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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 University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.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.
Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.ORCID iD: 0000-0003-0078-5904
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2017 (English)In: Biotechnology for Biofuels, ISSN 1754-6834, E-ISSN 1754-6834, Vol. 10, no 1, article id 126Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
BioMed Central, 2017. Vol. 10, no 1, article id 126
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
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
Note

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

Available from: 2017-05-17 Created: 2017-05-17 Last updated: 2018-08-31Bibliographically approved
In thesis
1. Depolymerization of Lignocellulose by Lytic Polysaccharide MonoOxygenases
Open this publication in new window or tab >>Depolymerization of Lignocellulose by Lytic Polysaccharide MonoOxygenases
2018 (English)Licentiate thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Luleå: Luleå University of Technology, 2018
Series
Licentiate thesis / Luleå University of Technology, ISSN 1402-1757
National Category
Bioenergy Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
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 (English)
Opponent
Supervisors
Available from: 2018-08-16 Created: 2018-08-15 Last updated: 2018-08-31Bibliographically approved

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

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