Investigating Cellulase Producing Potential of Two Iranian Thermoascus aurantiacus Isolates in Submerged Fermentation

Document Type: Research Article


1 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran.

2 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran; Novel Diagnostics Therapeutics Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.

3 Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran.

4 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran; Stem Cell and Tissue Engineering Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran.


Cellulose is the most plentiful renewable biopolymer in nature which could be utilized by cellulolytic enzymes. Cellulases are among the most important groups of industrial enzymes which are widely consumed in biofuel production, pulp and paper, textile, and detergent industries. These enzymes can support a cleaner environment through reducing chemical processes in mentioned industries and agro-industrial waste management. Thermophilic filamentous fungi produce thermostable types of the enzymes with the property of hydrolysis the cellulose in higher temperatures with higher rates of reaction, decreased amounts of enzyme quantities and reduced risk of contamination by the mesophilic microorganisms. The cellulolytic capacity of two Thermoascus aurantiacus isolates (from Mashhad, Iran) was examined in a simple liquid state fermentation in different carbon and nitrogen sources, in comparison to the Thermoascus aurantiacus DSM 1831 as a reference fungus. Among different the tested sources, wheat bran and peptone led to the highest level of endoglucanase production by the isolated thermophilic fungi. The isolates showed higher cellulase activities, including endoglucanase, avicelase, and FPase, of the crude enzymes from the isolates in comparison to the reference fungus. Gene expression profiling revealed that changes in the cellulase mRNA levels are not correlated with the changes in protein activities during a 12-day period. This observation might be due to a complex process of enzymatic regulation of cellulases in response to the environmental signals.


Anwar Z, Gulfraz M, Irshad M. 2014. Agro-industrial lignocellulosic biomass a key to unlock the future bio-energy: a brief review. J Radiat Res Appl Sci 7: 163-173.

Brienzo M, Arantes V, Milagres AM. 2008. Enzymology of the thermophilic ascomycetous fungus Thermoascus aurantiacus. Fungal Biol Rev 22: 120-130.

Colagar AH, Motallebi M, Zamani MR. 2004. Isolation, cloning, and partial characterization of the gene encoding the polygalacturonase inhibiting protein of Phaseolus vulgaris cv. Naz. Pak J Biotechnol 1(2): 1-11

Coral G, Arikan B, Ünaldi MN, Guvenmez H. 2002. Some properties of crude carboxymethyl cellulase of Aspergillus niger Z10 wild-type strain. Turkish J Biol 26: 209-213.

Deshpande MV, Eriksson KE, Pettersson LG. 1984. An assay for selective determination of exo-1, 4,-β-glucanases in a mixture of cellulolytic enzymes. Anal Biochem 138: 481-487.

Gielkens MM, Dekkers E, Visser J, de Graaff LH. 1999. Two cellobiohydrolase-encoding genes from Aspergillus niger require D-xylose and the xylanolytic transcriptional activator XlnR for their expression. Appl Environ Microbiol 65: 4340-4345.

Gomes D, Gomes J, Steiner W .1994. Factors influencing the induction of endo-xylanase by Thermoascus aurantiacus. ‎J Biotechnol 33: 87-94.

Grajek W. 1986. Temperature and pH optima of enzyme activities produced by cellulolytic thermophilic fungi in batch and solid-state cultures. Biotechnol Lett 8: 587-590.

Hart T, De Leij F, Kinsey G, Kelley J, Lynch J. 2002. Strategies for the isolation of cellulolytic fungi for composting of wheat straw. World J Microbiol Biotechnol 18: 471-480.

Himmel ME, Ruth MF, Wyman CE. 1999. Cellulase for commodity products from cellulosic biomass. Curr Opin Biotechnol 10: 358-364.

Hong J, Tamaki H, Kumagai H. 2006. Unusual hydrophobic linker region of β-glucosidase (BGLII) from Thermoascus aurantiacus is required for hyperactivation by organic solvents. Appl Microbiol Biotechnol 73: 80-88.

