| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Zerva, Anastasia, et al.
(författare)
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A novel thermophilic laccase-like multicopper oxidase from Thermothelomyces thermophila and its application in the oxidative cyclization of 2′,3,4-trihydroxychalcone
- 2019
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Ingår i: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 49, s. 10-18
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Tidskriftsartikel (refereegranskat)abstract
- Laccase-like multicopper oxidases (LMCOs) are a heterogeneous group of oxidases, acting mainly on phenolic compounds and which are widespread among many microorganisms, including Basidiomycetes and Ascomycetes. Here, we report the cloning, heterologous expression, purification and characterization of a novel LMCO from the thermophilic fungus Thermothelomyces thermophila. The 1953 bp lmco gene sequence comprises of 3 exons interrupted by 2 introns and according to the LccED database the translated sequence belongs to superfamily 6 of multicopper oxidases. After removal of the introns, the gene was transformed into Pichia pastoris, under the control of the alcohol oxidase (AOX1) promoter. The heterologous enzyme was purified with an apparent molecular weight of 80 kDa. TtLMCO1 displayed optimum activity at pH 4 and 50 °C and appeared thermostable up to 50 °C. A variety of phenolic compounds were oxidized by TtLMCO1, including standard laccase substrates such as ABTS and 2,6 dimethoxyphenol. The UV/Vis spectrum of purified TtLMCO1 indicates that it belongs to yellow laccase-like oxidases. The enzyme was used for the bioconversion of 2′,3,4-trihydroxychalcone to 3′,4′-dihydroxy-aurone, a bioactive aurone recently shown to possess inhibitory activity against several isoforms of the histone deacetylase complex (HDAC). Overall, the thermophilic yellow LMCO TtLMCO1 presents a number of superior properties with potential use in industrial biocatalysis.
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| 3. |
- Karnaouri, Anthi C., et al.
(författare)
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Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products : Structural insights and future perspectives for esterases and oxidative catalysts
- 2019
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Ingår i: Bioresource Technology. - : Elsevier. - 0960-8524 .- 1873-2976. ; 279, s. 362-372
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Tidskriftsartikel (refereegranskat)abstract
- Thermophilic enzyme systems are of major importance nowadays in all industrial processes due to their great performance at elevated temperatures. In the present review, an overview of the current knowledge on the properties of thermophilic and thermotolerant carbohydrate esterases and oxidative enzymes with great thermostability is provided, with respect to their potential use in biotechnological applications. A special focus is given to the lytic polysaccharide monooxygenases that are able to oxidatively cleave lignocellulose through the use of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. Structural characteristics of the enzymes, including active site conformation and surface properties are discussed and correlated with their substrate specificity and thermostability properties.
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| 4. |
- Karnaouri, Anthi, et al.
(författare)
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Screening of Recombinant Lignocellulolytic Enzymes Through Rapid Plate Assays
- 2021. - 2
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Ingår i: Protein Downstream Processing. - New York, NY : Springer Nature. ; , s. 479-503
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- In the search for novel biomass-degrading enzymes through mining microbial genomes, it is necessary to apply functional tests during high-throughput screenings, which are capable of detecting enzymatic activities directly by way of plate assay. Using the most efficient expression systems of Escherichia coli and Pichia pastoris, the production of a high amount of His-tagged recombinant proteins could be thrived, allowing the one-step isolation by affinity chromatography. Here, we describe simple and efficient assay techniques for the detection of various biomass-degrading enzymatic activities on agar plates, such as cellulolytic, hemicellulolytic, and ligninolytic activities and their isolation using immobilized-metal affinity chromatography.
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| 5. |
- Papaspyridi, Lefki Maria, et al.
