Abstract
A gene encoding a putative β-glucosidase was isolated from Thermoascus aurantiacus IFO9748 and designated as bgl2. The recombinant enzyme showed β-glucosidase activity when p-nitrophenyl-β-glucose (pNP-Glc) was used as substrate. We also found that the enzyme activity was increased in the presence of organic solvents. An addition of 20 % (v/v) 1-octanol resulted in 54-fold higher activity of pNP-Glc hydrolysis, and transglycosylation activity was also found to be activated. The results of tryptophan fluorescence spectral analysis revealed the changes in the tertiary structure of the enzyme in the presence of 1-hexanol that may cause increased enzyme activity. BGLII has a distinctive hydrophobic linker region between N- and C-terminal domains. A chimeric enzyme in which the linker region was substituted by the corresponding region of another β-glucosidase failed to be activated by organic solvents, suggesting that the hydrophobic linker region may act as a molecular switch in BGLII.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Explore related subjects
Discover the latest articles and news from researchers in related subjects, suggested using machine learning.References
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
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal Biochem 162:156–159
Christakopoulos P, Goodenough PW, Kekos D, Macris BJ, Claeyssens M, Bhat MK (1994) Purification and characterisation of an extracellular beta-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum. Eur J Biochem 224:379
Dan S, Marton I, Dekel M, Bravdo BA, He S, Withers SG, Shoseyov O (2000) Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase. J Biol Chem 275:4973–4980
Decker CH, Visser J, Schreier P (2001) beta-Glucosidase multiplicity from Aspergillus tubingensis CBS 643.92: purification and characterization of four beta-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition and acid tolerance. Appl Microbiol Biotechnol 55:157–163
Gomes I, Gomes J, Gomes DJ, Steiner W (2000) Simultaneous production of high activities of thermostable endoglucanase and beta-glucosidase by the wild thermophilic fungus Thermoascus aurantiacus. Appl Microbiol Biotechnol 53:461–468
Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263–269
Gueguen Y, Chemardin P, Arnaud A, Galzy P (1995) Purification and characterization of an intracellular beta-glucosidase from Botrytis cinerea. Enzyme Microb Technol 17:900–906
Gunata Z, Vallier MJ (1999) Production of a highly glucose-tolerant extracellular beta-glucosidase by three Aspergillus strains. Biotechnol Lett 21:219–223
Harvey AJ, Hrmova M, De Gori R, Varghese JN, Fincher GB (2000) Comparative modeling of the three-dimensional structures of family 3 glycoside hydrolases. Proteins 41:257–269
Henrissat B, Claeyssens M, Tomme P, Lemesle L, Mornon JP (1989) Cellulase families revealed by hydrophobic cluster-analysis. Gene 81:83–95
Kawamori M, Takayama K, Takasawa S (1987) Production of cellulases by a thermophilic fungus, Thermoascus aurantiacus a-131. Agric Biol Chem 51:647–654
Laemmli UK (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Millqvist-Fureby A, Gao C, Vulfson EN (1998) Regioselective synthesis of ethoxylated glycoside esters using beta-glucosidase in supersaturated solutions and lipases in organic solvents. Biotechnol Bioeng 59:747–753
Ogino H, Ishikawa H (2001) Enzymes which are stable in the presence of organic solvents. J Biosci Bioeng 91:109–116
Ozaki H, Yamada K (1991) Isolation of Streptomyces sp producing glucose-tolerant beta-gucosidases and properties of the enzymes. Agric Biol Chem 55:979–987
Parry NJ, Beever DE, Owen E, Vandenberghe I, Van Beeumen J, Bhat MK (2001) Biochemical characterization and mechanism of action of a thermostable beta-glucosidase purified from Thermoascus aurantiacus. Biochem J 353:117–127
Perezpons JA, Rebordosa X, Querol E (1995) Properties of a novel glucose-enhanced beta-glucosidase purified from Streptomyces sp (Atcc-11238). Biochim Biophys Acta 1251:145–153
Riou C, Salmon JM, Vallier MJ, Gunata Z, Barre P (1998) Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae. Appl Environ Microbiol 64:3607–3614
Saha BC, Bothast RJ (1996) Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata. Appl Environ Microbiol 62:3165–3170
Simon LM, Kotorman M, Garab G, Laczko I (2001) Structure and activity of alpha-chymotrypsin and trypsin in aqueous organic media. Biochem Biophys Res Commun 280:1367–1371
Tanaka A, Kawamoto T (1991) Immobilized enzymes in organic solvents. Bioprocess Technol 14:183–208
Varghese JN, Hrmova M, Fincher GB (1999) Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure 7:179–190
Wallecha A, Mishra S (2003) Purification and characterization of two beta-glucosidases from a thermo-tolerant yeast Pichia etchellsii. Biochim Biophys Acta 1649:74–84
Wehbi H, Feng J, Roberts MF (2003) Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity. Biochim Biophys Acta 1613:15–27
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 beta-glucosidase expressed in Escherichia coli. Appl Environ Microbiol 58:3455–3465
Yan TR, Lin CL (1997) Purification and characterization of a glucose-tolerant beta-glucosidase from Aspergillus niger CCRC 31494. Biosci Biotechnol Biochem 61:965–970
Ying L, Kitaoka M, Hayashi K (2004) Effects of truncation at the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens. Biosci Biotechnol Biochem 68:1113–1118
Acknowledgements
We thank Kenji Yamamoto, Graduate School of Biostudies, Bunzo Mikami and Noboyuki Takahashi, Graduate School of Agriculture, Kyoto University, for their invaluable suggestions. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan, the New Energy and Industrial Technology Development Organization, Japan Society for the Promotion of Science, and the Research Institute of Innovative Technology for the Earth.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hong, J., Tamaki, H. & Kumagai, H. Unusual hydrophobic linker region of β-glucosidase (BGLII) from Thermoascus aurantiacus is required for hyper-activation by organic solvents. Appl Microbiol Biotechnol 73, 80–88 (2006). https://doi.org/10.1007/s00253-006-0428-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-006-0428-0