Characterization of Endoglucanase (F-I-III) Purified from Trichoderma sp. C-4

새로운 섬유소분해 균주 Trichoderma sp. C-4에서 분리한 Endoglucanase (F-I-III)에 대한 연구

  • Sul Ok Ju (Department of Biological Science, University of Ulsan) ;
  • Chung Dae Kyun (Institute and Department of Genetic Engineering, College of Natural Sciences, Kyung-Hee University) ;
  • Han In Seob (Department of Biological Science, University of Ulsan) ;
  • Jeong Choon Soo (Department of Biological Science, University of Ulsan)
  • 설옥주 (울산대학교 자연대학 생명과학부) ;
  • 정대균 (경희대학교 자연대학 유전공학과) ;
  • 한인섭 (울산대학교 자연대학 생명과학부) ;
  • 정춘수 (울산대학교 자연대학 생명과학부)
  • Published : 2005.03.01


One of the endoglucanases, F-I-III, was purified from the culture filtrate of T. sp. C-4 through procedures including chromatography on Sephacryl S-200, DEAE-Sepharose A-50, and Chromatofocusing on Mono-P (FPLC). The molecular weight of the enzyme was determined to be about 56,000 Da by SDS-PAGE, and pI of 4.9 by analytical isoelectric focusing. F-I-III showed the highest enzyme activity at $55^{\circ}C$, and the pH optimum of the enzyme was 5.0. There was no loss of activity when the enzyme was incubated at $50^{\circ}C$ for 24 hours. The specific activity of the enzyme F-I-III toward the CMC was 315.4 U/mg. The Km value for $PNPG_2$ of F-I-III was 2.69 mM. N-terminal sequence of F-I-III was analyzed to be QPGTSTPEVHPKKLTTYK. It showed $95\%$ of homology to that of EGI from T. reesei. The presence of some metal ions (1 mM) had only a little effect on CMCase activity. The treatment of the reducing agents resulted in the increase of endoglucanase activity.


C-4;characterization;protein sequencing;Trichoderma sp.


