Applied Microbiology and Biotechnology

, Volume 98, Issue 10, pp 4409–4420 | Cite as

Investigating commercial cellulase performances toward specific biomass recalcitrance factors using reference substrates

  • Xiaohui Ju
  • Mark Bowden
  • Mark Engelhard
  • Xiao Zhang
Biotechnologically relevant enzymes and proteins


Three commercial cellulase preparations, Novozymes Cellic® Ctec2, Dupont Accellerase® 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulase enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulase performance. While the degree of polymerization of cellulose in the reference substrates did not present a major recalcitrance factor to Novozymes Cellic® Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.


Cellulase Hydrolytic efficiency Reference substrates Lignin Nanocrystalline cellulose PMOs 

Supplementary material

253_2013_5450_MOESM1_ESM.pdf (267 kb)
ESM 1(PDF 266 kb)


  1. Arantes V, Saddler JN (2010) Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis. Biotechol Biofuels 3:4CrossRefGoogle Scholar
  2. ASTM D4243–99, (2009) Standard test method for measurement of average viscometric degree of polymerization of new and aged electrical papers and boards. ASTM International, West ConshohockenGoogle Scholar
  3. Bansal P, Hall M, Realff MJ, Lee JH, Bommarius AS (2010) Multivariate statistical analysis of X-ray data from cellulose: a new method to determine degree of crystallinity and predict hydrolysis rates. Bioresour Technol 101:4461–4471PubMedCrossRefGoogle Scholar
  4. Beeson WT, Phillips CM, Cate JHD, Marletta MA (2012) Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases. J Am Chem Soc 134:890–892PubMedCrossRefGoogle Scholar
  5. Berlin A, Gilkes N, Kilburn D, Bura R, Markov A, Skomarovsky A, Okunev O, Gusakov A, Maximenko V, Gregg D, Sinitsyn A, Saddler J (2005a) Evaluation of novel fungal cellulase preparations for ability to hydrolyze softwood substrates—evidence for the role of accessory enzymes. Enzym Microb Technol 37:175–184CrossRefGoogle Scholar
  6. Berlin A, Gilkes N, Kurabi A, Bura R, Tu M, Kilburn D, Saddler J (2005b) Weak lignin-binding enzymes: a novel approach to improve activity of cellulases for hydrolysis of lignocellulosics. Appl Biochem Biotechnol 121–124:163–170PubMedCrossRefGoogle Scholar
  7. Berlin A, Balakshin M, Gilkes N, Kadla J, Maximenko V, Kubo S, Saddler J (2006) Inhibition of cellulase, xylanase, and β-glucosidase activities by softwood lignin preparations. J Biotechnol 125:198–209PubMedCrossRefGoogle Scholar
  8. Berlin A, Maximenko V, Gilkes N, Saddler J (2007) Optimization of enzyme complexes for lignocellulose hydrolysis. Biotechnol Bioeng 97:287–296PubMedCrossRefGoogle Scholar
  9. Brown EE, Hu DH, Abu Lail N, Zhang X (2013) Potential of nanocrystalline cellulose-fibrin nanocomposites for artificial vascular graft applications. Biomacromolecules 14:1063–1071PubMedCrossRefGoogle Scholar
  10. Browning BL (1967) Methods of wood chemistry. John Wiley & Sons Inc, New YorkGoogle Scholar
  11. Chang VS, Holtzapple MT (2000) Fundamental factors affecting biomass enzymatic reactivity. Appl Biochem Biotechnol 84–86:5–37PubMedCrossRefGoogle Scholar
  12. Chen Y, Stipanovic AJ, Winter WT, Wilson DB, Kim YJ (2007) Effect of digestion by pure cellulases on crystallinity and average chain length for bacterial and microcrystalline celluloses. Cellulose 14:283–293CrossRefGoogle Scholar
  13. Coughlan MP (1985) The properties of fungal and bacterial cellulases with comment on their production and application. Biotechnol Genet Eng Rev 3:39–109CrossRefGoogle Scholar
