Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena

  • Oscar A. Rojas-Rejón
  • Héctor M. Poggi-Varaldo
  • Ana C. Ramos-Valdivia
  • Alfredo Martínez-Jiménez
  • Eliseo Cristiani-Urbina
  • Mayra de la Torre Martínez
  • Teresa Ponce-Noyola
Original Paper


Derepressed mutant PR-22 was obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg−1 and cellulolytic activity from 1.9 to 4 U mg−1; this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20–32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l−1 of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.


Cellulomonas flavigena Mutant Derepressed Saccharification Sugarcane bagasse Cellulase Xylanase 



This work was supported by Consejo Nacional de Ciencia y Tecnología México (CONACYT) (Grant 104333).


  1. 1.
    Ali S, Sayed A (1992) Regulation of cellulase biosynthesis in Aspergillus terreus. World J Microbiol Biotechnol 8(1):73–75. doi: 10.1007/BF01200691 CrossRefGoogle Scholar
  2. 2.
    Creuzet N, Berenguer J, Frixon C (1983) Characterization of exoglucanase and synergistic hydrolysis of cellulose in Clostridium stercorarium. FEMS Microbiol Lett 20(3):347–350CrossRefGoogle Scholar
  3. 3.
    Cuskey SM, Schamhart DHJ, Chase T, Montenecourt BS, Eveleigh DE (1980) Screening for beta-glucosidase mutants of Trichoderma reesei with resistance to end-product inhibition. Dev Ind Microbiol 21:471–480Google Scholar
  4. 4.
    De la Torre M, Casas-Campillo C (1984) Isolation and characterization of a symbiotic cellulolytic mixed bacterial culture. Appl Microbiol Biotechnol 19(6):430–434. doi: 10.1007/BF00454383 CrossRefGoogle Scholar
  5. 5.
    Halliwell G, Griffin M (1973) The nature and mode of action of the cellulolytic component C1 of Trichoderma koningii on native cellulose. Biochem J 135(4):587–594PubMedGoogle Scholar
  6. 6.
    Huang L, Forsberg C (1990) Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85. Appl Environ Microbiol 56(5):1221–1228PubMedGoogle Scholar
  7. 7.
    Johnson E, Bouchot F, Demain A (1985) Regulation of cellulase formation in Clostridium thermocellum. J Gen Microbiol 131:2303–2308. doi: 10.1099/00221287-131-9-2303 Google Scholar
  8. 8.
    Kalogiratou Z, Simos TE (2003) Newton-cotes formulae for long-time integration. J Comp Appl Math 158:75–82. doi: 10.1016/S0377-0427(03)00479-5 CrossRefGoogle Scholar
  9. 9.
    Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with folin-phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  10. 10.
    Lynd L, Weimer P, van Zyl W, Pretorius I (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol R 66(3):506–577. doi: 10.1128/MMBR.66.3.506-577.2002 CrossRefGoogle Scholar
  11. 11.
    Mach-Aigner AR, Pucher ME, Mach RL (2010) D-xylose as repressor or inducer of xylanase expression in Hypocrea jecorina (Trichoderma reesei). Appl Environ Microbiol 76(6):1770–1776. doi: 10.1128/AEM.02746-09 CrossRefPubMedGoogle Scholar
  12. 12.
    Mandels M, Reese ET (1960) Induction of cellulases in fungi by cellobiose. J Bacteriol 79(6):816–826PubMedGoogle Scholar
  13. 13.
    Manning K, Wood D (1983) Production and regulation of extracellular endocellulase by Agaricus bisporus. J Gen Microbiol 129:1839–1847. doi: 10.1099/00221287-129-6-1839 Google Scholar
  14. 14.
    Mansfield SD, Mooney C, Saddler JN (1999) Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol Progr 15:804–816. doi: 10.1021/bp9900864 CrossRefGoogle Scholar
  15. 15.
    Mayorga-Reyes L, Ponce-Noyola T (1998) Isolation of a hyperxylanolytic Cellulomonas flavigena mutant growing on continuous culture on sugarcane bagasse. Biotechnol Lett 20(5):443–446. doi: 10.1023/A:1005423509856 CrossRefGoogle Scholar
