Skip to main content
NCBI home page
Indian Journal of Microbiology logoLink to Indian Journal of Microbiology
. 2009 Jun 3;49(2):188–195. doi: 10.1007/s12088-009-0030-0

Strain improvement of Trichoderma reesei Rut C-30 for increased cellulase production

He Jun 1,2, Yu Bing 1,2, Zhang Keying 1,2, Ding Xuemei 1,2, Chen Daiwen 1,2,✉
PMCID: PMC3450144  PMID: 23100767

Abstract

The strain of Trichoderma reesei Rut C-30 was subjected to mutation after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) for 6 h followed by UV irradiation for 15 min. Successive mutants showed enhanced cellulase production, clear hydrolysis zone and rapid growth on Avicel-containing plate. Particularly, the mutant NU-6 showed approximately two-fold increases in activity of both FPA and CMCase in shake flask culture when grown on basal medium containing peptone (1%) and wheat bran (1%). The enzyme production was further optimized using eight different media. When a mixture of lactose and yeast cream was used as cellulase inducer, the mutant NU-6 yielded the highest enzyme and cell production with a FPase activity of 6.2 U ml−1, a CMCase activity of 54.2 U ml−1, a β-glucosidase activity of 0.39 U ml−1, and a fungal biomass of 12.6 mg ml−1. It deserved noting that the mutant NU-6 also secreted large amounts of xylanases (291.3 U ml−1). These results suggested that NU-6 should be an attractive producer for both cellulose and xylanase production.

Keywords: Cellulase, Trichoderma reesei, NTG and ultraviolet mutation, Fermentation

Full Text

The Full Text of this article is available as a PDF (862.5 KB).