  14. Dai Z, Aryal UK, Shukla A, Qian W, Smith RD, Magnuson JK, Adney WS, Beckham GT, Brunecky R, Himmel ME, Decker SR, Ju X, Zhang X, Baker SE (2013) Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger. Fungal Genet Biol. doi:10.1016/j.fgb.2013.09.004 Google Scholar
  15. Drissen RET, Maas RHW, Van Der Maarel MJEC, Kabel MA, Schols HA, Tramper J, Beeftink HH (2007) A generic model for glucose production from various cellulose sources by a commercial cellulase complex. Biocatal Biotransform 25:419–429CrossRefGoogle Scholar
  16. Fox JM, Levine SE, Clark DS, Blanch HW (2012) Initial- and processive-cut products reveal cellobiohydrolase rate limitations and the role of companion enzymes. Biochemistry 51:442–452PubMedCrossRefGoogle Scholar
  17. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268Google Scholar
  18. Gregg DJ, Saddler JN (1996) Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process. Biotechnol Bioeng 51:375–383PubMedCrossRefGoogle Scholar
  19. Gupta R, Lee YY (2009) Mechanism of cellulase reaction on pure cellulosic substrates. Biotechnol Bioeng 102:1570–1581PubMedCrossRefGoogle Scholar
  20. Hall M, Bansal P, Lee JH, Realff MJ, Bommarius AS (2010) Cellulose crystallinity—a key predictor of the enzymatic hydrolysis rate. FEBS J 277:1571–1582PubMedCrossRefGoogle Scholar
  21. Hamad WY, Hu TQ (2010) Structure-process-yield interrelations in nanocrystalline cellulose extraction. Can J Chem Eng 88:392–402Google Scholar
  22. 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 49:3305–3316PubMedCrossRefGoogle 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 315:804–807PubMedCrossRefGoogle Scholar
  24. Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VGH (2012) Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 5:45PubMedCentralPubMedCrossRefGoogle Scholar
  25. Johansson LS, Campbell JM, Koljonen K, Stenius P (1999) Evaluation of surface lignin on cellulose fibers with XPS. Appl Surf Sci 144–45:92–95CrossRefGoogle Scholar
  26. Ju X, Engelhard M, Zhang X (2013a) An advanced understanding of the specific effects of xylan and surface lignin contents on enzymatic hydrolysis of lignocellulosic biomass. Bioresour Technol 132:137–145PubMedCrossRefGoogle Scholar
  27. Ju X, Grego C, Zhang X (2013b) Specific effects of fiber size and fiber swelling on biomass substrate surface area and enzymatic digestibility. Bioresour Technol 144:232–239PubMedCrossRefGoogle Scholar
  28. Kabel MA, van der Maarel MJEC, Klip G, Voragen AGJ, Schols HA (2006) Standard assays do not predict the efficiency of commercial cellulase preparations towards plant materials. Biotechnol Bioeng 93:56–63PubMedCrossRefGoogle Scholar
  29. Kerff F, Amoroso A, Herman R, Sauvage E, Petrella S, Filee P, Charlier P, Joris B, Tabuchi A, Nikolaidis N, Cosgrove DJ (2008) Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization. PNAS 105:16876–16881PubMedCentralPubMedCrossRefGoogle Scholar
  30. Klein-Marcuschamer D, Oleskowicz-Popiel P, Simmons BA, Blanch HW (2012) The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol Bioeng 109:1083–1087PubMedCrossRefGoogle Scholar
  31. Krassig HA (1993) Cellulose, structure, accessibility, and reactivity, vol 11. Gordon and Breach Science publisher, New YorkGoogle Scholar
  32. Kumar D, Murthy GS (2013) Stochastic molecular model of enzymatic hydrolysis of cellulose for ethanol production. Biotechnol Biofuels 6:63PubMedCentralPubMedCrossRefGoogle Scholar