  16. 16.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31(3):426–428. doi: 10.1021/ac60147a030 CrossRefGoogle Scholar
  17. 17.
    Ponce-Noyola T, de la Torre M (1995) Isolation of a high-specific-growth-rate mutant of Cellulomonas flavigena on sugarcane bagasse. Appl Microbiol Biotechnol 42(5):709–712. doi: 10.1007/BF00171949 CrossRefGoogle Scholar
  18. 18.
    Ponce-Noyola T, de la Torre M (2001) Regulation of cellulases and xylanases from a derepressed mutant of Cellulomonas flavigena growing on sugar-cane bagasse in continuous culture. Bioresour Technol 78(3):285–291. doi: 10.1016/S0960-8524(00)00181-4 CrossRefPubMedGoogle Scholar
  19. 19.
    Rajoka M, Bashir A, Kause A (1997) Mutagenesis of Cellulomonas biazotea for enhanced production of xylanases. Bioresour Technol 62(3):99–108. doi: 10.1016/S0960-8524(97)00116-8 CrossRefGoogle Scholar
  20. 20.
    Robson L, Chambliss G (1984) Characterization of the cellulolytic activity of a Bacillus isolate. Appl Environ Microbiol 47(5):1039–1046PubMedGoogle Scholar
  21. 21.
    Roche C, Dibble C, Stickel J (2009) Laboratory-scale method for enzymatic saccharification of lignocellulosic biomass at high-solids loadings. Biotechnol Biofuels 2:28. doi: 10.1186/1754-6834-2-28 CrossRefPubMedGoogle Scholar
  22. 22.
    Schimdhalter DR, Canevascini G (1992) Characterization of the cellulolytic enzyme system from the brown-rot fungus Coniophora puteana. Appl Microbiol Biotechnol 37(4):431–436. doi: 10.1007/BF00180963 Google Scholar
  23. 23.
    Segel I (1975) Ezyme kinetics: Behaviour and analysis of rapid equilibrium and steady-state enzyme systems. Wiley, New YorkGoogle Scholar
  24. 24.
    Suto M, Tomita F (2001) Induction and catabolite repression mechanisms of cellulase in fungi. J Biosci Bioeng 92(4):305–311. doi: 10.1016/S1389-1723(01)80231-0 CrossRefPubMedGoogle Scholar
  25. 25.
    Teather R, Wood P (1982) Use of congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from bovine rumen. Appl Environ Microbiol 43(4):777–780PubMedGoogle Scholar
  26. 26.
    Thomas KNG, Zeikus JG (1981) Comparison of extracellular cellulase activities of Clostridium thermocellum LQRI and Trichoderma reesei QM9414. Appl Environ Microbiol 42(2):231–240Google Scholar
  27. 27.
    Warzywoda M, Vandecasteele JP, Pourquié J (1983) A comparison of genetically improved strains of the cellulolytic fungus Trichoderma reseei. Biotechnol Lett 5(4):243–246. doi: 10.1007/BF00161123 CrossRefGoogle Scholar
  28. 28.
    Zhang P, Himmel M, Mielenz J (2006) Outlook for cellulase improvement: screening and selection strategies. Biotechnol Adv 24(5):452–481. doi: 10.1016/j.biotechadv.2006.03.003 CrossRefGoogle Scholar
  29. 29.
    Zhang P, Lynd L (2005) Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum. J Bacteriol 187(1):99–106. doi: 10.1128/JB.187.1.99-106.2005 CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2010

Authors and Affiliations

  • Oscar A. Rojas-Rejón
    • 1
  • Héctor M. Poggi-Varaldo
    • 1
  • Ana C. Ramos-Valdivia
    • 1
  • Alfredo Martínez-Jiménez
    • 2
  • Eliseo Cristiani-Urbina
    • 3
  • Mayra de la Torre Martínez
    • 4
  • Teresa Ponce-Noyola
    • 1
  1. 1.Departamento de Biotecnología y BioingenieríaCentro de Investigación y de Estudios Avanzados del Instituto Politécnico NacionalZacatencoMexico
  2. 2.Instituto de Biotecnología UNAMCuernavacaMexico
  3. 3.Escuela Nacional de Ciencias Biológicas del IPN, Prolongación Carpio y Plan de AyalaPlutarco Elías CallesMexico
  4. 4.Centro de Investigación en Alimentación y DesarrolloHermosilloMexico

Personalised recommendations