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Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae

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Abstract

Neotermes koshunensis is a lower termite that secretes endogenous β-glucosidase in the salivary glands. This β-glucosidase (G1NkBG) was successfully expressed in Aspergillus oryzae. G1NkBG was purified to homogeneity from the culture supernatant through ammonium sulfate precipitation and anion exchange, hydrophobic, and gel filtration chromatographies with a 48-fold increase in purity. The molecular mass of the native enzyme appeared as a single band at 60 kDa after gel filtration analysis, indicating that G1NkBG is a monomeric protein. Maximum activity was observed at 50 °C with an optimum pH at 5.0. G1NkBG retained 80% of its maximum activity at temperatures up to 45 °C and lost its activity at temperatures above 55 °C. The enzyme was stable from pH 5.0 to 9.0. G1NkBG was most active towards laminaribiose and p-nitrophenyl-β-d-fucopyranoside. Cellobiose, as well as cello-oligosaccharides, was also well hydrolyzed. The enzyme activity was slightly stimulated by Mn2+ and glycerol. The Km and Vmax values were 0.77 mM and 16 U/mg, respectively, against p-nitrophenyl-β-d-glucopyranoside. An unusual finding was that G1NkBG was stimulated by 1.3-fold when glucose was present in the reaction mixture at a concentration of 200 mM. These characteristics, particularly the stimulation of enzyme activity by glucose, make G1NkBG of great interest for biotechnological applications, especially for bioethanol production.

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References

  • Balat M, Balat H, Öz C (2008) Progress in bioethanol processing. Prog Energy Comb Sci 34:551–573

    Article  CAS  Google Scholar 

  • Béguin P, Aubert JP (1994) The biological degradation on cellulose. FEMS Microbiol Rev 13:25–58

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Bothast RJ, Saha BC (1997) Ethanol production from agricultural biomass substrates. Adv Appl Microbiol 44:261–286

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Carroll A, Somerville C (2009) Cellulosic biofuels. Annu Rev Plant Biol 60:165–182

    Article  CAS  Google Scholar 

  • Christakopoulos P, Goodenough PW, Kekos D, Macris BJ, Claeyssens M, Bhat MK (1994) Purification and characterisation of an extracellular β-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum. Eur J Biochem 224:379–385

    Article  CAS  Google Scholar 

  • Dashtban M, Schraft H, Qin W (2009) Fungal bioconversion of lignocellulosic residues; opportunities & perspectives. Int J Biol Sci 5:578–595

    CAS  Google Scholar 

  • Günata Z, Vallier MJ (1999) Production of a highly glucose-tolerant extracellular β-glucosidase by three Aspergillus strains. Biotechnol Lett 21:219–223

    Article  Google Scholar 

  • Karnchanatat A, Petsom A, Sangvanich P, Piaphukiew J, Whalley AJ, Reynolds CD, Sihanonth P (2007) Purification and biochemical characterization of an extracellular β-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm. FEMS Microbiol Lett 270:162–170

    Article  CAS  Google Scholar 

  • Kaur J, Chadha BS, Kumar BA, Kaur GS, Saini HS (2007) Purification and characterization of β-glucosidase from Melanocarpus sp. MTCC 3922. Eletron J Biotechnol 10:260–270

    CAS  Google Scholar 

  • Kitamoto K (2002) Molecular biology of the Koji molds. Adv Appl Microbiol 51:129–153

    Article  CAS  Google Scholar 

  • Klotz IM, Campbell BJ (1962) Copper-induced hydrolysis of the disulfide bond. Arch Biochem Biophys 96:92–99

    Article  CAS  Google Scholar 

  • Kotaka A, Bando H, Kaya M, Kato-Murai M, Kuroda K, Sahara H, Hata Y, Kondo A, Ueda M (2008) Direct ethanol production from barley β-glucan by sake yeast displaying Aspergillus oryzae β-glucosidase and endoglucanase. J Biosci Bioeng 105:622–627

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev 66:506–577

    Article  CAS  Google Scholar 

  • Mabashi Y, Kikuma T, Maruyama J, Arioka M, Kitamoto K (2006) Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria. Biosci Biotechnol Biochem 70:1882–1889

    Article  CAS  Google Scholar 

  • Nascimento CV, Souza FH, Masui DC, Leone FA, Peralta RM, Jorge JA, Furriel RP (2010) Purification and biochemical properties of a glucose-stimulated β-d-glucosidase produced by Humicola grisea var. thermoidea grown on sugarcane bagasse. J Microbiol 48:53–62

    Article  CAS  Google Scholar 

  • Nemoto T, Watanabe T, Mizogami Y, Maruyama J, Kitamoto K (2009) Isolation of Aspergillus oryzae mutants for heterologous protein production from a double proteinase gene disruptant. Appl Microbiol Biotechnol 82:1105–1114

    Article  CAS  Google Scholar 

  • Ni J, Tokuda G, Takehara M, Watanabe H (2007) Heterologous expression and enzymatic characterization of β-glucosidase from the drywood-eating termite, Neotermes koshunensis. Appl Entomol Zool 42:457–463

    Article  CAS  Google Scholar 

  • Pérez-Pons JA, Rebordosa X, Querol E (1995) Properties of a novel glucose-enhanced β-glucosidase purified from Streptomyces sp. (ATCC 11238). Biochim Biophys Acta 1251:145–153

