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Publications (10 of 18) Show all publications
Katsimpouras, C., Dedes, G., Thomaidis, N. S. & Topakas, E. (2019). A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity. Biotechnology for Biofuels, 12, Article ID 120.
Open this publication in new window or tab >>A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity
2019 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 12, article id 120Article in journal (Refereed) Published
Abstract [en]

Background:

The main representatives of hemicellulose are xylans, usually decorated β-1,4-linked d-xylose polymers, which are hydrolyzed by xylanases. The efficient utilization and complete hydrolysis of xylans necessitate the understanding of the mode of action of xylan degrading enzymes. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity.

Results:

The expression of a putative GH30_7 xylanase from the fungus Thermothelomyces thermophila (synonyms Myceliophthora thermophila, Sporotrichum thermophile) in Pichia pastoris resulted in the production and isolation of a novel xylanase with unique catalytic properties. The novel enzyme designated TtXyn30A, exhibited an endo- mode of action similar to that of bacterial GH30 xylanases that require 4-O-methyl-d-glucuronic acid (MeGlcA) decorations, in contrast to most characterized fungal ones. However, TtXyn30A also exhibited an exo-acting catalytic behavior by releasing the disaccharide xylobiose from the non-reducing end of XOS. The hydrolysis products from beechwood glucuronoxylan were MeGlcA substituted XOS, and xylobiose. The major uronic XOS (UXOS) were the aldotriuronic and aldotetrauronic acid after longer incubation indicating the ability of TtXyn30A to cleave linear parts of xylan and UXOS as well.

Conclusions:

Hereby, we reported the heterologous production and biochemical characterization of a novel fungal GH30 xylanase exhibiting endo- and exo-xylanase activity. To date, considering its novel catalytic properties, TtXyn30A shows differences with most characterized fungal and bacterial GH30 xylanases. The discovered xylobiohydrolase mode of action offers new insights into fungal enzymatic systems that are employed for the utilization of lignocellulosic biomass. The recombinant xylanase could be used for the production of X2 and UXOS from glucuronoxylan, which in turn would be utilized as prebiotics carrying manifold health benefits.

Keywords
GH30 xylanase, Glucuronoxylan, Thermothelomyces thermophila, Xylobiohydrolase, Xylooligosaccharides
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-74533 (URN)10.1186/s13068-019-1455-2 (DOI)000467550500002 ()31110561 (PubMedID)2-s2.0-85065661255 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-06-14 (oliekm)

Available from: 2019-06-14 Created: 2019-06-14 Last updated: 2019-06-25Bibliographically approved
Zerva, A., Koutroufini, E., Kostopoulou, I., Detsi, A. & Topakas, E. (2019). A novel thermophilic laccase-like multicopper oxidase from Thermothelomyces thermophila and its application in the oxidative cyclization of 2′,3,4-trihydroxychalcone. New Biotechnology, 49, 10-18
Open this publication in new window or tab >>A novel thermophilic laccase-like multicopper oxidase from Thermothelomyces thermophila and its application in the oxidative cyclization of 2′,3,4-trihydroxychalcone
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2019 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 49, p. 10-18Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Laccase-like multicopper oxidase, Thermophile biocatalysts, Thermothelomyces thermophila, Chalcone oxidative cyclization
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-72494 (URN)10.1016/j.nbt.2018.12.001 (DOI)000454542600002 ()30529567 (PubMedID)2-s2.0-85058170054 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-01-17 (johcin)

Available from: 2019-01-17 Created: 2019-01-17 Last updated: 2019-01-17Bibliographically approved
Mandic, M., Djokic, L., Nikolaivits, E., Prodanovic, R., O’Connor, K., Jeremic, S., . . . Nikodinovic-Runic, J. (2019). Identification and Characterization of New Laccase Biocatalysts from Pseudomonas Species Suitable for Degradation of Synthetic Textile Dyes. Catalysts, 9(7), Article ID 629.
Open this publication in new window or tab >>Identification and Characterization of New Laccase Biocatalysts from Pseudomonas Species Suitable for Degradation of Synthetic Textile Dyes
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2019 (English)In: Catalysts, ISSN 2073-4344, Vol. 9, no 7, article id 629Article in journal (Refereed) Published
Abstract [en]