(författare)
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Biocatalytic synthesis of fungal β-glucans
- 2018
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Ingår i: Catalysts. - : MDPI. - 2073-4344. ; 8:7
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Tidskriftsartikel (refereegranskat)abstract
- Glucans are the dominant polysaccharide constituents of fungal cell walls. Remarkably, these major bioactive polysaccharides account for the beneficial effects that have been observed by many mushrooms of medicinal interest. Accordingly, the prevailing tendency is the use of bioactive mushroom β-glucans mainly in pharmaceutical industries or as food additives, since it seems that they can be involved in meeting the overall growing demand for food in the future, but also in medical and material sectors. β-(1,3)-Glucan synthase (GLS) is the responsible enzyme for the synthesis of these important polysaccharides, which is a member of the glycosyl transferase (GT) family. For optimizing the production of such natural polymers of great interest, the comprehension of the fungal synthetic mechanism, as well as the biochemical and molecular characteristics of the key enzyme GLS and its expression seem to be crucial. Overall, in this review article, the fungal β-glucans biosynthesis by GLS is summarized, while the in vitro synthesis of major polysaccharides is also discussed, catalyzed by glycoside hydrolases (GHs) and GTs. Possible future prospects of GLS in medicine and in developing other potential artificial composite materials with industrial applications are also summarized
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| 6. |
- Zerva, Anastasia, et al.
(författare)
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Bioconversion of Biomass-Derived Phenols Catalyzed by Myceliophthora thermophila Laccase
- 2016
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Ingår i: Molecules. - : MDPI AG. - 1431-5157 .- 1420-3049. ; 21:5
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Tidskriftsartikel (refereegranskat)abstract
- Biomass-derived phenols have recently arisen as an attractive alternative for building blocks to be used in synthetic applications, due to their widespread availability as an abundant renewable resource. In the present paper, commercial laccase from the thermophilic fungus Myceliophthora thermophila was used to bioconvert phenol monomers, namely catechol, pyrogallol and gallic acid in water. The resulting products from catechol and gallic acid were polymers that were partially characterized in respect to their optical and thermal properties, and their average molecular weight was estimated via solution viscosity measurements and GPC. FT-IR and 1H-NMR data suggest that phenol monomers are connected with ether or C–C bonds depending on the starting monomer, while the achieved molecular weight of polycatechol is found higher than the corresponding poly(gallic acid). On the other hand, under the same condition, pyrogallol was dimerized in a pure red crystalline compound and its structure was confirmed by 1H-NMR as purpurogallin. The herein studied green synthesis of enzymatically synthesized phenol polymers or biological active compounds could be exploited as an alternative synthetic route targeting a variety of applications.
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| 7. |
- Zerva, Anastasia, et al.
(författare)
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Characterization and application of a novel class II thermophilic peroxidase from Myceliophthora thermophila in biosynthesis of polycatechol
- 2015
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Ingår i: Enzyme and microbial technology. - : Elsevier BV. - 0141-0229 .- 1879-0909. ; 75-76, s. 49-56
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Tidskriftsartikel (refereegranskat)abstract
- A peroxidase from the thermophilic fungus Myceliophthora thermophila that belongs to ascomycete Class II based on PeroxiBase classification was functionally expressed in methylotrophic yeast Pichia pastoris. The putative peroxidase from the genomic DNA was successfully cloned in P. pastoris X-33 under the transcriptional control of the alcohol oxidase (AOX1) promoter. The heterologous production was greatly enhanced by the addition of hemin with a titer of 0.41 U mL−1 peroxidase activity at the second day of incubation. The recombinant enzyme was purified to homogeneity (50 kDa) and characterized using a series of phenolic substrates that indicated similar characteristics with those of generic peroxidases. In addition, the enzyme was found thermostable, retaining its activity for temperatures up to 60 oC after eight hours incubation. Moreover, the enzyme displayed remarkable H2O2 stability, retaining more than 80% of its initial activity after 24 hours incubation in 5000- fold molar excess of H2O2. The ability of the peroxidase to polymerize catechol at high superoxide concentrations, together with its high thermostability and substrate specificity, indicate a potential commercial significance of the enzyme.
