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.
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Bhat MK, Bhat S (1997) Cellulose degrading enzymes and their potential industrial applications. Biotechnol Adv 15:583–620
Bhatia Y, Mishra S, Bisaria VS (2002) Microbial β-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol 22:375–407
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
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
Desai JD, Ray RM, Pate NP (1983) Purification and properties of extracellular β-glucosidase from Scytalidium lignicola. Biotechnol Bioeng 25:307–313
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
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
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
Kim S, Dale BE (2004) Global potential bioethanol production from wasted crops and crop residues. Biomass Bioenergy 26:361–375
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
Kumar R, Singh S, Singh OV (2008) Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives. J Ind Microbiol Biotechnol 35:377–391
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
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
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
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
Rudick MJ, Elbein AD (1975) Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second β-glucosidase. J Bacteriol 124:534–541
Sakamoto R, Arai M, Murao S (1985) Enzymatic properties three β-glucosidases from Aspergillus aculeatus No. F-50. Agric Biol Chem 49:1283–1290
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
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
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
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
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
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
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
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
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
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
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
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.
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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). https://doi.org/10.1007/s00253-013-5210-5