Purification and characterization of a native lytic polysaccharide monooxygenase from Thermoascus aurantiacus

Abstract

Lytic polysaccharide monooxygenases (LPMOs) have emerged as key proteins for depolymerization of cellulose. These copper-containing enzymes oxidize C-1 and/or C-4 bonds in cellulose, promoting increased hydrolysis of the oxidized cellulose chains. The LPMO from Thermoascus aurantiacus, a thermophilic ascomycete fungus, has been extensively studied and has served as a model LPMO. A method was developed to purify the LPMO from culture filtrates of T. aurantiacus along with its native cellobiohydrolase and endoglucanase. The activity of the purified LPMO was measured with a colorimetric assay that established the Topt of the native LPMO at 60 °C. Purification of the components of the T. aurantiacus cellulase mixture also enabled quantification of the amounts of cellobiohydrolase, endoglucanase and LPMO present in the T. aurantiacus culture filtrate, establishing that the LPMO was the most abundant protein in the culture supernatants. The importance of the LPMO to activity of the mixture was demonstrated by saccharifications with Avicel and acid-pretreated corn stover.

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References

  1. Bissaro B, Røhr ÅK, Müller G et al (2017) Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2. Nat Chem Biol 13:1123–1128. https://doi.org/10.1038/nchembio.2470

    CAS  Article  PubMed  Google Scholar 

  2. Blanch HW, Simmons BA, Klein-Marcuschamer D (2011) Biomass deconstruction to sugars. Biotechnol J 6:1086–1102. https://doi.org/10.1002/biot.201000180

    CAS  Article  PubMed  Google Scholar 

  3. Breslmayr E, Hanžek M, Hanrahan A et al (2018) A fast and sensitive activity assay for lytic polysaccharide monooxygenase. Biotechnol Biofuels 11:79. https://doi.org/10.1186/s13068-018-1063-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Chylenski P, Petrović DM, Müller G et al (2017) Enzymatic degradation of sulfite-pulped softwoods and the role of LPMOs. Biotechnol Biofuels 10:177. https://doi.org/10.1186/s13068-017-0862-5

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Forsberg Z, Sørlie M, Petrović D et al (2019) Polysaccharide degradation by lytic polysaccharide monooxygenases. Curr Opin Struct Biol 59:54–64. https://doi.org/10.1016/j.sbi.2019.02.015

    CAS  Article  PubMed  Google Scholar 

  6. Gladden JM, Allgaier M, Miller CS et al (2011) Glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass. Appl Environ Microbiol 77:5804–5812. https://doi.org/10.1128/AEM.00032-11

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Hamre AG, Strømnes A-GS, Gustavsen D et al (2019) Treatment of recalcitrant crystalline polysaccharides with lytic polysaccharide monooxygenase relieves the need for glycoside hydrolase processivity. Carbohydr Res 473:66–71. https://doi.org/10.1016/j.carres.2019.01.001

    CAS  Article  PubMed  Google Scholar 

  8. Harris PV, Welner D, McFarland KC et al (2010) Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family. Biochemistry 49:3305–3316. https://doi.org/10.1021/bi100009p

    CAS  Article  PubMed  Google Scholar 

  9. Khandke KM, Vithayathil PJ, Murthy SK (1989) Purification of xylanase, β-glucosidase, endocellulase, and exocellulase from a thermophilic fungus, Thermoascus aurantiacus. Arch Biochem Biophys 274:491–500

    CAS  Article  Google Scholar 

  10. Kjaergaard CH, Qayyum MF, Wong SD et al (2014) Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases. Proc Natl Acad Sci USA 111:8797–8802. https://doi.org/10.1073/pnas.1408115111

    CAS  Article  PubMed  Google Scholar 

  11. Kracher D, Scheiblbrandner S, Felice AKG et al (2016) Extracellular electron transfer systems fuel cellulose oxidative degradation. Science 352:1098–1101. https://doi.org/10.1126/science.aaf3165

    CAS  Article  PubMed  Google Scholar 

  12. Kuhad RC, Deswal D, Sharma S et al (2016) Revisiting cellulase production and redefining current strategies based on major challenges. Renew Sustain Energy Rev 55:249–272. https://doi.org/10.1016/j.rser.2015.10.132