References

  • 1.Ryu D.D.Y., Mandels M. Cellulases: biosynthesis and applications. Enzyme Microbiol Technol. 1980;2:91–102. doi: 10.1016/0141-0229(80)90063-0. [DOI] [Google Scholar]
  • 2.Galante Y, De Conti A and Monteverdi R (1998) Application of Trichoderma enzymes in the textile industry. In: Harman G. and Kubicek C.(eds.) Trichoderma and Gliocladium. Enzymes, biological control and commercial applications, Vol. 2. pp. 311–326
  • 3.Nierstrasz V., Warmoeskerken M. Process engineering and industrial enzyme applications. In: Cavaco-Paulo A., Gubitz G., editors. Textile processing with enzymes. England: Woodhead Publishing Ltd.; 2003. pp. 120–157. [Google Scholar]
  • 4.Ogel Z.B., Yarangumeli K., Dundar H., Ifrij I. Submerged cultivation of Scytalidium thermophilum on complex lignocellulosic biomass for endoglucanase production. Enz Microb Technol. 2001;28:689–695. doi: 10.1016/S0141-0229(01)00315-5. [DOI] [PubMed] [Google Scholar]
  • 5.Ikeda Y., Park E.Y., Okida N. Bioconversion of waste office paper to gluconic acid in a turbine blade reactor by the filamentous fungus Aspergillus niger. Bioresour Technol. 2006;97:1030–1035. doi: 10.1016/j.biortech.2005.04.040. [DOI] [PubMed] [Google Scholar]
  • 6.Sun Y., Cheng J. Hydrolysis of lignocellulosic materials for ethanol production. a review. Bioresour Technol. 2002;83:1–11. doi: 10.1016/S0960-8524(01)00212-7. [DOI] [PubMed] [Google Scholar]
  • 7.Zaldivar M, Velasquez JC, Contreras I and Perez LM (2001) Trichoderma aureoviride 7-121, a mutant with enhanced production of lytic enzymes: its potential use in waste cellulose degradation and biocontral. Ele J Biotechnol 1–7
  • 8.Adsul M.G., Bastawde K.B., Varma A.J., Gokhale D.V. Strain improvement of Penicillium janthinellum NCIM 1171 for increased cellulase production. Bioresour Technol. 2007;98:1467–1473. doi: 10.1016/j.biortech.2006.02.036. [DOI] [PubMed] [Google Scholar]
  • 9.Pothiraj C., Balaji P., Eyini M. Enhanced production of cellulases by various fungal cultures in solid state fermentation of cassava waste. Afr J Biotechnol. 2006;5:1882–1885. [Google Scholar]
  • 10.Gritzali M., Brown R.D., Jr The cellulase system of Trichoderma. The relationship between purified extracellular enzymes from induced or cellulose grown cells. Adv Chem Ser. 1979;191:237–260. doi: 10.1021/ba-1979-0181.ch012. [DOI] [Google Scholar]
  • 11.Muthuvelayudham R., Viruthagiri T. Fermentative production and kinetics of cellulose protein on Trichoderma reesei using sugar cane bagasse and rice straw. Afri J Biotechnol. 2006;5:1873–1881. [Google Scholar]
  • 12.Bailey M.J., Nevalainene K.M.H. Induction, isolation and testing of stable Trichoderma reesei mutants with improved production of solubilizing cellulose. Enzyme Microb Technol. 1981;3:153–157. doi: 10.1016/0141-0229(81)90076-4. [DOI] [Google Scholar]
  • 13.Cherry J.R., Fidantsef A.L. Directed evolution of industrial enzymes: an update. Curr Opin Biotech. 2003;14:438–443. doi: 10.1016/S0958-1669(03)00099-5. [DOI] [PubMed] [Google Scholar]
  • 14.Mendels M., Weber J., Parizek R. Enhanced cellulase production by a mutant of Trichoderma viride. Appl Microbiol. 1971;21:152–154. doi: 10.1128/am.21.1.152-154.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Johnwesly B., Virupakshi S., Patil G.N., Ramalingam Naik G.R. Cellulase-free thermostable alkaline xylanases from thermophillic and alkalophillic Bacillus sp. JB-99. J Microbiol Biotechnol. 2002;12:153–156. [Google Scholar]
  • 16.Bailey M.J., Tahtiharju J. Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer controlled feeding strategy. Appl Microbiol Biotechnol. 2003;62:156–162. doi: 10.1007/s00253-003-1276-9. [DOI] [PubMed] [Google Scholar]
  • 17.Gokhale D.V., Puntambekar U.S., Deobagkar D.N., Peberdy J.F. Production of cellulolytic enzymes by mutant of Aspergillus niger NCIM 1207. Enzyme Microb Technol. 1988;10:442–445. doi: 10.1016/0141-0229(88)90040-3. [DOI] [Google Scholar]
  • 18.Mandels M., Hontz I., Nystrom J. Enzymatic hydrolysis of waste cellulose. Biotechnol Bioeng. 1974;16:1471–1493. doi: 10.1002/bit.260161105. [DOI] [PubMed] [Google Scholar]
  • 19.Bailey M.J., Biely P., Pouanen K. Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol. 1992;23:257–270. doi: 10.1016/0168-1656(92)90074-J. [DOI] [Google Scholar]
  • 20.Miller G.L., Blum R., Glennon W.E., Burton A.L. Measurement of carboxymethylcellulase activity. Anal Biochem. 1960;2:127–132. doi: 10.1016/0003-2697(60)90004-X. [DOI] [Google Scholar]
  • 21.Bradford M.M. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  • 22.Ahamed A., Vermette P. Culture-based strategies to enhance cellulase enzyme production from Trichoderma reesei RUT-C30 in bioreactor culture conditions. Biochem Eng. 2008;40:399–407. doi: 10.1016/j.bej.2007.11.030. [DOI] [Google Scholar]
  • 23.Oksanen T., Pere J., Paavilainen L., Buchert J., Viilkari L. Treatment of recycled kraft pulps with Trichoderma reesei hemicellulases and cellulases. J Biotechnol. 2000;78:39–48. doi: 10.1016/S0168-1656(99)00232-1. [DOI] [PubMed] [Google Scholar]
  • 24.Shin C.S., Lee J.P., Lee J.S., Park S.C. Enzyme production of Trichoderma reesei Rut C-30 on various lignocellulosic substrates. Appl Biochem Biotechnol. 2000;84–86:237–245. doi: 10.1385/ABAB:84-86:1-9:237. [DOI] [PubMed] [Google Scholar]
  • 25.Grange D.C., Pretorius I.S., Zyl W.H. Expression of a Trichoderma reesei β-xylanase gene (XYN2) in Saccharomyces cerevisiae. Appl Environ Microbiol. 1996;62:1036–1044. doi: 10.1128/aem.62.3.1036-1044.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Chand P., Aruna A., Maqsood A.M., Rao L.V. Novel mutation method for increased cellulase production. J Appl Microbiol. 2005;98:318–323. doi: 10.1111/j.1365-2672.2004.02453.x. [DOI] [PubMed] [Google Scholar]
  • 27.Sheir-Neiss G., Montenecourt B.S. Characterization of the secreted cellulases of Trichoderma reesei wild type and mutants during controlled fermentations. Appl Microbiol Biotechnol. 1984;20:46–53. doi: 10.1007/BF00254645. [DOI] [Google Scholar]
  • 28.Mandels M., Reese E.T. Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. J Bacteriol. 1957;73:269–278. doi: 10.1002/path.1700730133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Mandels M. Cellulose as a chemical and energy source. Biotechnol Bioeng Symp NY. 1975;5:81. [PubMed] [Google Scholar]
  • 30.Muthuvelayudham R, Viruthagiri T and Selvapandian T (2005) Biosynthesis of cellulase protein on substrates like cellulose, xylose and lactose using Trichoderma reesei. Annamalai Univeristiy. J Eng Technol 118–121
  • 31.Harikrishna S., Rao K.C.S., Babu J.S., Reddy D.S. Studies on the production and application of cellulase from Trichoderma reesei QM9414. Bioprocess Eng. 2000;22:467–470. doi: 10.1007/s004490050760. [DOI] [Google Scholar]
  • 32.Mandels M., Parrish F.W., Reese E.T. Soporose as an inducer of cellulase in Trichoderma viride. J Bacteriol. 1962;83:400–408. doi: 10.1128/jb.83.2.400-408.1962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Sternberg D., Mandels G.R. Induction of cellulolytic enzymes in Trichoderma reesei by sophorose. J Bacteriol. 1979;139:761–769. doi: 10.1128/jb.139.3.761-769.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Zaldivar J., Nielsen J., Olsson L. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol. 2001;56:17–34. doi: 10.1007/s002530100624. [DOI] [PubMed] [Google Scholar]
  • 35.Bailey M.J., Buchert J., Viikari L. Effect of pH on production of xylanase by Trichoderma ressei on xylan- and cellulose-based media. Appl Microbiol Biotechnol. 1993;40:224–229. doi: 10.1007/BF00170371. [DOI] [Google Scholar]
  • 36.Saddler J.N., Hogan C.M., Louis-Seize G. A comparison between the cellulase systems of Trichoderma harzianum E58 and Trichoderma reesei C30. Appl Microbiol Biotechnol. 1985;22:139–145. doi: 10.1007/BF00250034. [DOI] [Google Scholar]
  • 37.Domingues F.C., Queiroz J.A., Cabral J.M.S., Fonseca L.P. The influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30. Enzyme Microbiol Technol. 2000;26(5–6):394–401. doi: 10.1016/S0141-0229(99)00166-0. [DOI] [PubMed] [Google Scholar]

Articles from Indian Journal of Microbiology are provided here courtesy of Springer

RESOURCES