Abstract
Thermostable enzymes offer potential benefits in the hydrolysis of lignocellulosic substrates; higher specific activity decreasing the amount of enzymes, enhanced stability allowing improved hydrolysis performance and increased flexibility with respect to process configurations, all leading to improvement of the overall economy of the process. New thermostable cellulase mixtures were composed of cloned fungal enzymes for hydrolysis experiments. Three thermostable cellulases, identified as the most promising enzymes in their categories (cellobiohydrolase, endoglucanase and β-glucosidase), were cloned and produced in Trichoderma reesei and mixed to compose a novel mixture of thermostable cellulases. Thermostable xylanase was added to enzyme preparations used on substrates containing residual hemicellulose. The new optimised thermostable enzyme mixtures were evaluated in high temperature hydrolysis experiments on technical steam pretreated raw materials: spruce and corn stover. The hydrolysis temperature could be increased by about 10–15 °C, as compared with present commercial Trichoderma enzymes. The same degree of hydrolysis, about 90% of theoretical, measured as individual sugars, could be obtained with the thermostable enzymes at 60 °C as with the commercial enzymes at 45 °C. Clearly more efficient hydrolysis per assayed FPU unit or per amount of cellobiohydrolase I protein used was obtained. The maximum FPU activity of the novel enzyme mixture was about 25% higher at the optimum temperature at 65 °C, as compared with the highest activity of the commercial reference enzyme at 60 °C. The results provide a promising basis to produce and formulate improved enzyme products. These products can have high temperature stability in process conditions in the range of 55–60 °C (with present industrial products at 45–50 °C) and clearly improved specific activity, essentially decreasing the protein dosage required for an efficient hydrolysis of lignocellulosic substrates. New types of process configurations based on thermostable enzymes are discussed.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Abrha B, Gashe BA (1992) Cellulase production and activity in a species of Cladosporium. World J Microbiol Biotech 8:164–166
Barnett C Berka R, Fowler T (1991) Cloning and amplification of the gene encoding an extracellular β-glucose from Trichoderma reesei: evidence for improved rates of saccharification of cellulosic substrates. Biotechnology 9:562–567
Barr B Hsieh, Ganem B, Wilson BD (1996) Identification of two functionally different classes of exocellulases. Biochemistry 35:586–592
Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing methods for assay of xylanase activity. J Biotechnol 23:257–270
Bailey M, Nevalainen H (1981) Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of solubilizing cellulase. Enzyme Microb Technol 3:153–157
Bailey MJ, Tähtiharju J (2003) Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer-controlled feeding strategy. Appl Microbiol Biotechnol 62:156–162
Bergquist P, Gibbs M, Morris D, Te'o V, Jsaul D, Morgan H (1999) Molecular diversity of thermophilic cellulolytic and hemicellulolytic bacteria. FEMS Microbiol Ecol 28:99–110
Bergquist PL, Te'o VSJ, Gibbs MD, Curah NC, Nevalainen KMH (2004) Recombinant enzymes from thermophilic micro-organisms expressed in fungal hosts. Biochem Soc Trans 32(2):293–297
Berlin A, Gilkes N, Kilburn D, Bura R, Markov A, Skomarovsky A, Okunev O, Gusakov A, Maximenko V, Gregg D, Sinitsyn A, Saddler J (2005) Evaluation of novel fungal cellulase preparations for ability to hydrolyze softwood substrates – evidence for the role of accessory enzymes. Enzyme Microb Technol 37:175–184
Bernfeld P (1955) Amylases α and β. In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol 1. Academic, New York, pp 149–158
Bok JD, Yernool DA, Eveleigh DE (1998) Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana. Appl Environ Microbiol 64:4774–4781
Bronnenmeier K, Rücknagel K, Staudenbauer W (1991) Purification and properties of a novel type exo-1,4-β-glucanase (Avicelase II) from the cellulolytic thermophile Clostridium stercorarium. Eur J Biochem 200:379–385
Bronnenmeier K, Staudenbauer W (1990) Cellulose hydrolysis by a highly thermostable endo-1,4-glucanase (Avicelase I) from Clostridium stercorarium. Enzyme Microbial Technol 12:431–436