  33. Le Bourvellec C, Renard CMGC (2012) Interactions between polyphenols and macromolecules: quantification methods and mechanisms. Crit Rev Food Sci Nutr 52:213–248PubMedCrossRefGoogle Scholar
  34. Lee SB, Kim IH, Ryu DDY, Taguchi H (1983) Structural-properties of cellulose and cellulase reaction-mechanism. Biotechnol Bioeng 25:33–51PubMedCrossRefGoogle Scholar
  35. Levine SE, Fox JM, Clark DS, Blanch HW (2011) A mechanistic model for rational design of optimal cellulase mixtures. Biotechnol Bioeng 108:2561–2570PubMedCrossRefGoogle Scholar
  36. Li H, Liu H, Fu SY, Zhan HY (2011) Surface hydrophobicity modification of cellulose fibers by layer-by-layer self-assembly of lignosulfonates. Bioresource 6:1681–1695Google Scholar
  37. Mansfield SD, Mooney C, Saddler JN (1999) Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol Prog 15:804–816PubMedCrossRefGoogle Scholar
  38. Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, Chapman J, Chertkov O, Coutinho PM, Cullen D, Danchin EGJ, Grigoriev IV, Harris P, Jackson M, Kubicek CP, Han CS, Ho I, Larrondo LF, de Leon AL, Magnuson JK, Merino S, Misra M, Nelson B, Putnam N, Robbertse B, Salamov AA, Schmoll M, Terry A, Thayer N, Westerholm-Parvinen A, Schoch CL, Yao J, Barbote R, Nelson MA, Detter C, Bruce D, Kuske CR, Xie G, Richardson P, Rokhsar DS, Lucas SM, Rubin EM, Dunn-Coleman N, Ward M, Brettin TS (2008) Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol 26:553–560PubMedCrossRefGoogle Scholar
  39. Mcqueenmason S, Cosgrove DJ (1994) Disruption of hydrogen-bonding between plant-cell wall polymers by proteins that induce wall extension. PNAS 91:6574–6578CrossRefGoogle Scholar
  40. Merino ST, Cherry J (2007) Progress and challenges in enzyme development for biomass utilization. Biofuels 108:95–120CrossRefGoogle Scholar
  41. Moon RJ, Martini A, Nairn J, Simonsen J, Youngblood J (2011) Cellulose nanomaterials review: structure, properties, and nanocomposites. Chem Soc Rev 40:3941–3994PubMedCrossRefGoogle Scholar
  42. Ozdal T, Capanoglu E, Altay F (2013) A review on protein-phenolic interactions and associated changes. Food Res Int 51:954–970CrossRefGoogle Scholar
  43. Pan X (2008) Role of functional groups in lignin inhibition of enzymatic hydrolysis of cellulose to glucose. J Biobased Mater Bioenergy 2:25–32CrossRefGoogle Scholar
  44. Pan XJ, Gilkes N, Kadla J, Pye K, Saka S, Gregg D, Ehara K, Xie D, Lam D, Saddler J (2006) Bioconversion of hybrid poplar to ethanol and co-products using an organosolv fractionation process: Optimization of process yields. Biotechnol Bioeng 94:851–861PubMedCrossRefGoogle Scholar
  45. Pandey A, Selvakumar P, Soccol CR, Nigam P (1999) Solid state fermentation for the production of industrial enzymes. Curr Sci 77:149–162Google Scholar
  46. Persson I, Tjerneld F, Hahn-Hägerdal B (1991) Fungal cellulolytic enzyme-production - a review. Process Biochem 26:65–74CrossRefGoogle Scholar
  47. Rosgaard L, Pedersen S, Cherry JR, Harris P, Meyer AS (2006) Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose. Biotechnol Prog 22:493–498PubMedCrossRefGoogle Scholar
  48. Rouxhet PG, Genet MJ (2011) XPS analysis of bio-organic systems. Surf Interface Anal 43:1453–1470CrossRefGoogle Scholar
  49. Saloheimo M, Paloheimo M, Hakola S, Pere J, Swanson B, Nyyssönen E, Bhatia A, Ward M, Penttilä M (2002) Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. Eur J Biochem 269:4202–4211PubMedCrossRefGoogle Scholar
