Skip to main content
SpringerLink
Log in

Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A) from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins. Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity of EG I-CD at 15°C, EG II-CD at 20°C and EG III at 37°C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and 15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3–1,4-β-d-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3–1,4-β-d-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3–1,4-β-d-glucan, xyloglucan, xylan, and mannan.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Aho S (1991) Structural and functional analysis of Trichoderma reesei endoglucanase I expressed in yeast Saccharomyces cerevisiae. FEBS Lett 291:45–49

    Article  CAS  PubMed  Google Scholar 

  • Aho S, Arffman A, Korhola M (1996) Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases. Appl Microbiol Biotechnol 46:36–45

    Article  CAS  PubMed  Google Scholar 

  • Bailey MJ, Siika-Aho M, Valkeajärvi A, Penttilä ME (1993) Hydrolytic properties of two cellulases of Trichoderma reesei expressed in yeast. Biotechnol Appl Biochem 17:65–76

    CAS  PubMed  Google Scholar 

  • Biely P, Vršanska M, Claeyssens M (1991) The endo-1,4-b-glucanase I from Trichoderma reesei. Eur J Biochem 200:157–163

    Article  CAS  PubMed  Google Scholar 

  • Boer H, Teeri TT, Koivula A (2000) Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters. Biotechnol Bioeng 69:486–494

    Article  CAS  PubMed  Google Scholar 

  • Day AG (Genencor International IU) (2003) Novel variant Hypocrea jecorina CBH I cellulases, their production with recombinant cells, and their uses. Patent 2,004,016,760

  • Farkas V, Liskova M, Biely P (1985) Novel media for detection of microbial producers of cellulase and xylanase. FEMS Microbiol Lett 28:137–140

    Article  CAS  Google Scholar 

  • Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, Dunn-Coleman NS, Goedegebuur F, Houfek TD, England GJ, Kelley AS, Meerman HJ, Mitchell T, Mitchinson C, Olivares HA, Teunissen PJM, Yao J, Ward M (2003) Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei. J Biol Chem 278:31988–31997

    Article  PubMed  Google Scholar 

  • Gehant RL (Genentech Inc) (2003) Process for protein extraction. Patent 6,967,241

    Google Scholar 

  • Hui JPM, White TC, Thibault P (2002) Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanase I and II from Trichoderma reesei. Glycobiol 12:837–849

    Article  CAS  Google Scholar 

  • Il K, Ekino K, Goto M, Furukawa K (1999) Heterologous expression and characterization of endoglucanase I (EGI) from Trichoderma viride HK-75. Biosci Biotechnol Biochem 63:1714–1720

    Article  Google Scholar 

  • Karlsson J, Momcilovic D, Wittgren B, Schülein M, Tjerneld F, Brinkmalm G (2002a) Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei. Biopolymers 63:32–40

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Kleywegt GJ, Zou J, Divne C, Davies GJ, Sinning I, Ståhlberg J, Reinikainen T, Srisodsuk M, Teeri TT, Alwyn Jones T (1997) The crystal structure of the catalytic core domain of endoglucanase I from at 3.6 Å resolution, and a comparison with related enzymes. J Mol Biol 272:383–397

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Liu D, Schmid RD, Rusnak M (2006) Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm-a screening system for a frequently used biocatalyst. Appl Microbiol Biotechnol 72:1024–1032

    Article  CAS  PubMed  Google Scholar 

  • Lorenzo LD, Aurelio H, Michael H, Uwe BT (2005) Heterologous production of forms of Rhizopus oryzae lipase in Escherichia coli. Appl Environ Microbial 17:8974–8977

    Article  Google Scholar 

  • Macarrón R, Acebel C, Castillón MP, Domínguez JM, Mata LDL, Pettersson G, Tomme P, Claeyssens M (1993) Mode of action of endoglucanase III from Trichoderma reesei. Biochem J 289:867–873

    Article  PubMed  PubMed Central  Google Scholar 

  • Nelson NJ (1984) A photometric adaptation of the Somogyi method for determination of glucose. J Biol Chem 375:375–380

    Google Scholar 

  • Okada H, Sekiya T, Yokoyama K, Tohda H, Kumagai H, Morikawa Y (1998a) Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products. Appl Microbiol Biotechnol 49:301–308

    Article  CAS  PubMed  Google Scholar 

  • Okada H, Tada K, Sekiya T, Yokoyama K, Takahashi A, Tohda H, Kumagai H, Morikawa Y (1998b) Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414. Appl Environ Microbial 64:555–563

    Article  CAS  Google Scholar 

  • Okada H, Mori K, Tada K, Nogawa M, Morikawa Y (2000) Identification of active site carboxylic residues in Trichoderma reesei endoglucanase Cel12A by site-directed mutagenesis. J Mol Cat B, Enzymatic 10:249–255

    Article  CAS  Google Scholar 

  • Penttilä ME, André L, Saloheimo M, Lehtovaara P, Knowles JKC (1987) Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae. Yeast 3:175–185

    Article  PubMed  Google Scholar 

  • Pentillä ME, André L, Lehtovaara P, Bailey M, Teeri TT, Knowles JKC (1988) Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae. Gene 63:103–113