  1. Eriksson K.E., and S.G. Hamp. 1978. Regulation of endo-1,4-$\beta$-glucanase production in Sporotrichum pulverulentum. Eur. J. Biochem. 90, 183-190
  2.  Mishra, C., and M. Rao. 1988. Mode of action and synergism of cellulases from Penicillium funiculosum. Appl. Biochem. Biotechnol. 19, 139-150
  3.  Kleman-Leyer, K.M., M. Siika-Aho, T.T. Teeri, and T.K. Kerk. 1996. The cellulases Endoglucanase I and cellobiohydrolase II of T. reesei act synergistically to solubilize native cotton cellulose but not to decrease its molecular size. Appl. Environ. Microbiol. 62, 2883-2887
  4.  Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-68
  5.  Maccaron, R., C. Acebal, M.P. Castillon, J.M. Dominguez, I. Mata, G. Pettersson, P. Tomme, and M. Claeyssens. 1993. Mode of action of endoglucanase III from Trichoderma reesei. Biochem. J. 289, 867-873
  6.  Somogyi, M. 1952. Note on sugar determination. J. Biol. Chem. 195, 19-23
  7.  Son, Y.J., O.J. Sul, D.K. Chung, I.S. Han, Y.J. Choi, and C.S. Jeong. 1997. Isolation and characterization of Trichoderma sp. C-4 producing celluloses. Kor. J. Appl. Microbiol. Biotechnol. 25, 346-353
  8. Sul, O.J., J.H. Kim, S.J. Park, Y.J. Son, B.R. Park, D.K. Chung, C.S. Jeong, and I.S. Han. 2004. Characterization and molecular cloning of a novel endoglucanase from Trichoderma sp. C-4. Appl. Microbiol. Biotechnol., 66, 63-70
  9. Wood, T.M., and S.I. McCrae. 1978. The cellulase of Trichoderma koningii: Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase. Biochem. J. 171: 61- 72
  10. Yun, S.I., C. S. Jeong, D. K. Jeong, and H. S. Choi. 2001. Purification and some properties of a $\beta$-glucosidase from Trichoderma harzianum type C-4. Biosci. Biotechnol. Biochem. 65, 2028-2032
  11. Chirico, W.T. and R.D. Brown. 1987. $\beta$-Glucosidase from T. reesei; subtrate-binding region and mode of action on $[1-^3H] $ cellooligosaccharide. Eur. J. Biochem. 165, 343-351
  12.  Henrissat, B., H. Driguez, C. Viet, M. Shulein. 1985. Synergism on cellulose from Trichoderma reesei in the degradation of cellulose. Bio/Techol. 3, 722-726
  13. Irwin, D.C., M. Spezio, L.P. Walker, and D.B. Wilson. 1993. Activity studies of eight purified cellulases: specificity, synergism, and binding domain effects. Biotechnol. Bioengineering 42, 1002-1013
  14.  Wood, T.M., and S.I. McCrae. 1982 Purification and some properties of the extracellular $\beta$-D-glucosidase of the cellulolytic fungus Trichoderma koningii. J. Gen. Microbiol. 128: 2973-2982
  15.  Mach, R.L., and S. Zeilinger. 2003. Regulation of gene expression in industrial fungi: Trichoderma. Appl. Microbiol. Biotechnol. 60, 515-522
  16. Deshpande, V., K.E. Eriksson, and B. Pettersson. 1978. Production, purification and partial characterization of 1,4-$\beta$-glucosidase enzymes from Sporotrichum pulverulentum. Eur. J. Biochem. 90, 191-198
  17.  Miettinen-Oinonen, A., and P. Suominen. 2002. Enhanced production of Trichoderma reesei endoglucanases and use of the new cellulose preparations in producing the stonewashed effect on denim fabric. App. Environ. Microbiol. 68, 3956-3964
  18.  Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951. Protein Measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275
  19. Durand, H. 1984. Comparative study of cellulases and hemicellulases from four fungi: mesophiles Trichoderma reesei and Penicillium sp. and thermophiles Thielavia terrestris and Sporotrichum cellulophilum. Enzyme Microb. Technol. 6, 175-180
  20.  Ng, T.K., and J.G. Zeikus. 1981. Comparison of extracellular cellulase activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414. Appl. Environ. Microbiol. 42, 231- 240
  21. Sul, O.J., J.Y. Choi, Y.J. Son, J.W. Shin, I.S. Han, D.K. Chung, and C.S. Jeong. 2000. Characterization of endoglucanase (F-II-II) from Trichoderma sp. C-4. Kor. J. Microbiol. 36, 20-25
  22. Woodward, J., M. Lima, and N.E. Lee. 1988. The role of cellulase concentration in determining the degree of synergism in the hydrolysis of microcrystalline cellulose. Biochem. J. 255: 895-899
  23. Ait, N., N. Creuzet, and J. Catteno. 1982. Properties of $\beta$-glucosidase purified from Clostridium thermocellum. J. Gen. Microbiol. 128, 569-577
  24. Berghem, L.E.R., L.G. Pettersson, and U.B. Axio-Fredriksson. 1975. The mechanism of enzymatic cellulose degradation: Characterization and enzymatic properties of a-1,4-$\beta$-glucan cellobiohydrolase from Trichoderma viride. Eur. J. Biochem. 53, 55-62
  25. Towbin, H., T. Staehelin and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc. Natl. Acad. Sci. USA 76, 4350-4354
  26. Ait, N., N. Creuzet, and J. Catteno. 1979. Characterization and purification of thermostable $\beta$-glucosidase from Clostridium thermocellum. Biochem. Biophys. Res. Commun. 90, 537-546
  27.  Mandels, M., and E.T. Reese. 1960. Induction of cellulase in fungi by cellobiose. J. Bacteriol. 79, 816-826
  28. Berghem, L.E.R. and L.G. Pettersson. 1974. The mechanism of enzymatic cellulose degradation: Isolation and some properties of $\hat{a}$-glucosidase of Trichoderma viride. Eur. J. Biochem. 46, 295- 305
  29. Berghem, L.E.R. and L. G. Pettersson. 1973. The mechanism of enzymatic cellulose degradation: Purification of a cellulolytic enzyme from Trichoderma viride active on highly ordered cellulose. Eur. J. Biochem. 37, 21-30
  30. Sul, O.J. 2000. Molecular and biochemical characterization of cellulase from Trichoderma type C-4 (Ph.D. Thesis in Department of Biological Sciences, University of Ulsan)
  31.  Lin, E., and D.B. Wilson. 1987. Regulation of $\beta$-1,4-endoglucanase synthesis in Thermomonospora fusca. Appl. Environ. Microbiol. 53, 1352-1357
  32. Halliwell ,G., and M. Griffin. 1973. The nature and mode of action of the cellulolytic component C1 of T. koningii on the native cellulose. Biochem. J. 135, 587-594