  50. Segal L, Creely JJ, Martin AE Jr, Conrad CM (1962) An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Tex Res J 29:786–794CrossRefGoogle Scholar
  51. Seiji N, Richard PC, Jack NS (2001) The influence of lignin on the enzymatic hydrolysis of pretreated biomass substrates. In: Zhu J, Zhang X, Pan X (eds) Sustainable production of fuels, chemicals, and fibers from forest biomass. American Chemical Society, Washington, pp 145–167Google Scholar
  52. Sewalt VJH, Glasser WG, Beauchemin KA (1997) Lignin impact on fiber degradation 3. Reversal of inhibition of enzymatic hydrolysis by chemical modification of lignin and by additives. J Agric Food Chem 45:1823–1828CrossRefGoogle Scholar
  53. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D (2006) Determination of sugars, byproducts, and degradation products in liquid fraction process samples. NREL/TP-510-42623 NREL Laboratory Analytical Procedure. National Renewable Energy Laboratory, Golden, CO.
  54. TAPPI, Peachtree Corners, GA (2007) T204 cm-07 “Solvent extractives of wood and pulp”.Google Scholar
  55. TAPPI, Peachtree Corners, GA (2009) T249 cm-09 “Carbohydrate composition of extractive-free wood and wood pulp”.Google Scholar
  56. TAPPI, Peachtree Corners, GA (2011) T222 om-11 “Acid-Insoluble Lignin in Wood and Pulp”.Google Scholar
  57. TAPPI, Peachtree Corners, GA (2012) T211 om-12 “Ash in Wood, Pulp, Paper, and Paperboard”.Google Scholar
  58. TAPPI, Peachtree Corners, GA (2013) T236 om-13 “Kappa number of pulp”.Google Scholar
  59. Tejirian A, Xu F (2011) Inhibition of enzymatic cellulolysis by phenolic compounds. Enzym Microb Technol 48:239–247CrossRefGoogle Scholar
  60. Tu MB, Chandra RP, Saddler JN (2007) Recycling cellulases during the hydrolysis of steam exploded and ethanol pretreated lodgepole pine. Biotechnol Prog 23:1130–1137PubMedCrossRefGoogle Scholar
  61. Wyman CE (1999) Biomass ethanol: Technical progress, opportunities, and commercial challenges. Annu Rev Energy Environ 24:189–226CrossRefGoogle Scholar
  62. Xiao Z, Zhang X, Gregg D, Saddler J (2004) Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates. Appl Biochem Biotechnol 115:1115–1126CrossRefGoogle Scholar
  63. Ximenes E, Kim Y, Mosier N, Dien B, Ladisch M (2010) Inhibition of cellulases by phenols. Enzym Microb Technol 46:170–176CrossRefGoogle Scholar
  64. Ximenes E, Kim Y, Mosier N, Dien B, Ladisch M (2011) Deactivation of cellulases by phenols. Enzym Microb Technol 48:54–60CrossRefGoogle Scholar
  65. Zhang YHP, Lynd LR (2004) Toward an aggregated understanding of enzymatic hydrolysis of cellulose: Noncomplexed cellulase systems. Biotechnol Bioeng 88:797–824PubMedCrossRefGoogle Scholar
  66. Zhang YHP, Himmel ME, Mielenz JR (2006) Outlook for cellulase improvement: Screening and selection strategies. Biotechnol Adv 24:452–481CrossRefGoogle Scholar
  67. Zhang X, Qin WJ, Paice MG, Saddler JN (2009) High consistency enzymatic hydrolysis of hardwood substrates. Bioresour Technol 100:5890–5897PubMedCrossRefGoogle Scholar
  68. Zifcakova L, Baldrian P (2012) Fungal polysaccharide monooxygenases: new players in the decomposition of cellulose. Fungal Ecol 5:481–489CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Xiaohui Ju
    • 1
  • Mark Bowden
    • 2
  • Mark Engelhard
    • 2
  • Xiao Zhang
    • 1
  1. 1.Voiland School of Chemical Engineering and Bioengineering, Bioproducts, Science and Engineering LaboratoryWashington State UniversityRichlandUSA
  2. 2.Environmental Molecular Science Laboratory, Pacific Northwest National LaboratoryRichlandUSA

Personalised recommendations