Hong J, Tamaki H, Yamamoto K, Kumagai H. 2003. Cloning of a gene encoding thermostable cellobiohydrolase from Thermoascus aurantiacus and its expression in yeast. Appl Microbiol Biotechnol 63: 42-50.

Ilmen M, Saloheimo A, Onnela ML, Penttilä ME. 1997. Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. Appl Environ Microbiol 63: 1298-1306.

Irshad MN, Anwar Z, But H.I, Afroz A, Ikram N, Rashid U. 2012. The industrial applicability of purified cellulase complex indigenously produced by Trichoderma viride through solid-state bio-processing of agro-industrial and municipal paper wastes. BioResources 8, 145-157.

Jain KK, Dey TB, Kumar S, Kuhad RC. 2015. Production of thermostable hydrolases (cellulases and xylanase) from Thermoascus aurantiacus RCKK: a potential fungus. Bioprocess Biosyst Eng 38: 787-796.

Kalogeris E, Christakopoulos P, Katapodis P, Alexiou A, Vlachou S, Kekos D, Macris B. 2003. Production and characterization of cellulolytic enzymes from the thermophilic fungus Thermoascus aurantiacus under solid state cultivation of agricultural wastes. Process Biochem 38: 1099-1104.

Kalogeris E, Christakopoulos P, Kekos D, Macris B. 1998. Studies on the solid-state production of thermostable endoxylanases from Thermoascus aurantiacus: characterization of two isozymes. ‎J Biotechnol 60: 155-163.

Kawamori M, Takayama KI, Takasawa S. 1987. Production of cellulases by a thermophilic fungus, Thermoascus aurantiacus A-131. Agric Biol Chem 51: 647-654.

Khandke KM, Vithayathil P, Murthy S. 1989. Purification of xylanase, β-glucosidase, endocellulase, and exocellulase from a thermophilic fungus, Thermoascus aurantiacus. Arch Biochem Biophys 274: 491-500.

Kim YK, Lee SC, Cho YY, Oh HJ, Ko YH. 2012. Isolation of cellulolytic Bacillus subtilis strains from agricultural environments. ISRN Microbiol 2012, 650563.

Krogh KB, Harris PV, Olsen CL, Johansen KS, Hojer-Pedersen J, Borjesson J, Olsson L. 2010. Characterization and kinetic analysis of a thermostable GH3 β-glucosidase from Penicillium brasilianum. Appl Microbiol Biotechnol 86: 143-154.

Kuhad RC, Gupta R, Khasa YP, Singh A. 2010. Bioethanol production from lantanacamara (red sage): pretreatment, saccharification and fermentation. Bioresour Technol 101, 8348-8354.

Kuhad RC, Gupta R, Singh A. 2011. Microbial cellulases and their industrial applications. Enzyme Res 2011, 280696.

Llanos A, François JM, Parrou JL. 2015. Tracking the best reference genes for RT-qPCR data normalization in filamentous fungi. BMC genomics 16: 71.

Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66: 506-577.

Mandels M, Parrish FW, Reese ET. 1962. Sophorose as an inducer of cellulase in Trichoderma viride. J Bacteriol 83: 400-408.

Mandels M, Reese ET. 1960. Induction of cellulase in fungi by cellobiose. J Bacteriol 79: 816-26.

McClendon SD, Batth T, Petzold CJ, Adams PD, Simmons BA, Singer SW. 2012. Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions. Biotechnol Biofuels 5 (1): 54. doi: 10.1186/1754-6834-5-54.

Michielse CB, Hooykaas PJ, van den Hondel CA, Ram AF. 2008. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc 3: 1671-1678.

Miller G. 1959. Use of DNS reagent for the measurement of reducing sugar. Anal Chem 31: 426-428.

Mitchell DA, Lonsane B. 1992. Definition, characteristics and potential. In: Doelle HW, Mitchell DA, Rolz CE, eds. Solid substrate cultivation. London and New York, Elsiever Sci Publ ltd, 1-13.