    Article  Google Scholar 

  • Prins RA, Kreulen DA (1991) Comparative aspects of plant cell wall digestion in insects. Anim Feed Sci Technol 32:101–118

    Article  Google Scholar 

  • Riou C, Salmon JM, Vallier MJ, Gunata Z, Barre P (1998) Purification, characterization, and substrate specificity of a novel highly glucose-tolerant β-glucosidase from Aspergillus oryzae. Appl Environ Microbiol 64:3607–3614

    CAS  Google Scholar 

  • Saha BC, Bothast RJ (1996) Production, purification, and characterization of a highly glucose-tolerant novel β-glucosidase from Candida peltata. Appl Environ Microbiol 62:3165–3170

    CAS  Google Scholar 

  • Saha BC, Freer SN, Bothast RJ (1994) Production, purification, and properties of a thermostable β-glucosidase from a color variant strain of Aureobasidium pullulans. Appl Environ Microbiol 60:3774–3780

    CAS  Google Scholar 

  • Scharf ME, Kovaleva ES, Jadhao S, Campbell JH, Buchman GW, Boucias DG (2010) Functional and translational analyses of a β-glucosidase gene (glycosyl hydrolase family 1) isolated from the gut of the lower termite Reticulitermes flavipes. Insect Biochem Mol Biol 40:611–620

    Article  CAS  Google Scholar 

  • Sonia KG, Chadha BS, Badhan AK, Saini HS, Bhat MK (2008) Identification of glucose tolerant acid active β-glucosidases from thermophilic and thermotolerant fungi. World J Microbiol Biotechnol 24:599–604

    Article  CAS  Google Scholar 

  • Tokuda G, Saito H, Watanabe H (2002) A digestive β-glucosidase from the salivary glands of the termite, Neotermes koshunensis (Shiraki): distribution, characterization and isolation of its precursor cDNA by 5′- and 3′-RACE amplifications with degenerate primers. Insect Biochem Mol Biol 32:1681–1689

    Article  CAS  Google Scholar 

  • Tokuda G, Miyagi M, Makiya H, Watanabe H, Arakawa G (2009) Digestive β-glucosidases from the wood-feeding higher termite, Nasutitermes takasagoensis: intestinal distribution, molecular characterization, and alteration in sites of expression. Insect Biochem Mol Biol 39:931–937

    Article  CAS  Google Scholar 

  • Watanabe H, Tokuda G (2001) Animal cellulases. Cell Mol Life Sci 58:1167–1178

    Article  CAS  Google Scholar 

  • Watanabe H, Tokuda G (2010) Cellulolytic systems in insects. Annu Rev Entomol 55:609–632

    Article  CAS  Google Scholar 

  • Wright RM, Yablonsky MD, Shalita ZP, Goyal AK, Eveleigh DE (1992) Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable β-glucosidase expressed in Escherichia coli. Appl Environ Microbiol 58:3455–3465

    CAS  Google Scholar 

  • Yan TR, Lin CL (1997) Purification and characterization of a glucose-tolerant β-glucosidase from Aspergillus niger CCRC 31494. Biosci Biotechnol Biochem 61:965–970

    Article  CAS  Google Scholar 

  • Yang S, Jiang Z, Yan Q, Zhu H (2008) Characterization of a thermostable extracellular β-glucosidase with activities of exoglucanase and transglycosylation from Paecilomyces thermophila. J Agric Food Chem 56:602–608

    Article  CAS  Google Scholar 

  • Yoon JJ, Kim KY, Cha CJ (2008) Purification and characterization of thermostable β-glucosidase from the brown-rot basidiomycete Fomitopsis palustris grown on microcrystalline cellulose. J Microbiol 46:51–55

    Article  CAS  Google Scholar 

  • Zanoelo FF, Polizeli ML, Terenzi HF, Jorge JA (2004) β-Glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose. FEMS Microbiol Lett 240:137–143

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported in part by a grant from Bio-oriented Technology Research Advancement Institution (BRAIN), Japan. We thank the Ministry of Education, Culture, Sports, Science, and Technology of Japan for the scholarship received by C.A.U. The authors are also thankful to the anonymous reviewers for their constructive comments, which helped us to improve the manuscript.

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Authors and Affiliations

  1. Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Cristiane Akemi Uchima, Katsuhiko Kitamoto & Manabu Arioka

  2. Center of Molecular Biosciences, University of the Ryukyus, Nishihara, Okinawa, 903-0213, Japan

    Gaku Tokuda

  3. National Institute of Agrobiological Science, Owashi, Tsukuba, Ibaraki, 305-8634, Japan

    Hirofumi Watanabe

Authors
  1. Cristiane Akemi Uchima
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  2. Gaku Tokuda
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  3. Hirofumi Watanabe
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  4. Katsuhiko Kitamoto
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  5. Manabu Arioka
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Corresponding author

Correspondence to Manabu Arioka.

Electronic supplementary material

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Online Resource 1

Heterologous protein production in a time course analyses (DOC 666 kb)

Online Resource 2

TLC analysis from cello-oligosaccharides hydrolyzed by the purified G1NkBG (DOC 256 kb)

Online Resource 3

Comparative table between the properties of β-glucosidases from different microorganisms (DOC 45 kb)

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Uchima, C.A., Tokuda, G., Watanabe, H. et al. Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae . Appl Microbiol Biotechnol 89, 1761–1771 (2011). https://doi.org/10.1007/s00253-010-2963-y

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  • DOI: https://doi.org/10.1007/s00253-010-2963-y

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