Applied Microbiology and Biotechnology

, Volume 97, Issue 19, pp 8455–8465 | Cite as

Recalcitrant polysaccharide degradation by novel oxidative biocatalysts

  • Maria Dimarogona
  • Evangelos Topakas
  • Paul ChristakopoulosEmail author


The classical hydrolytic mechanism for the degradation of plant polysaccharides by saprophytic microorganisms has been reconsidered after the recent landmark discovery of a new class of oxidases termed lytic polysaccharide monooxygenases (LPMOs). LPMOs are of increased biotechnological interest due to their implication in lignocellulosic biomass decomposition for the production of biofuels and high-value chemicals. They act on recalcitrant polysaccharides by a combination of hydrolytic and oxidative function, generating oxidized and non-oxidized chain ends. They are copper-dependent and require molecular oxygen and an external electron donor for their proper function. In this review, we present the recent findings concerning the mechanism of action of these oxidative enzymes and identify issues and questions to be addressed in the future.


Lytic polysaccharide monooxygenases CBM33 Cellobiose dehydrogenase GH61 Bioethanol Cellulose 


  1. Aachmann FL, Sorlie M, Skjak-Braek G, Eijsink VG, Vaaje-Kolstad G (2012) NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions. Proc Natl Acad Sci U S A. 109: 18779–19784Google Scholar
  2. Arantes V, Saddler JN (2010) Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis. Biotechnol Biofuels 3:4PubMedCrossRefGoogle Scholar
  3. Baldrian P, Valaskova V (2008) Degradation of cellulose by basidiomycetous fungi. FEMS Microbiol Rev 32(3):501–521PubMedCrossRefGoogle Scholar
  4. Beeson WT, Phillips CM, Cate JH, Marletta MA (2012) Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases. J Am Chem Soc 134(2):890–892PubMedCrossRefGoogle Scholar
  5. Beeson WTT, Iavarone AT, Hausmann CD, Cate JH, Marletta MA (2011) Extracellular aldonolactonase from Myceliophthora thermophila. Appl Environ Microbiol 77(2):650–656PubMedCrossRefGoogle Scholar
  6. Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan MC, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LD, Baker SE, Magnuson J, Laboissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A (2011) Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29(10):922–927PubMedCrossRefGoogle Scholar
  7. Bey M, Zhou S, Poidevin L, Henrissat B, Coutinho PM, Berrin JG, Sigoillot JC (2012) Comparison of two lytic polysaccharide monooxygenases (GH61) from Podospora anserina reveals differences upon cello-oligosaccharides oxidation. Appl Environ Microbiol in Press Google Scholar
  8. Cannella D, Hsieh CW, Felby C, Jorgensen H (2012) Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content. Biotechnol Biofuels 5(1):26PubMedCrossRefGoogle Scholar
  9. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238PubMedCrossRefGoogle Scholar
  10. Carugo O, Djinovic Carugo K (2005) When X-rays modify the protein structure: radiation damage at work. Trends Biochem Sci 30(4):213–219PubMedCrossRefGoogle Scholar
  11. Dahiya N, Tewari R, Hoondal GS (2006) Biotechnological aspects of chitinolytic enzymes: a review. Appl Microbiol Biotechnol 71(6):773–782PubMedCrossRefGoogle Scholar
  12. Dimarogona M, Topakas E, Olsson L, Christakopoulos P (2012a) Lignin boosts the cellulase performance of a GH-61 enzyme from Sporotrichum thermophile. Bioresour Technol 110:480–487PubMedCrossRefGoogle Scholar
  13. Dimarogona M, Topakas E, Christakopoulos P (2012b) Cellulose degradation by oxidative enzymes. Comput Struct Biotechnol J 2(3):e201209015Google Scholar
  14. Ding SY, Liu YS, Zeng Y, Himmel ME, Baker JO, Bayer EA (2012) How does plant cell wall nanoscale architecture correlate with enzymatic digestibility? Science 338(6110):1055–1060PubMedCrossRefGoogle Scholar