    CAS  Article  Google Scholar 

  13. McClendon SD, Batth T, Petzold CJ et al (2012) Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions. Biotechnol Biofuels 5:54. https://doi.org/10.1186/1754-6834-5-54

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Müller G, Chylenski P, Bissaro B et al (2018) The impact of hydrogen peroxide supply on LPMO activity and overall saccharification efficiency of a commercial cellulase cocktail. Biotechnol Biofuels 11:209. https://doi.org/10.1186/s13068-018-1199-4

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Müller G, Várnai A, Johansen KS et al (2015) Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions. Biotechnol Biofuels 8:187. https://doi.org/10.1186/s13068-015-0376-y

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Park JI, Steen EJ, Burd H et al (2012) A thermophilic ionic liquid-tolerant cellulase cocktail for the production of cellulosic biofuels. PLoS ONE 7:e37010. https://doi.org/10.1371/journal.pone.0037010

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Payne CM, Knott BC, Mayes HB et al (2015) Fungal cellulases. Chem Rev 115:1308–1448

    CAS  Article  Google Scholar 

  18. Petrović DM, Bissaro B, Chylenski P et al (2018) Methylation of the N-terminal histidine protects a lytic polysaccharide monooxygenase from auto-oxidative inactivation. Protein Sci 27:1636–1650. https://doi.org/10.1002/pro.3451

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Quinlan RJ, Sweeney MD, Lo Leggio L et al (2011) Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci USA 108:15079–15084. https://doi.org/10.1073/pnas.1105776108

    Article  PubMed  Google Scholar 

  20. Rosgaard L, Pedersen S, Cherry JR et al (2006) Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose. Biotechnol Prog 22:493–498. https://doi.org/10.1021/bp050361o

    CAS  Article  PubMed  Google Scholar 

  21. Scarlat N, Dallemand J-F, Monforti-Ferrario F, Nita V (2015) The role of biomass and bioenergy in a future bioeconomy: Policies and facts. Environ Dev 15:3–34. https://doi.org/10.1016/j.envdev.2015.03.006

    Article  Google Scholar 

  22. Schuerg T, Gabriel R, Baecker N et al (2017a) Thermoascus aurantiacus is an intriguing host for the industrial production of cellulases. CBIOT 6:89–97. https://doi.org/10.2174/2211550105666160520123504

    CAS  Article  Google Scholar 

  23. Schuerg T, Prahl J-P, Gabriel R et al (2017b) Xylose induces cellulase production in Thermoascus aurantiacus. Biotechnol Biofuels 10:271. https://doi.org/10.1186/s13068-017-0965-z

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Singh RK, Blossom BM, Russo DA et al (2020) Detection and characterization of a novel copper-dependent intermediate in a lytic polysaccharide monooxygenase. Chem Eur J 26:454–463. https://doi.org/10.1002/chem.201903562

    CAS  Article  PubMed  Google Scholar 

  25. Taylor LE, Knott BC, Baker JO et al (2018) Engineering enhanced cellobiohydrolase activity. Nat Commun 9:1186. https://doi.org/10.1038/s41467-018-03501-8

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors are grateful for funding support from the DOE Joint BioEnergy Institute (https://www.jbei.org) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy. Novozymes is acknowledged for the generous gift of Cellic CTec2. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. The authors thank Melvin Tucker of the National Renewable Energy Laboratory for providing acid-pretreated corn stover. We thank Andy DiGiovanni of the Joint BioEnergy Institute for assistance with protein purification.

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SWS and RG. designed research; SF, CH JG and RG performed research.

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Correspondence to Raphael Gabriel or Steven W. Singer.

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Fritsche, S., Hopson, C., Gorman, J. et al. Purification and characterization of a native lytic polysaccharide monooxygenase from Thermoascus aurantiacus. Biotechnol Lett (2020). https://doi.org/10.1007/s10529-020-02942-w

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Keywords

  • Lytic polysaccharide monooxygenase
  • Cellulose
  • Biomass deconstruction