    Article  Google Scholar 

  • Robert AL, William SA, Ali M, Michael EH, Steven RT (1996) Cloning and expression of full-length Trichoderma reesei cellobiohydrolase I cDNAs in Escherichia coli. Appl Biochemi Biotechnol 57/58:389–397

    Article  Google Scholar 

  • Rose SH, Van Zyl WH (2002) Constitutive expression of the Trichoderma reesei b-1,4-xylanase gene (xyn2) and the b-1,4-endoglucanase gene (egl) in Aspergillus niger in molasses and defined glucose media. Appl Microbiol Biotechnol 58:461–468

    Article  CAS  PubMed  Google Scholar 

  • Saloheimo M, Nakari-Setälä T, Tenkanen M, Penttilä M (1997) cDNA cloning of a Trichoderma reesei cellulase and demonstration of endoglucanase activity by expression in yeast. Eur J Biochem 249:584–591

    Article  CAS  PubMed  Google Scholar 

  • Saloheimo M, Lehtovaara P, Penttilä M, Teeri TT, Ståhlberg J, Johansson G, Pettersson G, Claeyssens M, Tomme P, Knowles JKC (1998) EG III, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. Gene 63:11–21

    Article  Google Scholar 

  • Sandgren M, Shaw A, Ropp TH, Wu S, Bott R, Cameron AD, Ståhlberg J, Mitchinson C, Alwyn jones T (2001) The X-ray crystal structure of the Trichoderma reesei family12 endoglucanase 3, Cel12A, at 1.9 Å resolution. J Mol Biol 308:295–310

    Article  CAS  PubMed  Google Scholar 

  • Sandgren M, Gualfetti PJ, Shau A, Gross LS, Saldajeno M, Day AG, Jones TA, Mitchinson C (2003) Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability. Protein Science 12:848–860

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Somogyi MJ (1952) Notes on sugar determination. J Biol Chem 195:19–23

    CAS  Google Scholar 

  • Sul O, Kim J, Park S, Son Y, Park B, Chung DK, Jeong C, Han I (2004) Characterization and molecular cloning of a novel endoglucanase from Trichoderma sp. C-4. Appl Microbiol Biotechnol 66:63–70

    Article  CAS  PubMed  Google Scholar 

  • Takashima S, Iikura H, Nakamura A, Hidaka M, Masaki H, Uozumi T (1998) Overproduction of recombinant Trichoderma reesei cellulase by Aspergillus oryzae and their enzymatic properties. J Biotechnol 65:163–171

    Article  CAS  PubMed  Google Scholar 

  • Teter S, Cherry J, Ward C, Jones A, Harris P, Yi J (Novozymes Biotech IU) (2004) Variants of cellobiohydrolase I from Trichoderma reesei with improved properties. Patent 20050048619

  • Van Arsdell JN, Kwok S, Schweickart VL, Ladner MB, Gelfand DH, Innis MA (1987) Cloning, characterization, and expression in Saccharomyces cerevisiae of endoglucanase I from Trichoderma reesei. Bio/Technology 5:60–64

    Google Scholar 

  • Walseth CS (1952) Occurrence of cellulases in enzyme preparations from microorganisms. TAPPI 35:228–233

    CAS  Google Scholar 

  • Wang T, Liu X, Yu Q, Zhang X, Qu Y, Gao P, Wang T (2005) Directed evolution for engineering pH profile of endoglucanase III from Trichoderma reesei. Biomole Eng 22:89–94

    Article  CAS  Google Scholar 

  • William SA, Yat-chen C, Stephen RD, Shi-you D, John OB, Glenna K, Todd BV, Michael EH (2003) Heterologous expression of Trichoderma reesei 1,4-b-D-Glucan cellobiohydrolase (Cel7A). Am Chem Soc Chapter 23:403–437

    Google Scholar 

  • Yuan S, Wu Y, Cosgrove DJ (2001) A fungal endoglucanase with plant cell wall extension activity. Plant Physiol 127:324–333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Dainippon Sumitomo Pharma and Dr. Yaoi of Advanced Industrial Science and Technology (AIST) for providing tamarind-xyloglucan. This work was partly supported by a grant from the Research Institute for Innovative Technology for the Earth (RITE) Project.

Author information

Authors and Affiliations

  1. Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, 940-21, Japan

    Hikaru Nakazawa, Katsunori Okada, Ryota Kobayashi, Tetsuya Kubota, Tomoko Onodera, Nobuhiro Ochiai, Naoki Omata, Wataru Ogasawara, Hirofumi Okada & Yasushi Morikawa

Authors
  1. Hikaru Nakazawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katsunori Okada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ryota Kobayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tetsuya Kubota
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tomoko Onodera
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nobuhiro Ochiai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Naoki Omata
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wataru Ogasawara
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hirofumi Okada
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yasushi Morikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yasushi Morikawa.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nakazawa, H., Okada, K., Kobayashi, R. et al. Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli . Appl Microbiol Biotechnol 81, 681–689 (2008). https://doi.org/10.1007/s00253-008-1667-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-008-1667-z

Keyword

Search

Navigation