Laccases are multicopper-oxidases with variety of biotechnological applications. While predominantly used, fungal laccases have limitations such as narrow pH and temperature range and their production via heterologous protein expression is more complex due to posttranslational modifications. In comparison, bacterial enzymes, including laccases, usually possess higher thermal and pH stability, and are more suitable for expression and genetic manipulations in bacterial expression hosts. Therefore, the aim of this study was to identify, recombinantly express, and characterize novel laccases from Pseudomonas spp. A combination of approaches including DNA sequence analysis, N-terminal protein sequencing, and genome sequencing data analysis for laccase amplification, cloning, and overexpression have been used. Four active recombinant laccases were obtained, one each from P. putida KT2440 and P. putida CA-3, and two from P. putida F6. The new laccases exhibited broad temperature and pH range and high thermal stability, as well as the potential to degrade selection of synthetic textile dyes. The best performing laccase was CopA from P. putida F6 which degraded five out of seven tested dyes, including Amido Black 10B, Brom Cresol Purple, Evans Blue, Reactive Black 5, and Remazol Brilliant Blue. This work highlighted species of Pseudomonas genus as still being good sources of biocatalytically relevant enzymes.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
laccase, genome-mining, heterologous expression, biocatalysis, Pseudomonas
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-75724 (URN)10.3390/catal9070629 (DOI)000478652600029 ()
Note

Validerad;2019;Nivå 2;2019-08-28 (johcin)

Available from: 2019-08-28 Created: 2019-08-28 Last updated: 2019-08-28Bibliographically approved
Chalima, A., Hatzidaki, A., Karnaouri, A. & Topakas, E. (2019). Integration of a dark fermentation effluent in a microalgal-based biorefinery for the production of high-added value omega-3 fatty acids. Applied Energy, 241, 130-138
Open this publication in new window or tab >>Integration of a dark fermentation effluent in a microalgal-based biorefinery for the production of high-added value omega-3 fatty acids
2019 (English)In: Applied Energy, ISSN 0306-2619, E-ISSN 1872-9118, Vol. 241, p. 130-138Article in journal (Refereed) Published
Abstract [en]

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. 

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Biorefinery, Crypthecodinium cohnii, Dark fermentation effluent, Docosahexaenoic acid, Omega-3, Volatile fatty acids
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-73234 (URN)10.1016/j.apenergy.2019.03.058 (DOI)000465509500012 ()2-s2.0-85062628275 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-03-18 (svasva)

Available from: 2019-03-18 Created: 2019-03-18 Last updated: 2019-06-18Bibliographically approved
Karnaouri, A. C., Antonopoulou, I., Zerva, A., Dimarogona, M., Topakas, E., Rova, U. & Christakopoulos, P. (2019). Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products: Structural insights and future perspectives for esterases and oxidative catalysts. Bioresource Technology, 279, 362-372
Open this publication in new window or tab >>Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products: Structural insights and future perspectives for esterases and oxidative catalysts
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2019 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 279, p. 362-372Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Thermophilic enzymes, Thermostability, Esterases, Laccases, Peroxidases, LPMOs, Biotransformation, Lignocellulose valorization
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-73041 (URN)10.1016/j.biortech.2019.01.062 (DOI)000458999200043 ()30685134 (PubMedID)2-s2.0-85060269655 (Scopus ID)
Note

Validerad;2019;Nivå 2;2019-02-27 (johcin)

Available from: 2019-02-27 Created: 2019-02-27 Last updated: 2019-03-08Bibliographically approved
Katsimpouras, C., Dedes, G., Bistis, P., Kekos, D., Kalogiannis, K. G. & Topakas, E. (2018). Acetone/water oxidation of corn stover for the production of bioethanol and prebiotic oligosaccharides. Bioresource Technology, 270, 208-215
Open this publication in new window or tab >>Acetone/water oxidation of corn stover for the production of bioethanol and prebiotic oligosaccharides
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2018 (English)In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 270, p. 208-215Article in journal (Refereed) Published
Abstract [en]

Ethanol production at high-gravity promise to achieve concentrations over the threshold for an economical distillation process and concurrently reduce water consumption. However, a persisting limitation is the poor mass transfer conditions resulting in low ethanol yields and concentrations. Hereby, the combination of an acetone/water oxidation pretreatment process (AWO) with a liquefaction/saccharification step, using a free-fall mixer, before simultaneous saccharification and fermentation (SSF) can realize ethanol concentrations of up to ca. 74 g/L at a solids content of 20 wt.%. The free-fall mixer achieved a biomass slurry’s viscosity reduction by 87 % after only 2 h of enzymatic saccharification, indicating the efficiency of the mixing system. Furthermore, the direct enzymatic treatment of AWO pretreated corn stover (CS) by a GH11 recombinant xylanase, led to the production of xylooligosaccharides (XOS) with prebiotic potential and the removal of insoluble fibers of hemicellulose improved the glucose release of AWOCS by 22 %.

