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Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes

Abstract

Lignocellulolytic enzymes are among the most costly part in production of bioethanol. Therefore, recycling of enzymes is interesting as a concept for reduction of process costs. However, stability of the enzymes during the process is critical. In this work, focus has been on investigating the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65 °C and up to 5 % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55 °C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation.

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References

  1. Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 23(3):257–270

    Article  CAS  Google Scholar 

  2. Bailey MJ, Tahtiharju J (2003) Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer-controlled feeding strategy. Appl Microbiol Biotechnol 62(2–3):156–162. doi:10.1007/s00253-003-1276-9

    PubMed  Article  CAS  Google Scholar 

  3. Boer H, Koivula A (2003) The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A. Eur J Biochem 270(5):841–848. doi:10.1046/j.1432-1033.2003.03431.x

    PubMed  Article  CAS  Google Scholar 

  4. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ (2004) Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J 382(Pt 3):769–781. doi:10.1042/BJ20040892

    PubMed  CAS  Google Scholar 

  5. Claeyssens M, Aerts G (1992) Characterization of cellulolytic activities in commercial Trichoderma reesei preparations—an approach using small, chromogenic substrates. Bioresour Technol 39(2):143–146. doi:10.1016/0960-8524(92)90133-I

    Article  CAS  Google Scholar 

  6. Darias R, Villalonga R (2001) Functional stabilization of cellulase by covalent modification with chitosan. J Chem Technol Biotechnol 76(5):489–493

    Article  CAS  Google Scholar 

  7. Farinas CS, Loyo MM, Baraldo A, Tardioli PW, Neto VB, Couri S (2010) Finding stable cellulase and xylanase evaluation of the synergistic effect of pH and temperature. New Biotechnol 27(6):810–815

    Article  CAS  Google Scholar 

  8. Galbe M, Zacchi G (2002) A review of the production of ethanol from softwood. Appl Microbiol Biotechnol 59(6):618–628. doi:10.1007/s00253-002-1058-9

    PubMed  Article  CAS  Google Scholar 

  9. Genencor (2012) Datasheet for Accelerase Trio. Available via Genencor’s homepage http://www.genencor.com. Accessed 11 Dec 2012

  10. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59(2):257–268

    Article  CAS  Google Scholar 

  11. Ghosh P, Pamment NB, Martin WRB (1982) Simultaneous saccharification and fermentation of cellulose—effect of beta-d-glucosidase activity and ethanol inhibition of cellulases. Enzyme Microb Tech 4(6):425–430

    Article  CAS  Google Scholar 

  12. Gnansounou E (2010) Production and use of lignocellulosic bioethanol in Europe: current situation and perspectives. Bioresour Technol 101(13):4842–4850. doi:10.1016/j.biortech.2010.02.002

    PubMed  Article  CAS  Google Scholar 

  13. Heinzelman P, Snow CD, Wu I, Nguyen C, Villalobos A, Govindarajan S, Minshull J, Arnold FH (2009) A family of thermostable fungal cellulases created by structure-guided recombination. Proc Natl Acad Sci USA 106(14):5610–5615. doi:10.1073/pnas.0901417106

    PubMed  Article  CAS  Google Scholar 

  14. 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):804–807. doi:10.1126/science.1137016

    PubMed  Article  CAS  Google Scholar 

  15. Holtzapple M, Cognata M, Shu Y, Hendrickson C (1990) Inhibition of Trichoderma Reesei cellulase by sugars and solvents. Biotechnol Bioeng 36(3):275–287

    PubMed  Article  CAS  Google Scholar 

  16. Jørgensen H, Vibe-Pedersen J, Larsen J, Felby C (2007) Liquefaction of lignocellulose at high-solids concentrations. Biotechnol Bioeng 96(5):862–870. doi:10.1002/Bit.21115

    PubMed  Article  Google Scholar 

  17. Karlsson J, Siika-aho M, Tenkanen M, Tjerneld F (2002) Enzymatic properties of the low molecular mass endoglucanases Cel12A (EG III) and Cel45A (EG V) of Trichoderma reesei. J Biotechnol 99(1):63–78