Bronnenmeier K, Kern A, Liebl W, Staudenbauer W (1995) Purification of Thermotoga maritima enzymes for the degradation of cellulosic materials. Appl Environ Microbiol 61:1339–1407
Coughlan M, McHale A (1988) Purification of β-d-glucoside glucohydrolases of Talaromyces emersonii. Methods Enzymol 160:437–443
Coutinho PM, Henrissat B (1999) Carbohydrate active enzymes. Database available at http://www.cazy.org , last visited: 10 May 2007
Demain A, Newcomb M, Wu JHD (2005) Cellulase, clostridia and ethanol. Microb Molec Biol Rev 69:124–154
Ding S-Y (2006) Thermotolerant cellulase. Industrial Bioproc 28(7):3–4
Divne C, Ståhlberg J, Teeri T, Jones T (1998) High-resolution crystal structures reveal how a cellulose chain is bound in the 50 Å long tunnel of cellobiohydrolase I from Trichoderma reesei. J Mol Biol 275:309–325
Eklund R, Galbe M, Zacchi G (1990) Optimization of temperature and enzyme concentration in the enzymatic saccharification of steam pretreated willow. Enzyme Microb Technol 12:225–228
Evans B, Gilman A, Cordray K, Woodward J (2000) Mechanism of substrate hydrolysis by a thermophilic endoglucanase from Thermotoga maritima. Biotechnol Lett 22:735–740
Fauth U, Romaniec M, Kobayashi T, Demain A (1991) Purification and characterization of endoglucanase Ss from Clostridium thermocellum. Biochem J 279:67–73
Feldman KA, Lovett JS, Tsao GT (1988) Isolation of cellulase enzymes from the thermophilic fungus Thermoascus aurantiacus and regulation of enzyme production. Enzyme Microb Technol 10:262–272
Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, Dunn-Coleman NS, Goedegebuur F, Houfek T, England G, Kelley A, Meerman H, Mitchell T, Mitchinson C, Olivares H, Teunissen P, Yao J, Ward M (2003) Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei. J Biol Chem 278(34):31988–31997
Fullbrook PD (1996) Practical limits and prospects (kinetics). In: Godfrey T, West S (eds) Industrial enzymology, 2nd edn. MacMillan, London, pp 508–509
Gomes I, Gomes J, Gomes D, Steiner W (2000) Simultaneous production of high activities of thermostable endoglucanase and β-d-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus. Appl Microbiol Biotechnol 53:461–468
Grassick A, Murray P, Thompson R, Collins C, Byrnes L, Birrane G, Higgins T, Tuohy M (2004) Three-dimensional structure of a thermostable native cellobiohydrolase, CBH IB, and molecular characterization of the cel7 gene from the filamentous fungus, Talaromyces emersonii. Eur J Biochem 271:4495–4506
Grishutin S, Gusakov A, Markov A, Ustinov B, Semenova M, Sinitsyn A (2004) Specific xyloglucanases as a new class of polysaccharide-degrading enzymes. Biochim Biophys Acta 1674:268–281
Hakamada Y, Koike K, Yoshimatsu T, Mori H, Kobayashi T, Ito S (1997) Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237. Extremophiles 1:151–156
Harchand R, Singh S (1997) Characterization of cellulase complex of Streptomyces albaduncus; thermostable cellulase, cellobiohydrolase and beta-glucosidase characterization. J Basic Microbiol 37:93–103
Henrissat B, Driquez H, Viet C, Schülein M (1985) Synergism of cellulases from Trichoderma reesei in degradation of cellulose. Bio/Technology 3:722–726
Himmel M, Adney W, Tucker M, Grohmann K (1994) Thermostable purified endoglucanase from Acidothermus cellulolyticus ATCC 43068. US Patent 5275944
Hogsett DA, Ahn H-J, Bernardez TD, South CR, Lynd LR (1992) Direct microbial conversion. Prospects, progress and obstacles. Appl Biochem Biotechnol 34:527–541
Hreggvidsson G, Kaiste E, Holst O, Eggertsson G, Palsdottir A, Kristjansson A (1996) An extremely thermostable cellulase from the thermophilic eubacterium Rhodothermus marinus. Appl Environ Microbiol 62:3047–3049
Ishihara M, Shinkichi T, Toyama S (1999) Disintegration of uncooked rice by carboxymethyl cellulase from Sporotrichum sp. HG-I. J Biosci Bioeng 87:249–251
International Union of Pure and Applied Chemistry (IUPAC) (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268
Jang HD, Chen KS (2003) Production and characterization of thermostable cellulases from Streptomyces transformant T3-1. World J Microb Biotechnol 19:263–268
Karlsson J, Momcilovic D, Wittgren B, Schülein M, Tjerneld F, Brinkman G (2002) Enzymatic degradation of carboxymethyl cellulose hydrolysed by the endoglucanases Cel5A, Cel7B and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45A core from Trichoderma reesei. J Biotechnol 63:32–40