Place, publisher, year, edition, pages
Elsevier, 2018
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-70763 (URN)10.1016/j.biortech.2018.09.018 (DOI)000447384200025 ()30218937 (PubMedID)2-s2.0-85053040053 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-09-18 (svasva)

Available from: 2018-09-05 Created: 2018-09-05 Last updated: 2018-11-01Bibliographically approved
Papaspyridi, L. M., Zerva, A. & Topakas, E. (2018). Biocatalytic synthesis of fungal β-glucans. Catalysts, 8(7), Article ID 274.
Open this publication in new window or tab >>Biocatalytic synthesis of fungal β-glucans
2018 (English)In: Catalysts, ISSN 2073-4344, Vol. 8, no 7, article id 274Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
MDPI, 2018
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-70268 (URN)10.3390/catal8070274 (DOI)000440016400021 ()2-s2.0-85050008185 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-08-08 (andbra)

Available from: 2018-08-08 Created: 2018-08-08 Last updated: 2018-11-26Bibliographically approved
Muraleedharan, M. N., Zouraris, D., Karantonis, A., Topakas, E., Sandgren, M., Rova, U., . . . Karnaouri, A. C. (2018). Effect of lignin fractions isolated from different biomass sources on cellulose oxidation by fungal lytic polysaccharide monooxygenases. Biotechnology for Biofuels, 11(1), Article ID 296.
Open this publication in new window or tab >>Effect of lignin fractions isolated from different biomass sources on cellulose oxidation by fungal lytic polysaccharide monooxygenases
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2018 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 11, no 1, article id 296Article in journal (Refereed) Published
Abstract [en]

Background

Lytic 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.

Results

The 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.

Conclusions

Lignins 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.

Place, publisher, year, edition, pages
London: BMC, 2018
Keywords
Lytic polysaccharide monooxygenases, Lignin structural properties, Electron donor, Cyclic voltammetry, Redox potential, Forest biomass, Pretreatment
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-71479 (URN)10.1186/s13068-018-1294-6 (DOI)000448645700001 ()30386433 (PubMedID)2-s2.0-85055687969 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-11-07 (johcin) 

Available from: 2018-11-07 Created: 2018-11-07 Last updated: 2019-04-24Bibliographically approved
Muraleedharan, M. N., Zouraris, D., Karantonis, A., Topakas, E., Sandgren, M., Rova, U., . . . Karnaouri, A. C. (2018). Effect of structural properties of lignin isolated from different sources on its efficiency to serve as electron donor of fungal Lytic Polysaccharide Monooxygenases. Biotechnology for Biofuels
Open this publication in new window or tab >>Effect of structural properties of lignin isolated from different sources on its efficiency to serve as electron donor of fungal Lytic Polysaccharide Monooxygenases
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2018 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834Article in journal (Refereed) Submitted
Place, publisher, year, edition, pages
BMC, 2018
National Category
Bioenergy Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-70690 (URN)
Available from: 2018-08-31 Created: 2018-08-31 Last updated: 2018-08-31
Karnaouri, A. C., Topakas, E., Matsakas, L., Rova, U. & Christakopoulos, P. (2018). Fine-tuned enzymatic hydrolysis of organosolv pretreated forest materials for the efficient production of cellobiose. Frontiers in Chemistry, 6, Article ID 128.
Open this publication in new window or tab >>Fine-tuned enzymatic hydrolysis of organosolv pretreated forest materials for the efficient production of cellobiose
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2018 (English)In: Frontiers in Chemistry, E-ISSN 2296-2646, Vol. 6, article id 128Article in journal (Refereed) Published
Abstract [en]

Non-digestible oligosaccharides (NDOs) are likely prebiotic candidates that have been related to the prevention of intestinal infections and other disorders for both humans and animals. Lignocellulosic biomass is the largest carbon source in the biosphere, therefore cello-oligosacharides (COS), especially cellobiose, are potentially the most widely available choice of NDOs. Production of COS and cellobiose with enzymes offers numerous benefits over acid-catalyzed processes, as it is milder, environmentally friendly and produces fewer by-products. Cellobiohydrolases (CBHs) and a class of endoglucanases (EGs), namely processive EGs, are key enzymes for the production of COS, as they have higher preference toward glycosidic bonds near the end of cellulose chains and are able to release soluble products. In this work, we describe the heterologous expression and characterization of two CBHs from the filamentous fungus Thermothelomyces thermophila, as well as their synergism with proccessive EGs for cellobiose release from organosolv pretreated spruce and birch. The properties, inhibition kinetics and substrate specific activities for each enzyme are described in detail. The results show that a combination of EGs belonging to Glycosyl hydrolase families 5, 6 and 9, with a CBHI and CBHII in appropriate proportions, can enhance the production of COS from forest materials, underpinning the potential of these biocatalysts in the production of NDOs.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2018
Keywords
cellobiohydrolases, hydrolysis, enzymatic cocktail, cellobiose, experimental design, thermostable enzymes, prebiotics
National Category
Engineering and Technology Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
urn:nbn:se:ltu:diva-68257 (URN)10.3389/fchem.2018.00128 (DOI)000430398300001 ()2-s2.0-85047449575 (Scopus ID)
Projects
"Food-grade prebiotic oligosaccharide production, merging marine and forest resources for moving up the cellulose value-chain (ForceUpValue)"
Funder
VINNOVA, 158526
Note

Validerad;2018;Nivå 2;2018-05-03 (andbra)

Available from: 2018-04-10 Created: 2018-04-10 Last updated: 2018-12-14Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-0078-5904

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