The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay

Filandr, František; Man, Petr; Halada, Petr; Chang, Hui S.; Ludwig, Roland; Kracher, Daniel

doi:10.1186/s13068-020-01673-4

Cited by 55 publications

(67 citation statements)

References 58 publications

(103 reference statements)

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“…As recent studies reported that LPMOs are more efficient when fueled with H 2 O 2 as co-substrate instead of O 2 [ 18 , 28 , 57 , 58 ], we assessed the suitability of H 2 O 2 as co-substrate for Taus LPMO9B. First, PASC was incubated with Taus LPMO9B, H 2 O 2 and an excess of reductant (1-mM ascorbic acid).…”

Section: Resultsmentioning

confidence: 99%

Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions

Calderaro

Keser

Akeroyd

et al. 2020

Biotechnol Biofuels

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Background The discovery of lytic polysaccharide monooxygenases (LPMO) has changed our perspective on enzymatic degradation of plant biomass. Through an oxidative mechanism, these enzymes are able to cleave and depolymerize various polysaccharides, acting not only on crystalline substrates such as chitin and cellulose, but also on other polysaccharides, such as xyloglucan, glucomannan and starch. Despite their widespread use, uncertainties related to substrate specificity and stereospecificity, the nature of the co-substrate, in-process stability, and the nature of the optimal reductant challenge their exploitation in biomass processing applications. Results In this work, we studied the properties of a novel fungal LPMO from the thermophilic fungus Thielavia australiensis, TausLPMO9B. Heterologous expression of TausLPMO9B in Aspergillus niger yielded a glycosylated protein with a methylated N-terminal histidine showing LPMO activity. High sequence identity of the AA9 domain to that of MtLPMO9B (MYCTH_80312) from Myceliophthora thermophila (84%) indicated strictly C1-oxidizing activity on cellulose, which was confirmed experimentally by the analysis of products released from cellulose using HPAEC. The enzyme was stable and active at a pH ranging from 4 to 6, thus matching the conditions commonly used in industrial biomass processing, where a low pH (between 4 and 5) is used due to the pH-optima of commercial cellulases and a desire to limit microbial contamination. Conclusion While the oxidative cleavage of phosphoric acid swollen cellulose (PASC) by TausLPMO9B was boosted by the addition of H2O2 as a co-substrate, this effect was not observed during the saccharification of acid pretreated corn stover. This illustrates key differences between the lab-scale tests with artificial, lignin-free substrates and industrial settings with lignocellulosic biomass as substrate.

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Section: Resultsmentioning

confidence: 99%

Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions

Calderaro

Keser

Akeroyd

et al. 2020

Biotechnol Biofuels

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“…In particular, addition of fresh AscA will lead to increased generation of H 2 O 2 . Increased formation of H 2 O 2 would normally speed up the reaction, since H 2 O 2 is a good co-substrate for LPMOs [ 20 , 25 , 28 ]. However, in this case, one could envisage a situation where too much H 2 O 2 is produced due to excess AscA, which can damage the LPMO.…”

Section: Discussionmentioning

confidence: 99%

“…LPMO catalysis has generally been thought to be strictly dependent on molecular oxygen and a reductant that delivers two electrons and two protons for each catalytic cycle [9,23,24]. However, recent studies on LPMOs belonging to families AA9 and AA10, have shown that H 2 O 2 can drive LPMO reactions, and that these reactions are orders of magnitude faster than O 2 -driven reactions [20,[25][26][27][28][29]. The peroxygenase driven reaction only requires sub-stoichiometric amounts of reductant for an initial, "priming", reduction of the LPMO, after which the enzyme can catalyze multiple reactions.…”

Section: Introductionmentioning

confidence: 99%

A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range

Tuveng

Jensen

Fredriksen

et al. 2020

Biotechnol Biofuels

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Background Lytic polysaccharide monooxygenases (LPMOs) are oxidative, copper-dependent enzymes that function as powerful tools in the turnover of various biomasses, including lignocellulosic plant biomass. While LPMOs are considered to be of great importance for biorefineries, little is known about industrial relevant properties such as the ability to operate at high temperatures. Here, we describe a thermostable, cellulose-active LPMO from a high-temperature compost metagenome (called mgLPMO10). Results MgLPMO10 was found to have the highest apparent melting temperature (83 °C) reported for an LPMO to date, and is catalytically active up to temperatures of at least 80 °C. Generally, mgLPMO10 showed good activity and operational stability over a wide temperature range. The LPMO boosted cellulose saccharification by recombinantly produced GH48 and GH6 cellobiohydrolases derived from the same metagenome, albeit to a minor extent. Cellulose saccharification studies with a commercial cellulase cocktail (Celluclast®) showed that the performance of this thermostable bacterial LPMO is comparable with that of a frequently utilized fungal LPMO from Thermoascus aurantiacus (TaLPMO9A). Conclusions The high activity and operational stability of mgLPMO10 are of both fundamental and applied interest. The ability of mgLPMO10 to perform oxidative cleavage of cellulose at 80 °C and the clear synergy with Celluclast® make this enzyme an interesting candidate in the development of thermostable enzyme cocktails for use in lignocellulosic biorefineries.

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“…Perna et al showed that the observed effect is due to increased H 2 O 2 -production by reactions involving laccase-modified lignin [269]. For the successful exploitation of these effects in biomass conversion, however, further research is needed, addressing, for example, the interaction of lignin-active oxidoreductases with lignin, as well as the actual flow of electrons, the [67,104,180,190,330], 9F [180,190,335], 9E, and 9J [180,190] from N. crassa and PsAA9A and 9B from Pestalotiopsis sp. [263] NcCDH IIB + NcAA9C [180,190,330], 9E, 9F, and 9J [180,190] from N. crassa [190] Pyranose dehydrogenase (PDH), PQQ-dependent AA12 [266,312] Catalase from C. glutamicum…”

Section: Other Oxidoreductases In Biomass Conversionmentioning

confidence: 99%

“…from N. crassa [104] The tested enzyme pairs and the (putative) modes of interaction between them are listed for each type of oxidoreductase a The role and nature of the reduction step differs between catalytic scenarios, as outlined in the main text and Fig. 3 [37].…”

Section: Other Oxidoreductases In Biomass Conversionmentioning

confidence: 99%

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

Østby

Hansen

Horn

et al. 2020

Journal of Industrial Microbiology and Biotechnology

136

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Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.

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The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay

Cited by 55 publications

References 58 publications

Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions

Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions

A thermostable bacterial lytic polysaccharide monooxygenase with high operational stability in a wide temperature range

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

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