Klyosov A (1990) Trends in biochemistry and enzymology of cellulose degradation. Biochemistry 29:10577–10585
Lin J, Pillay B, Singh S (1999) Purification and biochemical characteristics of β-d-glucosidase from a thermophilic fungus, Thermomyces lanuginosus–SSBP. Biotechnol Appl Biochem 30:81–87
Lin S, Stutzenberger F (1995) Purification and characterization of the major beta-1,4-endoglucanase from Thermomonospora curvata. J Appl Bacteriol 79:447–53
Li D-C, Lu M, Li Y-A, Lu J (2003) Purification and characterization of an endocellulase from the thermophilic fungus Chaetomium thermophilum CT 2. Enzyme Microb Technol 33:932–937
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 193:265–75
Macarron R, Acebal C, Castillon M, Claeyssens M (1996) Mannanase activity of endoglucanase III from Trichoderma reesei QM9414. Biochem Lett 18:599–602
Maheshwari R, Bharadwaj G, Bhat M (2000) Thermophilic fungi: Their physiology and enzymes. Microbiol Mol Biol Rev 64:461–488
Medve J, Ståhlherg J, Tjerneld F (1994) Adsorption and synergism of cellobiohydrolase I and II of Trichoderma reesei during hydrolysis of microcrystalline cellulose. Biotechnol Bioeng 44:1064–1073
Miettinen-Oinonen A, Londesborough J, Vehmaanperä J, Haakana H, Mäntylä A, Lantto R, Elovainio M, Joutsjoki V, Paloheimo M, Suominen P (1996) New cellulases from fungi for use in pulp and textile processing and the genes encoding the enzymes. WO Patent no. 9714804
Millner PD (1977) Radial growth responses to temperature by 58 Chaetomium species and some taxonomic relationships. Mycologia 69:492–502
Mooney C, Mansfield S, Tuohy M, Saddler J (1998) The effect of the initial pore volume and lignin content on the enzymatic hydrolysis of softwoods. Biores Technol 64:113–119
Murray P, Grassick A, Laffey C, Cuffe M, Higgins T, Savage A, Planas A, Tuohy M (2001) Isolation and characterization of a thermostable endo-β-glucanase active on 1,3-1,4-β-d-glucans from the aerobic fungus Talaromyces emersonii CBS 814.70. Enzyme Microb Technol 29:90–98
Nidetzky B, Claeyssens M (1994) Specific quantification of Trichoderma reesei cellulases in reconstituted mixtures and its application to cellulose-cellulose binding studies. Biotechnol Bioeng 44:961–966
Nidetzky B, Hayn M, Macarron R, Steiner W (1993) Synergism of Trichoderma reesei cellulases while degrading different celluloses. Biotechnol Lett 151:71–76
Nidetzky B, Steiner W, Hayn M, Claeyssens M (1994) Cellulose hydrolysis by the cellulases from Trichoderma reesei: A new model for synergistic interaction. Biochem J 298:705–710
Nigam P, Prabhu KA (1991) Influence of sugars on the activity of cellulose systems from two basidomycetes cultures. J Basic Microbiol 31:279–283
Nutt A, Sild V, Petterson G, Johansson G (1998) Progress curves a mean for functional classification of cellulases. Eur J Biochem 258:200–206
Palonen H, Viikari L (2004) Role of oxidative enzymatic treatments on enzymatic hydrolysis of softwood. Biotechnol Bioeng 86:550–557
Park C, Kawaguchi T, Sumitani J, Arai M (2001) Purification and characterization of cellulases (CBH I and EGL 1) produced by thermophilic microorganism Streptomyces sp. M23. Appl Biol Sci 7(1):27–35
Parry N, Beever D, Owen E, Vandenbergh I, van Beeumen J, Bhat M (2001) Biochemical characterization and mechanism of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus. Biochem J 353:117–127
Parry N, Beever D, Owen E, Nerinckx W, Claeyssens M, Van Beeumen J, Bhat M (2002) Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus. Arch Biochem Biophys 404:243–253
Phillippidis GP, Smith TK, Wyman CE (1993) Study of the enzymatic hydrolysis of cellulose for production of fuel ethanol by the simultaneous saccharification and fermentation process. Biotechnol Bioeng 41:846–853
Romaniec M, Fauth U, Kobayashi T, Huskisson N, Barker P, Demain A (1992) Purification and characterization of a new endoglucanase from Clostridium thermocellum. Biochem J 283:69–73
Rosgaard L, Pedersen S, Cherry JR, Harris P, Meyer AS (2006) Efficiency of new fungal cellulose systems in boosting enzymatic degradation of barley straw lignocellulose. Biotechnol Progr 22:493–498
Roy S, Raha S, Dey S, Chakrabarty S (1990) Effect of temperature on the production and location of cellulose components in Myceliopthora thermophila D-14 (ATCC 48104). Enzyme Microb Technol 12:710–713
