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

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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.

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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.

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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

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  1. Hikaru Nakazawa
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  2. Katsunori Okada
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Correspondence to Yasushi Morikawa.

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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

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