Mycobiology

The Korean Society of Mycology (한국균학회)
권호 표지
DOI QR코드

DOI QR Code

Purification and Characterization of Intracellular Cellulase from Aspergillus oryzae ITCC-4857.01

  • Published : 2009.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was $45^{\circ}C$ and the highest activity was exhibited in 35 to $45^{\circ}C$. The enzyme lost their activities almost completely (95${\sim}$100%) at $80^{\circ}C$ or above and as well as bellow $25^{\circ}C$.

Keywords

References

  1. Akiba, S., Kimura, Y., Yamamoto, K. and Kumagai, H. 1995. Purification and characterization of a protease-resistant cellulase from Aspergillus niger. J. Ferment. Bioeng. 79:125-130. https://doi.org/10.1016/0922-338X(95)94078-6
  2. Amadioha, A. C. 1993. Production of cellulolytic enzymes by Rhizopus oryzae in culture and Rhizopus-infected tissues of potato tubers. Mycologia 88:574-578.
  3. Andrews, P. 1965. The gel filtration behavior of proteins related to their molecular weights over a wide range. Biochem. J. 96:595-605
  4. Beguin, P. 1990. Molecular biology of cellulose degradation. Annu. Rev. Microbiol. 44:219-248. https://doi.org/10.1146/annurev.mi.44.100190.001251
  5. Begum, M. F. 2005. Screening of aspergilli from cellulosic waste materials and studies on their cellulolytic properties. Ph.D. thesis. Department of Botany, University of Rajshahi. Bangladesh.
  6. Berghem, L., Pettersson, L. and Asio-Fredriksson, U. 1975. The mechanism of enzymatic ecllulose degradation, characterization and enzymatic properties of a β-1-4-glucan cellobiolydrolase from Trichoderma viride. Eur. J. Biochem. 53:55. https://doi.org/10.1111/j.1432-1033.1975.tb04041.x
  7. Bhadauria, A., Sodhi, H. S., Kapoor, S. and Phutela, R. P. 1997. Isolation and characterization of Volvariella diplasia enzyme mutants. Indian J. Exp. Biol. 35:516-519.
  8. Busto, M. D., Ortega, N. and Pereg, M. M. 1996. Location, kinetics. and stability of cellulases induced in Trichoderma rcesei cultures. Biores. Tech. 57:187-192. https://doi.org/10.1016/0960-8524(96)00073-9
  9. Cavazzoni, V. and Manzoni, M. 1994. Extracellular cellulytic complex from Morchella conica: Production and purification. Lebensmttel-Wissenschaft Technologie 27:73-77. https://doi.org/10.1006/fstl.1994.1015
  10. Coral, G., Arikan, B., Unaldi, M. N. and Guvenmes, H. 2002. Some properties of crude carboxymethyl cellulase of Aspergillus niger Z10 wild-type Strain. Turk. J. Biol. 26:209-213.
  11. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. and Smith, G. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:61-77.
  12. Enokibara, S., Mori, N. and Kitamoto, K. 1991. Purification and characterization of endoglucanases from Favouls arcularicus. J. Fermen. Bioeng. 73:230-232.
  13. Gielkens, M. M. C., Dekkers, E., Visser, J. and Graaff, L. H. 1999. Two cellubiohydrolase-encoding genes from Aspergillus niger require D-xylose and the xylanolytic transcriptional activator XlnR for their expression. Appl. Environ. Microbiol. 65:4340-4345.
  14. Godfrey, T. and West, S. 1996. Industrial Enzymology (2nd. ed), Macmillan Press. London
  15. Goma, M., Zein, G. N., Mahmoud, R. M., Gibriel, A. and Abouzied, M. 1982. Characteristics of cellulolytic enzymes of Aspergillus niger and Aspergillus terreus. Minufiya J. Agr. Res.5:299-317.
  16. Gupta, J. K. and Gupta, Y. P. 1979. Properties of cellulase from Trichodema viride. Folia Microbiol. 24:269-272. https://doi.org/10.1007/BF02926459
  17. Hofsten, B. V. 1972. Microbial conversion of cellulosic wastes products. Waste recovery by microorganisms. Khalalampur, UNESCO, May, 1-18
  18. Hong, J., Tamaki, H., Akiba, S., Yamamoto, K. and Kumaga, H. 2001. Cloning of a gene encoding a highly stable endo-β-1, 4-glucanase from Aspergillus niger and its expression in yeast. J. Biosci. Bioeng. 92:434-441. https://doi.org/10.1263/jbb.92.434
  19. Lowery, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.
