Skip to main content

Two new β-glucosidases from ethanol-fermenting fungus Mucor circinelloides NBRC 4572: enzyme purification, functional characterization, and molecular cloning of the gene

An Erratum to this article was published on 12 October 2013


Two β-glucosidases (BGLs 1 and 2) were purified to homogeneity from the extracellular enzyme preparations of the ethanol-fermenting Mucor circinelloides NBRC 4572 statically grown on rice straw. BGLs 1 and 2 are monomeric glycoproteins whose apparent molecular masses (Ms) are around 78 kDa, which decreased by approximately 10 kDa upon enzymatic deglycosylation. Both BGLs showed similar enzyme characteristics in optimal temperature and pH, stability, and inhibitors. They were active against a wide range of aryl-β-glucosides and β-linked glucose oligosaccharides. Their amino acid sequences shared 81 % identity and exhibited less than 60 % identity with the known family-3 BGLs. Considering properties such as reduced inhibition by ethanol, glucose, and cellobiose, low transglucosylation activity, wider substrate range, less binding affinity to lignocellulosic materials, and abundant expression, BGL1 is likely to be more suitable for bioethanol production than BGL2 via simultaneous saccharification and fermentation of rice straw with M. circinelloides.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  • Bhat MK, Bhat S (1997) Cellulose degrading enzymes and their potential industrial applications. Biotechnol Adv 15:583–620

    Article  PubMed  CAS  Google Scholar 

  • Bhatia Y, Mishra S, Bisaria VS (2002) Microbial β-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol 22:375–407

    Article  PubMed  CAS  Google Scholar 

  • Borgia PI, Mehnert DW (1982) Purification of a soluble and a wall-bound form of β-glucosidase from Mucor racemosus. J Bacteriol 149:515–522

    PubMed  CAS  Google Scholar 

  • Chahal DS (1983) Microorganisms in solid state fermentation for upgrading of protein values of lignocelluloses and cellulase production. In: Found Biochem Eng (ed) ACS Symp. Ser. 207, pp 421–442

  • De Crombrugghe B, Perlman RL, Varmus HE, Pastan I (1969) Regulation of inducible enzyme synthesis in Escherichia coli by cyclic adenosine 3′, 5′-monophosphate. J Biol Chem 244:5828–5835

    PubMed  Google Scholar 

  • Desai JD, Ray RM, Pate NP (1983) Purification and properties of extracellular β-glucosidase from Scytalidium lignicola. Biotechnol Bioeng 25:307–313

    Article  PubMed  CAS  Google Scholar 

  • Eyzaguirre J, Hidalgo M, Leschot A (2005) β-Glucosidases from filamentous fungi: properties, structure, and applications. In: Yarema KJ (ed) Handbook of carbohydrate engineering. CRC Press, Boca Raton, FL pp 645–686

  • Harvey AJ, Hrmova M, De Gori R, Varghese JN, Fincher GB (2000) Comparative modeling of the three-dimensional structures of GH3 proteins. Proteins 41:257–269

    Article  PubMed  CAS  Google Scholar 

  • Hong J, Ladisch MR, Gong C-S, Wankat PC, Tsao GT (1981) Combined product and substrate inhibition equation for cellobiase. Biotechnol Bioeng 23:2779–2788

    Article  CAS  Google Scholar 

  • Kawai T, Nakazawa H, Ida N, Okada H, Tani S, Sumitani J, Kawaguchi T, Ogasawara W, Morikawa Y, Kobayashi Y (2012) Analysis of the saccharification capability of high-functional cellulase JN11 for various pretreated biomasses through a comparison with commercially available counterpart. J Ind Microbiol Biotechnol 39:1741–1749

    Article  PubMed  CAS  Google Scholar 

  • Kim S, Dale BE (2004) Global potential bioethanol production from wasted crops and crop residues. Biomass Bioenergy 26:361–375

    Article  Google Scholar 

  • Krisch J, Bencsik O, Papp T, Vágvölgyi C, Takó M (2012) Characterization of a β-glucosidase with transgalactosylation capacity from the zygomycete Rhizomucor miehei. Bioresour Technol 114:555–560

    Article  PubMed  CAS  Google Scholar 

  • Kumar R, Singh S, Singh OV (2008) Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives. J Ind Microbiol Biotechnol 35:377–391

    Article  PubMed  CAS  Google Scholar 

  • Nakazawa H, Kawai T, Ida N, Shida Y, Kobayashi Y, Okada H, Tani S, Sumitani J, Kawaguchi T, Morikawa Y, Ogasawara W (2012) Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus β-glucosidase 1 for efficient biomass conversion. Biotechnol Bioeng 109:92–99