    PubMed  Article  CAS  Google Scholar 

  18. Kristensen JB, Felby C, Jørgensen H (2009) Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol Biofuels 2. doi:10.1186/1754-6834-2-11

  19. Larsen J, Petersen MØ, Thirup L, Li HW, Iversen FK (2008) The IBUS process—lignocellulosic bioethanol close to a commercial reality. Chem Eng Technol 31(5):765–772. doi:10.1002/ceat.200800048

    Article  CAS  Google Scholar 

  20. Lodish HBA, Matsudaira P, Kaiser CA, Krieger M, Scott MP, Zipursky SL, Darnell J (2004) Molecular cell biology, 5th edn. Freeman

  21. Lu YP, Yang B, Gregg D, Saddler JN, Mansfield SD (2002) Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam-exploded softwood residues. Appl Biochem Biotech 98:641–654

    Article  Google Scholar 

  22. Maheshwari R, Bharadwaj G, Bhat MK (2000) Thermophilic fungi: their physiology and enzymes. Microbiol Mol Biol R 64(3):461–488

    Google Scholar 

  23. Margeot A, Hahn-Hägerdal B, Edlund M, Slade R, Monot F (2009) New improvements for lignocellulosic ethanol. Curr Opin Biotech 20(3):372–380. doi:10.1016/j.copbio.2009.05.009

    PubMed  Article  CAS  Google Scholar 

  24. Merino ST, Cherry J (2007) Progress and challenges in enzyme development for biomass utilization. Biofuels 108:95–120. doi:10.1007/10_2007_066

    Article  CAS  Google Scholar 

  25. Novozymes (2012) Datasheet for Cellic CTec 3. Available via Novozymes homepage http://bioenergy.novozymes.com. Accessed 12 Dec 2012

  26. Ooshima H, Ishitani Y, Harano Y (1985) Simultaneous saccharification and fermentation of cellulose—effect of ethanol on enzymatic saccharification of cellulose. Biotechnol Bioeng 27(4):389–397

    PubMed  Article  CAS  Google Scholar 

  27. Podkaminer KK, Shao XJ, Hogsett DA, Lynd LR (2011) Enzyme inactivation by ethanol and development of a kinetic model for thermophilic simultaneous saccharification and fermentation at 50° C with Thermoanaerobacterium saccharolyticum ALK2. Biotechnol Bioeng 108(6):1268–1278. doi:10.1002/Bit.23050

    PubMed  Article  CAS  Google Scholar 

  28. Pribowo A, Arantes V, Saddler JN (2012) The adsorption and enzyme activity profiles of specific Trichoderma reesei cellulase/xylanase components when hydrolyzing steam pretreated corn stover. Enzyme Microb Tech 50(3):195–203. doi:10.1016/j.enzmictec.2011.12.004

    Article  CAS  Google Scholar 

  29. Qi BK, Chen XR, Su Y, Wan YH (2011) Enzyme adsorption and recycling during hydrolysis of wheat straw lignocellulose. Bioresour Technol 102(3):2881–2889. doi:10.1016/j.biortech.2010.10.092

    PubMed  Article  CAS  Google Scholar 

  30. Shao Q, Fan Y, Yang L, Gao YQ (2012) From protein denaturant to protectant: comparative molecular dynamics study of alcohol/protein interactions. J Chem Phys 136(11):115101. doi:10.1063/1.3692801

    PubMed  Article  Google Scholar 

  31. Steele E, Raj S, Nghiem J, Stowers M (2005) Enzyme recovery and recycling following hydrolysis of ammonia fiber explosion-treated corn stover. Appl Biochem Biotech 121:901–910

    Article  Google Scholar 

  32. Szczodrak J, Targonski Z (1989) Simultaneous saccharification and fermentation of cellulose—effect of ethanol and cellulases on particular stages. Acta Biotechnol 9(6):555–564

    Article  CAS  Google Scholar 

  33. Szijarto N, Horan E, Zhang JH, Puranen T, Siika-aho M, Viikari L (2011) Thermostable endoglucanases in the liquefaction of hydrothermally pretreated wheat straw. Biotechnol Biofuels 4. doi:10.1186/1754-6834-4-2

  34. Tu MB, Chandra RP, Saddler JN (2007) Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates. Biotechnol Progr 23(2):398–406. doi:10.1021/Bp060354f