  15. Forsberg Z, Vaaje-Kolstad G, Westereng B, Bunaes AC, Stenstrom Y, Mackenzie A, Sorlie M, Horn SJ, Eijsink VG (2011) Cleavage of cellulose by a CBM33 protein. Protein Sci 20(9):1479–1483PubMedCrossRefGoogle Scholar
  16. Gibson LJ (2012) The hierarchical structure and mechanics of plant materials. J R Soc Interface 9(76):2749–2766PubMedCrossRefGoogle Scholar
  17. Harreither W, Felice AK, Paukner R, Gorton L, Ludwig R, Sygmund C (2012) Recombinantly produced cellobiose dehydrogenase from Corynascus thermophilus for glucose biosensors and biofuel cells. Biotechnol J 7(11):1359–1366PubMedCrossRefGoogle Scholar
  18. Harreither W, Sygmund C, Augustin M, Narciso M, Rabinovich ML, Gorton L, Haltrich D, Ludwig R (2011) Catalytic properties and classification of cellobiose dehydrogenases from ascomycetes. Appl Environ Microbiol 77(5):1804–1815PubMedCrossRefGoogle Scholar
  19. Harris PV, Welner D, McFarland KC, Re E, Navarro Poulsen JC, Brown K, Salbo R, Ding H, Vlasenko E, Merino S, Xu F, Cherry J, Larsen S, Lo Leggio L (2010) Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family. Biochemistry 49(15):3305–3316PubMedCrossRefGoogle Scholar
  20. Hemsworth GR, Davies GJ, Walton PH (2013) Recent insights into copper-containing lytic polysaccharide mono-oxygenases. Curr Opin Struct Biol in Press Google Scholar
  21. Henriksson G, Johansson G, Pettersson G (2000) A critical review of cellobiose dehydrogenases. J Biotechnol 78(2):93–113PubMedCrossRefGoogle Scholar
  22. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316PubMedGoogle Scholar
  23. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315(5813):804–807PubMedCrossRefGoogle Scholar
  24. Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VG (2012) Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 5(1):45PubMedCrossRefGoogle Scholar
  25. Karkehabadi S, Hansson H, Kim S, Piens K, Mitchinson C, Sandgren M (2008) The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution. J Mol Biol 383(1):144–154PubMedCrossRefGoogle Scholar
  26. Karlsson J, Saloheimo M, Siika-Aho M, Tenkanen M, Penttila M, Tjerneld F (2001) Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei. Eur J Biochem 268(24):6498–6507PubMedCrossRefGoogle Scholar
  27. Kittl R, Kracher D, Burgstaller D, Haltrich D, Ludwig R (2012) Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay. Biotechnol Biofuels 5(1):79PubMedCrossRefGoogle Scholar
  28. Kopper SFS (2003) The composition of keto aldoses in aqueous solution as determined by NMR spectroscopy. Helv Chim Acta 86:827–843CrossRefGoogle Scholar
  29. Koseki T, Mese Y, Fushinobu S, Masaki K, Fujii T, Ito K, Shiono Y, Murayama T, Iefuji H (2008) Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii. Appl Microbiol Biotechnol 77(6):1279–1285PubMedCrossRefGoogle Scholar
  30. Kostylev MWD (2012) Synergistic interactions in cellulose hydrolysis. Biofuels 3(1):61–70CrossRefGoogle Scholar
  31. Langston JA, Shaghasi T, Abbate E, Xu F, Vlasenko E, Sweeney MD (2011) Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61. Appl Environ Microbiol 77(19):7007–7015PubMedCrossRefGoogle Scholar
  32. Levasseur A, Drula E, Lombard V, Coutinho PM, Henrissat B (2013) Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnol Biofuels 6(1):41PubMedCrossRefGoogle Scholar
  33. Li X, Beeson WTT, Phillips CM, Marletta MA, Cate JH (2012) Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases. Structure 20(6):1051–1061PubMedCrossRefGoogle Scholar
  34. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66(3):506–577PubMedCrossRefGoogle Scholar
  35. MacPherson IS, Murphy ME (2007) Type-2 copper-containing enzymes. Cell Mol Life Sci 64(22):2887–2899PubMedCrossRefGoogle Scholar