  20. Macris, B. J., Ketos, D., Evangelidou, X., Galiotou-Panayotou, M. and Rodis, P. 1987. Solid state fermentation of straw with Neurospora crassa for CMCase and β-glucosidase production. Biotech. Lett. 9:661-664. https://doi.org/10.1007/BF01033207
  21. Mahadevan, A. and Sridhar, R. 1982. Methods in physiological plant pathology. Sivakami publications. Madrus
  22. Mantyla, A., Paloheimo, M. and Suominen, P. 1998. Industrial mutants and recombinant strains of Trichoderma reesei. In: Harman, G. F. and Kubicek, C. P. (eds) Trichoderma & Gliocladium-Enzymes, biological control and commercial applications (2nd ed), Taylor & Francis, Ltd. London. pp.291-309
  23. Miller, G. L. 1959. Use of dinitrosalicylic acid for determination of reducing sugar. Anal. Chem. 31:426-428. https://doi.org/10.1021/ac60147a030
  24. Mishra, A. 1988. Studies an cellulolytic fungi and cellulolytic enzymes with special reference to Aspergilli. Ph.D. Thesis. Department of Botany, Gorakhpur Univ. India.
  25. Murai, T., Ueda, M., Kavaguchi, T., Arai, M. and Tanaka, M. 1998. Assimilation of cellooligosaccharides by a cell surfaceengineered yeast expressing β-glucosidase and carboxymethylcellulase from Aspergillus aculeatus. Appl. Environ. Microbiol. 64:4857-4861.
  26. Ogawa, K. 1989. Fractionation and purification of cellulases from Trichoderma viride. Bull. Fac. Agr. Myazaki Univ. 36: 271-280.
  27. Olama, Z. A., Hamza, M. A., El-Sayed, M. M. and Abdel-Fattah, M. 1993. Purification, properties and factors affecting the activity of Trichoderma viride cellulase. Food Chem. 47:221-226. https://doi.org/10.1016/0308-8146(93)90153-7
  28. Ornstein, L. 1964. Disc electrophoreses I - Background and theory. Ann. N.Y. Acad. Sci. 121:321-349. https://doi.org/10.1111/j.1749-6632.1964.tb14207.x
  29. Peij, N., Gielkens, M. M. C., Veries, R. P., Visser, J. and Graaff, L. H. 1998. The transcriptional activator XlnR Regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger. Appl. Environ. Microbiol. 64:3615-3619.
  30. Po-Jui, C., TaoChun, W., YaoTsung, C. and Liangling, L. 2004. Purification and characterization of carboxylmethyl cellulase from Sinorhigobium fredii. Bot. Bull. Acad. Sinica. 45:111-118.
  31. Robson, L. M. and Chambliss, G. H. 1989. Cellulases of bacterial origin. Enzyme Microb. Technol. 11:626-644. https://doi.org/10.1021/es60129a607
  32. Robyt, J. F. and White, B. J. 1990. Biochemical Techniques: Theory and practical. Waveland Press. Inc. Illinois, U.S.A.
  33. Sandhya, S. 1992. Effect of temperature on deactivation of cellulase. Asian Environ. 14:78-81.
  34. Shaojun, D., Wei, G. and Busuell, J. A. 2001. Endoglucanase 1 from the edible straw mushroom, Volvariella volvaeeae: Purification, characterization, cloning and expression. Eur. J. Biochem. 268:5687-5695. https://doi.org/10.1046/j.0014-2956.2001.02503.x
  35. Sultana, S. 1997. Isolation of cellulolytic microorganism and their activities. M. Phil Thesis. Biochemistry Department, Rajshahi Univ. Bangladesh.
  36. Uhlig, H. 1998. Industrial enzymes and their applications: John Wiley & Sons, New York. p 435
  37. Wang, J. S. 1999. Cellulase production by a mutant strain of Trichoderma reesei from bagasse. 23rd ISSCT Congress, New Delhi, India, 67-76.
  38. Weber, K. and Osborn, M. 1969. The reliability of molecular weight determination by SDS-PAGE. J. Biol. Chem. 244:4406-4412.

Cited by

  1. Endoglucanase and Total Cellulase from Newly Isolated Rhizopus oryzae and Trichoderma reesei: Production, Characterization, and Thermal Stability vol.172, pp.1, 2014, https://doi.org/10.1007/s12010-013-0518-2
  2. Cellulases: A New Approach to Degrading Lignocellulosic Material vol.39, pp.2, 2015, https://doi.org/10.1111/jfbc.12097
  3. Purification and Some Properties of Endo-1,4-<i>β</i>-Glucanases of <i>Trichoderma harzianum</i> UzCF-28 vol.06, pp.08, 2016, https://doi.org/10.4236/ojapps.2016.68051
  4. Prospecting Agro-waste Cocktail: Supplementation for Cellulase Production by a Newly Isolated Thermophilic B. licheniformis 2D55 vol.182, pp.4, 2017, https://doi.org/10.1007/s12010-017-2401-z