    Article  CAS  Google Scholar 

  35. Tu MB, Chandra RP, Saddler JN (2007) Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated lodgepole pine. Biotechnol Progr 23(5):1130–1137. doi:10.1021/Bp070129d

    CAS  Google Scholar 

  36. Varnai A, Siika-Aho M, Viikari L (2010) Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose. Enzyme Microb Tech 46(3–4):185–193. doi:10.1016/j.enzmictec.2009.12.013

    Article  CAS  Google Scholar 

  37. Varnai A, Viikari L, Marjamaa K, Siika-Aho M (2010) Adsorption of monocomponent enzymes in enzyme mixture analyzed quantitatively during hydrolysis of lignocellulose substrates. Bioresour Technol. doi:10.1016/j.biortech.2010.07.120

    PubMed  Google Scholar 

  38. Viikari L, Alapuranen M, Puranen T, Vehmaanpera J, Siika-Aho M (2007) Thermostable enzymes in lignocellulose hydrolysis. Biofuels 108:121–145. doi:10.1007/10_2007_065

    Article  CAS  Google Scholar 

  39. Voutilainen SP, Murray PG, Tuohy MG, Koivula A (2010) Expression of Talaromyces emersonii cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity. Protein Eng Des Sel 23(2):69–79. doi:10.1093/protein/gzp072

    PubMed  Article  CAS  Google Scholar 

  40. Voutilainen SP, Puranen T, Siika-Aho M, Lappalainen A, Alapuranen M, Kallio J, Hooman S, Viikri L, Vehmaanpera J, Koivula A (2008) Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases. Biotechnol Bioeng 101(3):515–528. doi:10.1002/Bit.21940

    PubMed  Article  CAS  Google Scholar 

  41. Wilson DB (2009) Cellulases and biofuels. Curr Opin Biotech 20(3):295–299. doi:10.1016/j.copbio.2009.05.007

    PubMed  Article  CAS  Google Scholar 

  42. Wood TM, Bhat KM (1988) Methods for measuring cellulase activities. Method Enzymol 160:87–112

    Article  CAS  Google Scholar 

  43. Wu Z, Lee YY (1997) Inhibition of the enzymatic hydrolysis of cellulose by ethanol. Biotechnol Lett 19(10):977–979. doi:10.1023/A:1018487015129

    Article  CAS  Google Scholar 

  44. Xiao ZZ, Storms R, Tsang A (2004) Microplate-based filter paper assay to measure total cellulase activity. Biotechnol Bioeng 88(7):832–837. doi:10.1002/Bit.20286

    PubMed  Article  CAS  Google Scholar 

  45. Yoshikawa H, Hirano A, Arakawa T, Shiraki K (2012) Effects of alcohol on the solubility and structure of native and disulfide-modified bovine serum albumin. Int J Biol Macromol 50(5):1286–1291. doi:10.1016/j.ijbiomac.2012.03.014

    PubMed  Article  CAS  Google Scholar 

  46. Yoshikawa H, Hirano A, Arakawa T, Shiraki K (2012) Mechanistic insights into protein precipitation by alcohol. Int J Biol Macromol 50(3):865–871. doi:10.1016/j.ijbiomac.2011.11.005

    PubMed  Article  CAS  Google Scholar 

  47. Zhang JH, Tuomainen P, Siika-aho M, Viikari L (2011) Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis. Bioresour Technol 102(19):9090–9095. doi:10.1016/j.biortech.2011.06.085

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

This research was financed by the EU project HYPE, grant agreement no. 213139. The authors thank Roal Oy and VTT (Finland) for providing thermostable enzymes and Novozymes (Denmark) for providing Celluclast and Novozym188.

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Correspondence to Henning Jørgensen.

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Skovgaard, P.A., Jørgensen, H. Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes. J Ind Microbiol Biotechnol 40, 447–456 (2013). https://doi.org/10.1007/s10295-013-1248-8

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  • DOI: https://doi.org/10.1007/s10295-013-1248-8

Keywords

  • Thermostability
  • Cellulases
  • Xylanases
  • Distillation
  • Enzyme recycling
  • Bioethanol