  36. Martinez D, Challacombe J, Morgenstern I, Hibbett D, Schmoll M, Kubicek CP, Ferreira P, Ruiz-Duenas FJ, Martinez AT, Kersten P, Hammel KE, Vanden Wymelenberg A, Gaskell J, Lindquist E, Sabat G, Bondurant SS, Larrondo LF, Canessa P, Vicuna R, Yadav J, Doddapaneni H, Subramanian V, Pisabarro AG, Lavin JL, Oguiza JA, Master E, Henrissat B, Coutinho PM, Harris P, Magnuson JK, Baker SE, Bruno K, Kenealy W, Hoegger PJ, Kues U, Ramaiya P, Lucas S, Salamov A, Shapiro H, Tu H, Chee CL, Misra M, Xie G, Teter S, Yaver D, James T, Mokrejs M, Pospisek M, Grigoriev IV, Brettin T, Rokhsar D, Berka R, Cullen D (2009) Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion. Proc Natl Acad Sci U S A 106(6):1954–1959PubMedCrossRefGoogle Scholar
  37. Mba Medie F, Davies GJ, Drancourt M, Henrissat B (2012) Genome analyses highlight the different biological roles of cellulases. Nat Rev Microbiol 10(3):227–234PubMedCrossRefGoogle Scholar
  38. Merino ST, Cherry J (2007) Progress and challenges in enzyme development for biomass utilization. Adv Biochem Eng Biotechnol 108:95–120PubMedGoogle Scholar
  39. Panwar NL, Kaushik SC, Surendra K (2011) Role of renewable energy sources in environmental protection: a review. Renew Sust Energ Rev 15(3):1513–1524CrossRefGoogle Scholar
  40. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612PubMedCrossRefGoogle Scholar
  41. Phillips CM, Beeson WT, Cate JH, Marletta MA (2011) Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. ACS Chem Biol 6(12):1399–1406PubMedCrossRefGoogle Scholar
  42. Quinlan RJ, Sweeney MD, Lo Leggio L, Otten H, Poulsen JC, Johansen KS, Krogh KB, Jorgensen CI, Tovborg M, Anthonsen A, Tryfona T, Walter CP, Dupree P, Xu F, Davies GJ, Walton PH (2011) Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci U S A 108(37):15079–15084PubMedCrossRefGoogle Scholar
  43. Raush E, Totrov M, Marsden BD, Abagyan R (2009) A new method for publishing three-dimensional content. PLoS One 4(10):e7394. doi: 10.1371/journal.pone.0007394 PubMedCrossRefGoogle Scholar
  44. Reese ET, Siu RG, Levinson HS (1950) The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. J Bacteriol 59(4):485–497PubMedGoogle Scholar
  45. Schrempf H (2001) Recognition and degradation of chitin by streptomycetes. Antonie Van Leeuwenhoek 79(3–4):285–289PubMedCrossRefGoogle Scholar
  46. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539PubMedCrossRefGoogle Scholar
  47. Sims RE, Mabee W, Saddler JN, Taylor M (2010) An overview of second generation biofuel technologies. Bioresour Technol 101(6):1570–1580PubMedCrossRefGoogle Scholar
  48. Subramaniam SS, Nagalla SR, Renganathan V (1999) Cloning and characterization of a thermostable cellobiose dehydrogenase from Sporotrichum thermophile. Arch Biochem Biophys 365(2):223–230PubMedCrossRefGoogle Scholar
  49. Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 83(1):1–11PubMedCrossRefGoogle Scholar
  50. Sweeney MDXF (2012) Biomass converting enzymes as industrial biocatalysts for fuels and chemicals: recent developments. Catalysts 2:244–263CrossRefGoogle Scholar
  51. Sygmund C, Kracher D, Scheiblbrandner S, Zahma K, Felice AK, Harreither W, Kittl R, Ludwig R (2012) Characterization of the two Neurospora crassa cellobiose dehydrogenases and their connection to oxidative cellulose degradation. Appl Environ Microbiol 78(17):6161–6171PubMedCrossRefGoogle Scholar
  52. Turbe-Doan A, Arfi Y, Record E, Estrada-Alvarado I, Levasseur A (2012) Heterologous production of cellobiose dehydrogenases from the basidiomycete Coprinopsis cinerea and the ascomycete Podospora anserina and their effect on saccharification of wheat straw. Appl Microbiol Biotechnol 97(11):4873–4885PubMedCrossRefGoogle Scholar
  53. Vaaje-Kolstad G, Bohle LA, Gaseidnes S, Dalhus B, Bjoras M, Mathiesen G, Eijsink VG (2012) Characterization of the chitinolytic machinery of Enterococcus faecalis V583 and high-resolution structure of its oxidative CBM33 enzyme. J Mol Biol 416(2):239–254PubMedCrossRefGoogle Scholar
  54. Vaaje-Kolstad G, Horn SJ, van Aalten DM, Synstad B, Eijsink VG (2005a) The non-catalytic chitin-binding protein CBP21 from Serratia marcescens is essential for chitin degradation. J Biol Chem 280(31):28492–28497PubMedCrossRefGoogle Scholar