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| 8. |
- Zerva, Anastasia, et al.
(författare)
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Cross-Linked Enzyme Aggregates of Feruloyl Esterase Preparations from Thermothelomyces thermophila and Talaromyces wortmannii
- 2018
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Ingår i: Catalysts. - : MDPI. - 2073-4344. ; 8:5
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Tidskriftsartikel (refereegranskat)abstract
- Cross-linked enzyme aggregates (CLEA®) technology is a well-established method in the current literature for the low-cost and effective immobilization of several enzymes. The main advantage of this particular method is the simplicity of the process, since it consists of only two steps. However, CLEA immobilization must be carefully designed for each desired enzyme, since the optimum conditions for enzymes can vary significantly, according to their physicochemical properties. In the present study, an investigation of the optimum CLEA immobilization conditions was carried out for eight feruloyl esterase preparations. Feruloyl esterases are a very important enzyme group in the valorization of lignocellulosic biomass, since they act in a synergistic way with other enzymes for the breakdown of plant biomass. Specifically, we investigated the type and concentration of precipitant and the crosslinker concentration, for retaining optimal activity. FAE68 was found to be the most promising enzyme for CLEA immobilization, since in this case, the maximum retained activity, over 98%, was observed. Subsequently, we examined the operational stability and the stability in organic solvents for the obtained CLEA preparations, as well as their structure. Overall, our results support that the maximum activity retaining and the stability properties of the final CLEAs can vary greatly in different FAE preparations. Nevertheless, some of the examined FAEs show a significant potential for further applications in harsh industrial conditions.
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| 9. |
- Zerva, Anastasia, et al.
(författare)
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Crosslinked Enzyme Aggregates (CLEAs) of Laccases from Pleurotus citrinopileatus Induced in Olive Oil Mill Wastewater (OOMW)
- 2020
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Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 25:9
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Tidskriftsartikel (refereegranskat)abstract
- The enzymatic factory of ligninolytic fungi has proven to be a powerful tool in applications regarding the degradation of various types of pollutants. The degradative potential of fungi is mainly due to the production of different types of oxidases, of which laccases is one of the most prominent enzymatic activities. In the present work, crude laccases from the supernatant of Pleurotus citrinopileatus cultures grown in olive oil mill wastewater (OOMW) were immobilized in crosslinked enzyme aggregates (CLEAs), aiming at the development of biocatalysts suitable for the enzymatic treatment of OOMW. The preparation of laccase CLEAs was optimized, resulting in a maximum of 72% residual activity. The resulting CLEAs were shown to be more stable in the presence of solvents and at elevated temperatures compared to the soluble laccase preparation. The removal of the phenolic component of OOMW catalyzed by laccase-CLEAs exceeded 35%, while they were found to retain their activity for at least three cycles of repetitive use. The described CLEAs can be applied for the pretreatment of OOMW, prior to its use for valorization processes, and thus, facilitate its complete biodegradation towards a consolidated process in the context of circular economy.
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| 10. |
- Zerva, Anastasia, et al.
(författare)
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Degradation of olive mill wastewater by the induced extracellular ligninolytic enzymes of two wood-rot fungi
- 2017
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Ingår i: Journal of Environmental Management. - : Elsevier. - 0301-4797 .- 1095-8630. ; 203:2, s. 791-798
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Tidskriftsartikel (refereegranskat)abstract
- Olive mill wastewater (OMWW) is a major problem in olive oil – producing countries, due to its high organic load and concentration in phenols that are toxic for marine life, plants and soil microorganisms. In the present study, two mushroom species were tested in regard to their OMWW's oxidative capacity, Pleurotus citrinopileatus LGAM 28684 and Irpex lacteus LGAM 238. OMWW (25% v/v) degradation was investigated for several culture conditions, namely pH, agitation speed, nitrogen-based supplements and their concentration. The selected values were pH 6, agitation rate 150 rpm, 30 g L−1 corn steep liquor as nitrogen source for P. citrinopileatus and 20 g L−1 diammonium tartrate for I. lacteus. The two strains performed well in cultures supplemented with OMWW, generating very high titers of oxidative enzymes and achieving more than 90% color and phenols reduction within a 24 days cultivation period. In addition, the amount of glucans present in the fungal biomass was assessed. Hence, P. citrinopileatus and I. lacteus appear as potent degraders of OMWW with the ability to use the effluent as a substrate for the production of biotechnologically important enzymes and valuable fungal glucans.
