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Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.

Bennati-Granier C, Garajova S, Champion C, Grisel S, Haon M, Zhou S, Fanuel M, Ropartz D, Rogniaux H, Gimbert I, Record E, Berrin JG - Biotechnol Biofuels (2015)

Bottom Line: Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products.Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, ESIL Polytech, F-13288 Marseille, France ; Polytech Marseille, Aix Marseille Université, F-13288 Marseille, France.

ABSTRACT

Background: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood.

Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).

Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

No MeSH data available.


Related in: MedlinePlus

PaLPMO9H activity on oligosaccharide substrates. a Generation of H2O2 by PaLPMO9H in the presence and/or absence of various oligosaccharides substrates. Glc4, cellotetraose; Glc5, cellopentaose; Glc6, cellohexaose; Lam6, laminarinhexaose; Man6, mannohexaose; β(1,3;1,4)Glc4, β(1,3;1,4)-glucotetraose (G4G3G4G); XXXG, xyloglucan-derived heptasaccharide. b HPAEC chromatogram of products released from cellohexaose by action of PaLPMO9H in the presence of ascorbic acid (in black) or PaCDHB (in red) with the same labeling of peaks as Fig. 2
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Fig5: PaLPMO9H activity on oligosaccharide substrates. a Generation of H2O2 by PaLPMO9H in the presence and/or absence of various oligosaccharides substrates. Glc4, cellotetraose; Glc5, cellopentaose; Glc6, cellohexaose; Lam6, laminarinhexaose; Man6, mannohexaose; β(1,3;1,4)Glc4, β(1,3;1,4)-glucotetraose (G4G3G4G); XXXG, xyloglucan-derived heptasaccharide. b HPAEC chromatogram of products released from cellohexaose by action of PaLPMO9H in the presence of ascorbic acid (in black) or PaCDHB (in red) with the same labeling of peaks as Fig. 2

Mentions: Among the set of PaLPMO9s studied, PaLPMO9H was the only one displaying inhibition of H2O2 production in the presence of cellohexaose (DP6), cellopentaose (DP5), and cellotetraose (DP4) with the residual H2O2 production of 4.8, 6.5, and 19.4 %, respectively (Fig. 5a). However, no decrease in H2O2 production was observed in the presence of non-cellulosic oligosaccharides [mannohexaose, laminarihexaose, xyloglucan-derived heptasaccharide (XXXG), and mixed linkage β-(1-3,1-4)-tetraose] (Fig. 5a) suggesting that PaLPMO9H is not active on these oligosaccharide substrates. To verify these findings, we used the HPAEC method to detect native and oxidized species using cellopentaose and cellohexaose as substrates. In the presence of PaCDHB or ascorbic acid, PaLPMO9H was able to oxidatively cleave cellohexaose since the peak decreased significantly with the concomitant apparition of oxidized and non-oxidized species (Fig. 5b). The ascorbate condition yielded the release of C1-oxidized oligosaccharides (DP3ox and DP4ox) as well as presumably C4-oxidized species observed at 27 and 38 min (Fig. 5b). The use of PaCDHB as a donor of electron led to an increase of C1-oxidized oligosaccharides and to the apparition of a major peak eluting at 39 min which may correspond to a double-oxidized DP2 (C1–C4) and to longer double-oxidized products (C1–C4) eluting around 42–44 min (Fig. 5b) as observed for cellulose (Fig. 2). A similar pattern of products was obtained when cellopentaose was used as substrate (Additional file 1: Figure S4).Fig. 5


Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.

Bennati-Granier C, Garajova S, Champion C, Grisel S, Haon M, Zhou S, Fanuel M, Ropartz D, Rogniaux H, Gimbert I, Record E, Berrin JG - Biotechnol Biofuels (2015)

PaLPMO9H activity on oligosaccharide substrates. a Generation of H2O2 by PaLPMO9H in the presence and/or absence of various oligosaccharides substrates. Glc4, cellotetraose; Glc5, cellopentaose; Glc6, cellohexaose; Lam6, laminarinhexaose; Man6, mannohexaose; β(1,3;1,4)Glc4, β(1,3;1,4)-glucotetraose (G4G3G4G); XXXG, xyloglucan-derived heptasaccharide. b HPAEC chromatogram of products released from cellohexaose by action of PaLPMO9H in the presence of ascorbic acid (in black) or PaCDHB (in red) with the same labeling of peaks as Fig. 2
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4487207&req=5

Fig5: PaLPMO9H activity on oligosaccharide substrates. a Generation of H2O2 by PaLPMO9H in the presence and/or absence of various oligosaccharides substrates. Glc4, cellotetraose; Glc5, cellopentaose; Glc6, cellohexaose; Lam6, laminarinhexaose; Man6, mannohexaose; β(1,3;1,4)Glc4, β(1,3;1,4)-glucotetraose (G4G3G4G); XXXG, xyloglucan-derived heptasaccharide. b HPAEC chromatogram of products released from cellohexaose by action of PaLPMO9H in the presence of ascorbic acid (in black) or PaCDHB (in red) with the same labeling of peaks as Fig. 2
Mentions: Among the set of PaLPMO9s studied, PaLPMO9H was the only one displaying inhibition of H2O2 production in the presence of cellohexaose (DP6), cellopentaose (DP5), and cellotetraose (DP4) with the residual H2O2 production of 4.8, 6.5, and 19.4 %, respectively (Fig. 5a). However, no decrease in H2O2 production was observed in the presence of non-cellulosic oligosaccharides [mannohexaose, laminarihexaose, xyloglucan-derived heptasaccharide (XXXG), and mixed linkage β-(1-3,1-4)-tetraose] (Fig. 5a) suggesting that PaLPMO9H is not active on these oligosaccharide substrates. To verify these findings, we used the HPAEC method to detect native and oxidized species using cellopentaose and cellohexaose as substrates. In the presence of PaCDHB or ascorbic acid, PaLPMO9H was able to oxidatively cleave cellohexaose since the peak decreased significantly with the concomitant apparition of oxidized and non-oxidized species (Fig. 5b). The ascorbate condition yielded the release of C1-oxidized oligosaccharides (DP3ox and DP4ox) as well as presumably C4-oxidized species observed at 27 and 38 min (Fig. 5b). The use of PaCDHB as a donor of electron led to an increase of C1-oxidized oligosaccharides and to the apparition of a major peak eluting at 39 min which may correspond to a double-oxidized DP2 (C1–C4) and to longer double-oxidized products (C1–C4) eluting around 42–44 min (Fig. 5b) as observed for cellulose (Fig. 2). A similar pattern of products was obtained when cellopentaose was used as substrate (Additional file 1: Figure S4).Fig. 5

Bottom Line: Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products.Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, Faculté des Sciences de Luminy, ESIL Polytech, F-13288 Marseille, France ; Polytech Marseille, Aix Marseille Université, F-13288 Marseille, France.

ABSTRACT

Background: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood.

Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan).

Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries.

No MeSH data available.


Related in: MedlinePlus