Ruttersmith L, Daniel R (1991) Thermostable cellobiohydrolase from the thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1: purification and properties. Biochem J 277:887–890
Saboto D, Nucci R, Rossi M, Gryczynski I, Gryczynski Z, Lakowicz J (1999) The β-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: enzyme activity and conformational dynamics at temperatures above 100 °C. Biophys Chem 81:23–31
Saha B, Freer S, Bothast R (1994) Production, purification and properties of a thermostable β-glucosidase from a color variant strain of Aureobasidium pullulans. Appl Env Microbiol 60:3774–3780
Sakon J, Adney W, Himmel M, Thomas S, Karplus P (1996) Crystal structure of thermostable family 5 endoglucanase EI from Acidothermus cellulolyticus in complex with cellotetraose. Biochemistry 35:10648–10660
Sarker M, Ilias M, Mozammel H (1998) Charaterization of xylanase and CMCase from Rhizomucor pusillus. Bangladesh J Microbiol 15(2):41–47
Sassner P, Galbe M, Zacchi G (2006) Bioethanol production based on simultaneous saccharification and fermentation of steam-pretreated salix at high dry-matter content. Enzyme Microb Technol 39:756–762
Shepherd M, Tong C, Cole A (1981) Substrate specificity and mode of action of the cellulases from the thermophilic fungus Thermoascus aurantiacus. Biochem J 193:67–84
Shoemaker S, Watt K, Tsikovsky G, Cox R (1983) Characterization and properties of cellulases purified from Trichoderma reesei strain L27. Bio/Technology 1:687–690
Stenberg K, Bollok M, Reczey K, Galbe M, Zacchi G (2000) Effect of substrate and cellulose concentration on simultaneous saccharification and fermentation of steam pretreated softwood for ethanol production. Biotechnol Bioeng 68:204–210
Ståhlberg J (1991) Functional organization of cellulases from Trichoderma reesei. PhD Thesis, Uppsala University, Sweden
Takashima S, Nakamura A, Hidaka M, Masaki H, Uozumi T (1999) Molecular cloning and expression of the novel fungal β-glucosidase genes from Humicola grisea and Trichoderma reesei. J Biochem 125:728–736
Tenkanen M, Siika-aho M (2000) An α-glucuronidase of Schizophyllum commune acting on polymeric xylan. J Biotechnol 75:149–161
Te'o V, Saul D, Bergquist P (1995) CelA, another gene coding for a multidomain cellulases from the extreme thermophile Caldocellum saccharolyticum. Appl Microbiol Biotechnol 43:291–296
Tomme P, Heriben V, Claeyssens M (1990) Adsorption of two cellobiohydrolases from Trichoderma reesei to Avicel: Evidence for exo–exo synergism and possible “loose complex” formation. Biotechnol Lett 127:525–530
Tuohy M, Walsh J, Murray P, Claeyssens M, Cuffe M, Savage A, Coughlan M (2002) Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii. Biochem Biophys Acta 1596:366–380
Venturi L, Polizeli M, Terenzi H, Furriel R, Jorge J (2002) Extracellular β-d-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some properties. J Basic Microbiol 42:55–66
Väljamäe P, Sild V, Petterson G, Johansson G (1998) The initial kinetics of hydrolysis by cellobiohydrolase I and II is consistent with a cellulose surface – erosion model. Eur J Biochem 253:469–475
Wright JD, Wyman CE, Grohmann K (1988) Simultaneous saccharification and fermentation of lignocellulose: process evaluation. Appl Biochem Biotechnol 18:75–90
Zverlov V, Mahr S, Riedel K, Bronnenmeier K (1998) Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile Anaerocellum thermophilum with separate glycosyl hydrolase family 9 and 48 catalytic domains. Microbiology 143:3537–3542
Öhgren K, Vehmaanperä J, Siika-aho M, Galbe M, Viikari L, Zacchi G (2007) High temperature enzymatic hydrolysis prior to simultaneous saccharification and fermentation of steam pretreated corn stover for ethanol production. Enzyme Microb Technol 40:607–613
Author information
Authors and Affiliations
Corresponding author
Editor information
Rights and permissions
Copyright information
© 2007 Springer-Verlag Berlin Heidelberg
About this chapter
Cite this chapter
Viikari, L., Alapuranen, M., Puranen, T., Vehmaanperä, J., Siika-aho, M. (2007). Thermostable Enzymes in Lignocellulose Hydrolysis . In: Olsson, L. (eds) Biofuels. Advances in Biochemical Engineering/Biotechnology, vol 108. Springer, Berlin, Heidelberg. https://doi.org/10.1007/10_2007_065
Download citation
DOI: https://doi.org/10.1007/10_2007_065
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-540-73650-9
Online ISBN: 978-3-540-73651-6
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)