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| 11. |
- 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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| 12. |
- Zerva, Anastasia, et al.
(författare)
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Recent advances on key enzymatic activities for the utilisation of lignocellulosic biomass
- 2021
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Ingår i: Bioresource Technology. - : Elsevier. - 0960-8524 .- 1873-2976. ; 342
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Forskningsöversikt (refereegranskat)abstract
- The field of enzymatic degradation of lignocellulose is actively growing and the recent updates of the last few years indicate that there is still much to learn. The growing number of protein sequences with unknown function in microbial genomes indicates that there is still much to learn on the mechanisms of lignocellulose degradation. In this review, a summary of the progress in the field is presented, including recent discoveries on the nature of the structural polysaccharides, new technologies for the discovery and functional annotation of gene sequences including omics technologies, and the novel lignocellulose-acting enzymes described. Novel enzymatic activities and enzyme families as well as on accessory enzymes and their synergistic relationships regarding biomass breakdown are described. Moreover, it is shown that all the valuable knowledge of the enzymatic decomposition of plant biomass polymers can be employed towards the decomposition and upgrading of synthetic polymers, such as plastics.
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| 13. |
- Zerva, Anastasia, et al.
(författare)
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Valorization of Olive Mill Wastewater for the Production of β-glucans from Selected Basidiomycetes
- 2017
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Ingår i: Waste and Biomass Valorization. - : Springer. - 1877-2641 .- 1877-265X. ; 8:5, s. 1721-1731
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Tidskriftsartikel (refereegranskat)abstract
- PurposeThe aim of the present study was to investigate the feasibility of polysaccharides production by selected basidiomycetes in submerged culture. Olive mill wastewater (OMWW) was also tested as a potential substrate for polysaccharides production by mushroom strains, focusing on the simultaneous degradation and valorization of the waste material.MethodsThe tested strains were grown in two different substrates, and after biomass harvesting, polysaccharides were isolated using two different methods. The extracellular polysaccharides were isolated from the culture broth, with ethanol precipitation. The isolated fractions were partially characterized with FT-IR spectroscopy.ResultsAll three strains performed well in both substrates. Maximum degradation performance of OMWW was achieved by Ganoderma lucidum, achieving 19.4% phenols reduction together with 47.56% decolorization. The extracellular polysaccharides (EPS) produced by all strains were found to be richer in total glucans during growth in semi-synthetic medium, compared to growth in OMWW-based medium. In regard to biomass polysaccharides, Pleurotus ostreatus biomass was found to be richer in glucans, reaching 8.68% (w/w) total glucan content when grown in semi-synthetic medium and 7.58% (w/w) when grown in OMWW-based medium. After purification of biomass polysaccharides with two methods, the fraction with the highest glucan content was found to be the one from G. lucidum after growth in semi-synthetic medium cultures, with 49.1% (w/w) total glucans. FT-IR spectra of the isolated samples revealed the bands corresponding to α- and β-glucosidic bonds, but also the existence of protein contamination.ConclusionsPurification of biomass polysaccharides with two distinct methods revealed that α-amylase and Sevag treatments failed to remove completely α-glucans and proteins respectively, leading to the suggestion that these two steps could be omitted without significant impact. Moreover, the results imply that the valorization of OMWW might be feasible with the use of mushroom strains, leading to the production of important products, such as glucans.
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