  55. Vaaje-Kolstad G, Houston DR, Riemen AH, Eijsink VG, van Aalten DM (2005b) Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21. J Biol Chem 280(12):11313–11319PubMedCrossRefGoogle Scholar
  56. Vaaje-Kolstad G, Westereng B, Horn SJ, Liu Z, Zhai H, Sorlie M, Eijsink VG (2010) An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science 330(6001):219–222PubMedCrossRefGoogle Scholar
  57. Vanden Wymelenberg A, Gaskell J, Mozuch M, Sabat G, Ralph J, Skyba O, Mansfield SD, Blanchette RA, Martinez D, Grigoriev I, Kersten PJ, Cullen D (2010) Comparative transcriptome and secretome analysis of wood decay fungi Postia placenta and Phanerochaete chrysosporium. Appl Environ Microbiol 76(11):3599–3610PubMedCrossRefGoogle Scholar
  58. Viikari L, Alapuranen M, Puranen T, Vehmaanpera J, Siika-Aho M (2007) Thermostable enzymes in lignocellulose hydrolysis. Adv Biochem Eng Biotechnol 108:121–145PubMedGoogle Scholar
  59. Westereng B, Agger JW, Horn SJ, Vaaje-Kolstad G, Aachmann FL, Stenstrom YH, Eijsink VG (2013) Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases. J Chromatogr A 1271(1):144–152PubMedCrossRefGoogle Scholar
  60. Westereng B, Ishida T, Vaaje-Kolstad G, Wu M, Eijsink VG, Igarashi K, Samejima M, Stahlberg J, Horn SJ, Sandgren M (2011) The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose. PLoS One 6(11):e27807. doi: 10.1371/journal.pone.0027807 PubMedCrossRefGoogle Scholar
  61. Wilson DB (2012) Processive and nonprocessive cellulases for biofuel production—lessons from bacterial genomes and structural analysis. Appl Microbiol Biotechnol 93(2):497–502PubMedCrossRefGoogle Scholar
  62. Wu M, Beckham GT, Larsson AM, Ishida T, Kim S, Payne CM, Himmel ME, Crowley MF, Horn SJ, Westereng B, Igarashi K, Samejima M, Stahlberg J, Eijsink VG, Sandgren M (2013) Crystal structure and computational characterization of the lytic polysaccharide monooxygenase GH61D from the Basidiomycota fungus Phanerochaete chrysosporium. J Biol Chem 288(18):12828–12839PubMedCrossRefGoogle Scholar
  63. Wymelenberg AVGJ, Mozuch M, Kersten P, Sabat G, Martinez D, Cullen D (2009) Transcriptome and secretome analyses of Phanerochaete chrysosporium reveal complex patterns of gene expression. Appl Environ Microbiol 75(12):4058–4068CrossRefGoogle Scholar
  64. Yakovlev I, Vaaje-Kolstad G, Hietala AM, Stefanczyk E, Solheim H, Fossdal CG (2012) Substrate-specific transcription of the enigmatic GH61 family of the pathogenic white-rot fungus Heterobasidion irregulare during growth on lignocellulose. Appl Microbiol Biotechnol 95(4):979–990PubMedCrossRefGoogle Scholar
  65. Zamocky M, Ludwig R, Peterbauer C, Hallberg BM, Divne C, Nicholls P, Haltrich D (2006) Cellobiose dehydrogenase—a flavocytochrome from wood-degrading, phytopathogenic and saprotropic fungi. Curr Protein Pept Sci 7(3):255–280PubMedCrossRefGoogle Scholar
  66. Zhang YH, Lynd LR (2004) Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng 88(7):797–824PubMedCrossRefGoogle Scholar
  67. Zhao XQ, Zi LH, Bai FW, Lin HL, Hao XM, Yue GJ, Ho NW (2012) Bioethanol from lignocellulosic biomass. Adv Biochem Eng Biotechnol 128:25–51PubMedGoogle Scholar
  68. Zifcakova L, Baldrian P (2012) Fungal polysaccharide monooxygenases: new players in the decomposition of cellulose. Fungal Ecology 5: 481–489Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Maria Dimarogona
    • 1
  • Evangelos Topakas
    • 1
  • Paul Christakopoulos
    • 2
    Email author
  1. 1.Biotechnology Laboratory, School of Chemical EngineeringNational Technical University of AthensAthensGreece
  2. 2.Biochemical and Chemical Process Engineering, Division of Sustainable Process Engineering, Department of Civil, Environmental and Natural Resources EngineeringLuleå University of TechnologyLuleåSweden

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