Moretti M, Bocchini-Martins DA, Silva RD, Rodrigues A, Sette LD, Gomes E. 2012. Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation. Braz J Microbiol 43: 1062-1071.

Parry NJ, Beever DE, Emyr O, Vandenberghe I, Van Beeumen J. 2001 Biochemical characterization and mechanism of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus. Biochem J 353: 117-127.

Payne SH. 2015. The utility of protein and mRNA correlation. Trends Biochem Sci 40: 1-3.

Prior BA, Du Preez JC, Rein PW. 1992. Environmental parameters. In: Doelle HW, Mitchell DA, Rolz CE, eds. Solid substrate cultivation. London and New York, Elsiever Sci Publ ltd, 65-86.

Rao M, Seeta R, Deshpande V. 1989. Comparative evaluation of cellulases: role of individual components in hydrolysis. Biotechnol Appl Biochem 11: 477-482.

Roche N, Desgranges C, Durand A. 1994. Study on the solid-state production of a thermostable α-L-arabinofuranosidase of Thermoascus aurantiacus on sugar beet pulp. ‎J Biotechnol 38: 43-50.

Sánchez C. 2009. Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnol Adv 27, 185-194.

Sazci A, Erenler K, Radford A. 1986. Detection of cellulolytic fungi by using congo red as an indicator: a comparative study with the dinitrosalicyclic acid reagent method. J Appl Microbiol 61: 559-562.

Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Bolchacova E, Voigt K, Crous PW. 2012. Nuclear ribosomal internal transcribed spacer region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci 109: 6241-6246.

Singh A, Kuhad RC, Ward OP. 2007. Industrial application of microbial cellulases. In: Kudah RC, Singh A, eds. Lignocellulose Biotechnology: Future Prospects. New Delhi, IK. International Pub House, 345-358.

Soliman, SA, El-Zawahry YA, El-Mougith AA. 2013. Fungal biodegradation of agro-industrial waste. In: van de Ven T, Kadla J, eds. Cellulose, biomass conversion, InTechOpen.

Sonnleitner B, Fiechter A. 1983. Advantages of using thermophiles in biotechnological processes: expectations and reality. Trends Biotechnol 1: 74-80.

Sukumaran RK, Singhania RR, Pandey A. 2005. Microbial cellulases-production, applications and challenges. J Sci Ind Res 64: 832-844.

Tian C, Beeson WT, Iavarone AT, Sun J, Marletta MA, Cate JH, Glass NL. 2009. Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa. Proc Natl Acad Sci 106: 22157-22162.

Uhlig H. 1998. Industrial enzymes and their applications. John Wiley & Sons. New York, NY, USA.

Viikari L, Alapuranen M, Puranen T, Vehmaanperä J, Siika-Aho M. 2007. Thermostable enzymes in lignocellulose hydrolysis. Adv Biochem Eng Biotechnol 108: 121-145.

Volossiouk T, Robb EJ, Nazar RN. 1995. Direct DNA extraction for PCR-mediated assays of soil organisms. Appl Environ Microbiol 61: 3972-3976.

Wiseman A. 1993. Designer enzyme and cell applications in industry and in environmental monitoring. J Chem Technol Biotechnol 56: 3-13.

Xiao Z, Storms R, Tsang A. 2004a. Microplate‐based filter paper assay to measure total cellulase activity. Biotechnol Bioeng 88: 832-837.

Xiao Z, Zhang X, Gregg DJ, Saddler JN. 2004b. Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates. Appl Biochem Biotechnol 115: 1115-1126.

Yoneda A, Kuo HWD, Ishihara M, Azadi P, Yu SM, Ho TD. 2014. Glycosylation variants of a β-glucosidase secreted by a Taiwanese fungus, Chaetomella raphigera, exhibit variant-specific catalytic and biochemical properties. PLoS One 9: e106306.

Zhang YP, Hong J, Ye X. 2009. Cellulase assays. Methods Mol Biol 581: 213-231.