproteins. The OMICs profiles of the audible sound and low frequency ultrasound treated samples had some similarities while showing significant differences with the high frequency ultrasound treated samples.
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| 9. |
- Paschos, T., et al.
(författare)
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Ethanol effect on metabolic activity of the ethalogenic fungus Fusarium oxysporum
- 2015
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Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Fusarium oxysporum is a filamentous fungus which has attracted a lot of scientific interest not only due to its ability to produce a variety of lignocellulolytic enzymes, but also because it is able to ferment both hexoses and pentoses to ethanol. Although this fungus has been studied a lot as a cell factory, regarding applications for the production of bioethanol and other high added value products, no systematic study has been performed concerning its ethanol tolerance levels. Results: In aerobic conditions it was shown that both the biomass production and the specific growth rate were affected by the presence of ethanol. The maximum allowable ethanol concentration, above which cells could not grow, was predicted to be 72 g/L. Under limited aeration conditions the ethanol-producing capability of the cells was completely inhibited at 50 g/L ethanol. The lignocellulolytic enzymatic activities were affected to a lesser extent by the presence of ethanol, while the ethanol inhibitory effect appears to be more severe at elevated temperatures. Moreover, when the produced ethanol was partially removed from the broth, it led to an increase in fermenting ability of the fungus up to 22.5%. The addition of F. oxysporum's system was shown to increase the fermentation of pretreated wheat straw by 11%, in co-fermentation with Saccharomyces cerevisiae. Conclusions: The assessment of ethanol tolerance levels of F. oxysporum on aerobic growth, on lignocellulolytic activities and on fermentative performance confirmed its biotechnological potential for the production of bioethanol. The cellulolytic and xylanolytic enzymes of this fungus could be exploited within the biorefinery concept as their ethanol resistance is similar to that of the commercial enzymes broadly used in large scale fermentations and therefore, may substantially contribute to a rational design of a bioconversion process involving F. oxysporum. The SSCF experiments on liquefied wheat straw rich in hemicellulose indicated that the contribution of the metabolic system of F. oxysporum in a co-fermentation with S. cerevisiae may play a secondary role.
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| 10. |
- Patel, Alok, Dr. 1989-, et al.
(författare)
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Volatile Fatty Acids (VFAs) Generated by Anaerobic Digestion Serve as Feedstock for Freshwater and Marine Oleaginous Microorganisms to Produce Biodiesel and Added-Value Compounds
- 2021
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Given an increasing focus on environmental sustainability, microbial oils have been suggested as an alternative to petroleum-based products. However, microbial oil production relies on the use of costly sugar-based feedstocks. Substrate limitation, elevated costs, and risk of contamination have sparked the search for alternatives to sugar-based platforms. Volatile fatty acids are generated during anaerobic digestion of organic waste and are considered a promising substrate for microbial oil production. In the present study, two freshwater and one marine microalga along with two thraustochytrids were evaluated for their potential to produce lipids when cultivated on volatile fatty acids generated from food waste via anaerobic digestion using a membrane bioreactor. Freshwater microalgae Auxenochlorella protothecoides and Chlorella sorokiniana synthesized lipids rich in palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2). This composition corresponds to that of soybean and jatropha oils, which are used as biodiesel feedstock. Production of added-value polyunsaturated fatty acids (PUFA) mainly omega-3 fatty acids was examined in three different marine strains: Aurantiochytrium sp. T66, Schizochytrium limacinum SR21, and Crypthecodinium cohnii. Only Aurantiochytrium sp. T66 seemed promising, generating 43.19% docosahexaenoic acid (DHA) and 13.56% docosapentaenoic acid (DPA) in total lipids. In summary, we show that A. protothecoides, C. sorokiniana, and Aurantiochytrium sp. T66 can be used for microbial oil production from food waste material.
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