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Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates
Luleå tekniska universitet, Institutionen för samhällsbyggnad och naturresurser, Kemiteknik.ORCID-id: 0000-0001-9164-7667
Luleå tekniska universitet, Institutionen för samhällsbyggnad och naturresurser, Kemiteknik.ORCID-id: 0000-0003-1336-2396
Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
Luleå tekniska universitet, Institutionen för samhällsbyggnad och naturresurser, Kemiteknik. Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.ORCID-id: 0000-0003-0078-5904
Visa övriga samt affilieringar
2017 (Engelska)Ingår i: Biotechnology for Biofuels, E-ISSN 1754-6834, Vol. 10, nr 1, artikel-id 126Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Background

Filamentous 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.

Results

In 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.

Conclusions

The 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.

Ort, förlag, år, upplaga, sidor
BioMed Central (BMC), 2017. Vol. 10, nr 1, artikel-id 126
Nationell ämneskategori
Bioprocessteknik
Forskningsämne
Biokemisk processteknik
Identifikatorer
URN: urn:nbn:se:ltu:diva-63401DOI: 10.1186/s13068-017-0813-1ISI: 000401621000002PubMedID: 28515785Scopus ID: 2-s2.0-85020628468OAI: oai:DiVA.org:ltu-63401DiVA, id: diva2:1096309
Anmärkning

Validerad; 2017; Nivå 2; 2017-05-17 (andbra)

Tillgänglig från: 2017-05-17 Skapad: 2017-05-17 Senast uppdaterad: 2024-04-04Bibliografiskt granskad
Ingår i avhandling
1. Depolymerization of Lignocellulose by Lytic Polysaccharide MonoOxygenases
Öppna denna publikation i ny flik eller fönster >>Depolymerization of Lignocellulose by Lytic Polysaccharide MonoOxygenases
2018 (Engelska)Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Lignocellulose biomass is considered as one of the most potential and sustainable sources for the production of value-added chemicals and fuels while replacing the traditional petroleum resources. In a biorefinery, by employing biochemical conversion processes,cellulose present in the biomass is broken down into monomeric sugars which can belater converted into fuels or chemicals. This process is done with the help of different cellulose digesting enzymes (cellulases), isolated from natural cellulolytic organisms suchas saprophytic fungi.

Lytic polysaccharide monooxygenases (LPMOs) are considered as one of the vital classesof enzymes in the bio-conversion of lignocellulose. They are copper active enzymes present naturally in cellulose degrading fungi. Unlike the traditional cellulases, they havea unique way of breaking cellulose using molecular oxygen or hydrogen peroxide as cosubstratein the presence of a reducing agent. Their ability to enhance the action of other cellulases in depolymerizing the cellulose, make them an integral part of today’s commercial cellulase cocktails.

This thesis comprises the study about the action of lytic polysaccharide monooxygenaseson various cellulose substrates, both model and natural. The first part of the thesis focuses on the ability of an LPMO (MtLPMO9) and a traditional cellulase (MtEG5A), to act insynergism. The evaluation was done based on the release of oxidized and non-oxidized sugars and also on the ability to liquefy the substrates. It was observed that together, these two enzymes resulted in enhanced release of oxidized and non-oxidized sugars. Both were able to reduce viscosity of the substrates but no further synergistic effect was observed when added together.

The second part focuses on the ability of LPMOs to accept electrons from lignins for their action of breaking cellulose chains. Three LPMOs, MtLPMO9, PcLPMO9D and NcLPMO9C, lignins from agricultural and forest biomass pretreated by various pretreatment methods were selected. It was demonstrated that lignins, both in isolatedand substrate bound form were able to act indirectly as reducing agents, by releasingsoluble low-molecular-weight molecules that act as mediators between enzyme and bulklignins. The structural and compositional properties of lignins also affected their ability toact as electron donors. In addition, the effect of biomass pretreatment methods on the lignin properties was also studied. The lignins from acid catalyzed organosolv pretreatment were found as the best candidates in supplying electrons to the enzymes.Interestingly, NcLPMO9C was not able to utilize lignins as electron donors requiring further investigation on their mechanism both in vivo and in vitro.

Ort, förlag, år, upplaga, sidor
Luleå: Luleå University of Technology, 2018
Serie
Licentiate thesis / Luleå University of Technology, ISSN 1402-1757
Nationell ämneskategori
Bioenergi Bioprocessteknik
Forskningsämne
Biokemisk processteknik
Identifikatorer
urn:nbn:se:ltu:diva-70406 (URN)978-91-7790-186-0 (ISBN)978-91-7790-187-7 (ISBN)
Presentation
2018-09-05, F341, Luleå University of Technology, Luleå, 13:30 (Engelska)
Opponent
Handledare
Tillgänglig från: 2018-08-16 Skapad: 2018-08-15 Senast uppdaterad: 2023-09-05Bibliografiskt granskad
2. Lytic Polysaccharide MonoOxygenases; their role for lignocellulose depolymerization and production of (functional) biobased compounds
Öppna denna publikation i ny flik eller fönster >>Lytic Polysaccharide MonoOxygenases; their role for lignocellulose depolymerization and production of (functional) biobased compounds
2021 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

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. 

Ort, förlag, år, upplaga, sidor
Luleå University of Technology, 2021
Serie
Doctoral thesis / Luleå University of Technology 1 jan 1997 → …, ISSN 1402-1544
Nationell ämneskategori
Industriell bioteknik Bioprocessteknik
Forskningsämne
Biokemisk processteknik
Identifikatorer
urn:nbn:se:ltu:diva-82232 (URN)978-91-7790-745-9 (ISBN)978-91-7790-746-6 (ISBN)
Disputation
2021-02-12, A109, 10:00 (Engelska)
Opponent
Handledare
Forskningsfinansiär
Energimyndigheten
Tillgänglig från: 2021-01-11 Skapad: 2021-01-10 Senast uppdaterad: 2023-09-05Bibliografiskt granskad

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Karnaouri, Anthi CMuraleedharan, Madhu NairTopakas, EvangelosRova, UlrikaChristakopoulos, Paul

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