    Article  PubMed  CAS  Google Scholar 

  • Nazir A, Soni R, Saini HS, Kaur A, Chadha BS (2010) Profiling differential expression of cellulases and metabolite footprints in Aspergillus terreus. Appl Biochem Biotechnol 162:538–547

    Article  PubMed  CAS  Google Scholar 

  • Nomura T, Ogita S, Kato Y (2012) A novel lactone-forming carboxylesterase: molecular identification of a tuliposide A-converting enzyme in tulip. Plant Physiol 159:565–578

    Article  PubMed  CAS  Google Scholar 

  • Petruccioli M, Brimer L, Cicalini AR, Federici F (1999) The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava. J Appl Microbiol 86:302–310

    Article  CAS  Google Scholar 

  • Rudick MJ, Elbein AD (1975) Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second β-glucosidase. J Bacteriol 124:534–541

    PubMed  CAS  Google Scholar 

  • Sakamoto R, Arai M, Murao S (1985) Enzymatic properties three β-glucosidases from Aspergillus aculeatus No. F-50. Agric Biol Chem 49:1283–1290

    Article  CAS  Google Scholar 

  • Silverstein RA, Chen Y, Sharma-Shivappa RR, Boyette MD, Osborne J (2007) A comparison of chemical pretreatment methods for improving saccharification of cotton stalks. Bioresour Technol 98:3000–3011

    Article  PubMed  CAS  Google Scholar 

  • Singhania RR, Patel AK, Sukumaran RK, Larroche C, Pandey A (2013) Role and significance of β-glucosidases in the hydrolysis of cellulose for bioethanol production. Bioresour Technol 127:500–507

    Article  PubMed  CAS  Google Scholar 

  • Takano M, Hoshino K (2012a) Production of biofuel from waste lignocellulosic biomass materials based on energy saving viewpoint. Adv Mater Dev Perform 6:715–720

    CAS  Google Scholar 

  • Takano M, Hoshino K (2012b) Direct ethanol production from rice straw by coculture with two high-performing fungi. Front Chem Sci Eng 6:139–145

    Article  CAS  Google Scholar 

  • Takii Y, Ikeda K, Sato C, Yano M, Sato T (2005) Production and characterization of β-glucosidase from Rhizopus oryzae MIBA348. Int J Biol Macromol 5:11–16

    CAS  Google Scholar 

  • Takó M, Farkas E, Lung S, Krisch J, Vágvölgyi C, Papp T (2010a) Identification of acid- and thermotolerant extracellular β-glucosidase activities in Zygomycetes fungi. Acta Biol Hung 6:101–110

    Article  Google Scholar 

  • Takó M, Tóth A, Nagy LG, Krisch J, Vágvölgyi C, Papp T (2010b) A new β-glucosidase gene from the zygomycete fungus Rhizomucor miehei. Antonie Leeuwenhoek 97:1–10

    Article  PubMed  Google Scholar 

  • Varghese JN, Hrmova M, Fincher GB (1999) Three-dimensional structure of a barley β-d-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure 15:179–190

    Google Scholar 

  • Willick GE, Seligy VL (1985) Multiplicity in cellulases of Schizophyllum commune. Derivation partly from heterogeneity in transcription and glycosylation. Eur J Biochem 151:89–96

    Article  PubMed  CAS  Google Scholar 

  • Wood TM, Mccrae SI (1982) Purification and some properties of the extracellular β-d-glucosidase of the cellulolytic fungus Trichoderma koningii. J Gen Microbiol 128:2973–2982

    CAS  Google Scholar 

  • Yoshioka H, Hayashida, S (1981) Relationship between carbohydrate moiety and thermostability of β-glucosidase from Mucor miehei YH-10. Agric Biol Chem 45:571–577

    Google Scholar 

Download references


This work was supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO) project. The authors thank Ms. Haruna Tsubata and Ms. Rina Sakaguchi of Toyama Prefectural University for their technical assistance in the optimization of the cultivation conditions of M. circinelloides.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Yasuo Kato.

Electronic supplementary material

Below is the link to the electronic supplementary material.


(PDF 318 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kato, Y., Nomura, T., Ogita, S. et al. Two new β-glucosidases from ethanol-fermenting fungus Mucor circinelloides NBRC 4572: enzyme purification, functional characterization, and molecular cloning of the gene. Appl Microbiol Biotechnol 97, 10045–10056 (2013).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: