Abstract
The filamentous fungus Stachybotrys microspora possess a rich β-glucosidase system composed of five β-glucosidases. Three of them were already purified to homogeneity and characterized. In order to isolate the β-glucosidase genes from S. microspora and study their regulation, a PCR strategy using consensus primers was used as a first step. This approach enabled the isolation of three different fragments of family 3 β-glucosidase gene. A representative genomic library was constructed and probed with one amplified fragment gene belonging to family 3 of β-glucosidase. After two rounds of hybridization, seven clones were obtained and the analysis of DNA plasmids leads to the isolation of one clone (CF3) with the largest insert of 7 kb. The regulatory region shows multiple TC-rich elements characteristic of constitutive promoter, explaining the expression of this gene under glucose condition, as shown by zymogram and RT-PCR analysis. The tertiary structure of the deduced amino acid sequence of Smbgl3 was predicted and has shown three conserved domains: an (α/β)8 triose phosphate isomerase (TIM) barrel, (α/β)5 sandwich, and fibronectin type III domain involved in protein thermostability. Zymogram analysis highlighted such thermostable character of this novel β-glucosidase.
Similar content being viewed by others
References
Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarity. Biochemical Journal, 280, 309–316.
Opassiri, R., Pomthong, B., Akiyama, T., Nakphaichit, M., Onkoksoong, T., Ketudat-Cairns, M., et al. (2007). A stress-induced rice b-glucosidase represents a new subfamily of glycosyl hydrolase family 5 containing a fascin-like domain. Biochemical Journal, 408, 241–249.
Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V., & Henrissat, B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Research, 37, D233–D238.
Rye, C. S., & Withers, S. G. (2000). Glycosidase mechanisms. Current Opinion in Chemical Biology, 4, 573–580.
Varghese, J. N., Hrmova, M., & Fincher, G. B. (1999). Three-dimensional structure of a barley β-d-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure, 7, 179–190.
Tomme, P., Warren, R. A., & Gilkes, N. R. (1995). Cellulose hydrolysis by bacteria and fungi. Advances in Microbial Physiology, 37, 1–81.
Spezio, M., Wilson, D. D., & Karplus, P. A. (1993). Crystal structure of the catalytic domain of a thermophilic endocellulase. Biochemistry, 32, 9906–9916.
Kleywegt, G., Zou, J. Y., Divine, C., Davies, G. J., Sinning, L., Stohlberg, J., et al. (1997). The crystal structures of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 Å resolution, and a comparison with related enzymes. Journal of Molecular Biology, 272, 383–397.
Saloheimo, A., Aro, N., Ilmén, M., & Pentilla, M. (2000). Isolation of the ace1 gene encoding a cys2-His2 transcription factor involved in regulation of activity of the cellulase promotor cbh1 of Trichoderma reesei. Journal of Biological Chemistry, 275, 5817–5825.
Zeilinger, S., Ebner, A., Marosits, T., Mach, R., & Kubicek, C. P. (2001). The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element. Molecular Genetics and Genomics, 266(1), 56–63.
IImem, M., Klemsdal, S., & Pentilla, M. (1996). Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei. Molecular Genetics and Genomics, 253, 303–314.
Esen, A. (1993). β-Glucosidase overview. In A. Esen (Ed.), β-Glucosidases: biochemistry and molecular biology (pp. 1–14). ACS Symposium Series 533. Washington, DC: American Chemical Society.
Gargouri, M., Smaali, I., Maugard, T., Legoy, M. D., & Marzouki, N. (2004). Fungus β-glycosidases. Immobilization and use in alkyl-β-glycoside synthesis. Journal of Molecular Catalysis B: Enzymatic, 29, 89–94.
Bhat, M. K. (2000). Cellulases and related enzymes in biotechnology. Biotechnology Advances, 18, 355–383.
Amouri, B., & Gargouri, A. (2006). Characterization of a novel β-glucosidase from a Stachybotrys strain. Biochemical Engineering Journal, 32, 191–197.
Saibi, W., Amouri, B., & Gargouri, A. (2007). Purification and biochemical characterization of a transglucosilating β-glucosidase of Stachybotrys strain. Applied Microbiology and Biotechnology, 77, 293–300.
Saibi, W., Abdeljalil, S., & Gargouri, A. (2011). Carbon source directs the differential expression of β-glucosidases in Stachybotrys microspora. World Journal of Microbiology & Biotechnology, 27, 1765–1774.
Mandels, M., & Weber, J. (1969). The production of cellulases. In G. D. Hajny & E. T. Reese (Eds.), Cellulases and their applications (pp. 391–414). Washington, DC: ACS.
Trigui-Lahiani, H., & Gargouri, A. (2007). Cloning, genomic organization and mRNA expression of a pectin lyase gene from a mutant strain of Penicillium occitanis. Gene, 388, 54–60.
Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press.
Matys, V., Kel-Margoulis, O. V., Fricke, E., Liebich, I., Land, S., Barre-Dirrie, A., et al. (2006). TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Research, 34, D108–D110.
Nielsen, H., Engelbrecht, J., Brunak, S., & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering, 10, 1–6.
Petersen, T. N., Brunak, S., von Heijne, G., & Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature Methods, 8, 785–786.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R., et al. (2005). Protein identification and analysis tools on the ExPASy server. In J. M. Walker (Ed.), The proteomic protocols handbook (pp. 571–607). Totowa, NJ: Humana Press.
Hopwood, D. A., Bibb, M. J., Chater, K. F., et al. (1985). Genetic manipulation of streptomyces: a laboratory manual (pp. 152–153). Norwich: John Innes Foundation.
Laemmli, U. K., & Favre, M. (1973). Maturation of the head of bacteriophage T4. I. DNA packaging events. Journal of Molecular Biology, 80, 575–599.
Arnold, K., Bordoil, L., Kopp, J., & Schwede, T. (2005). The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics, 22, 195–201.
Pozzo, T., Linares-Pasten, J., Nordberg Karlsson, E., & Logan, D. T. (2010). Structural and functional analyses of β-glucosidase 3B from Thermotoga neapolitana: a thermostable three-domain representative of glycoside hydrolase 3. Journal of Molecular Biology, 397, 724–739.
Guex, N., & Peitsch, M. C. (1997). SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis, 18, 2714–2723.
Yuen, K. Y., Pascal, G., Wong, S. S. Y., Glaser, P., Woo, P. C. Y., Kunst, F., et al. (2003). Exploring the Penicillium marneffei genome. Archives of Microbiology, 179(5), 339–353.
Kupfer, D. M., Drabenstot, S. D., Buchanan, K. L., Lai, H., Zhu, H., Dyer, D. W., et al. (2004). Introns and splicing elements of five diverse fungi. Eukaryotic Cell, 3, 1088–1100.
Kubicek, C. P., & Penttilä, M. E. (1998). Regulation of production of plant polysaccharide degrading enzymes by Trichoderma and Gliocladium. In G. E. Harman & C. P. Kubicek (Eds.), Enzymes, biological control and commercial applications (Vol. 2, pp. 49–72). London: Taylor and Francis Ltd.
Iyer, V., & Struhl, K. (1995). Mechanism of differential utilization of the his3 TR and TC TATA elements. Molecular and Cellular Biology, 15, 7059–7066.
Huisinga, K. L., & Pugh, B. F. (2004). A genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. Molecular Cell, 13, 573–585.
Martinez, E., Zhou, Q., L’Etoile, N. D., Oelgeschläger, T., Berk, A. J., & Roeder, R. G. (1995). Core promoter-specific function of a mutant transcription factor TFIID defective in TATA box binding. In Proceedings of the National Academy of Sciences, Washington, DC, vol. 92, pp. 11864–11868.
Sandelin, A., Carninci, P., Lenhard, B., Ponjavic, J., Hayashizaki, Y., & Hume, D. A. (2007). Mammalian RNA polymerase II core promoters: insights from genome-wide studies. Nature Reviews Genetics, 8, 424–436.
Bernard, V., Brunaud, V., & Lecharny, A. (2010). TC-motifs at the TATA-box expected position in plant genes: a novel class of motifs involved in the transcription regulation. BMC Genomics, 11, 166.
Broda, P., Birch, P. R. J., Brooks, P. R., & Sims, P. F. G. (1995). PCR mediated analysis of lignocellulolytic gene transcription by Phanerochaete chrysosporium: substrate-dependent differential expression within gene families. Applied Environmental Microbiology, 61, 2358–2364.
Tempelaars, C. A., Birch, P. R., Sims, P. F., & Broda, P. (1994). Isolation, characterization, and analysis of the expression of the cbhII gene of Phanerochaete chrysosporium. Applied Environmental Microbiology, 60, 4387–4393.
Workman, W. E., & Day, D. (1982). Purification and properties of beta-glucosidase from Aspergillus terreus. Applied and Environmental Microbiology, 44, 1289–1295.
Watanabe, T., Sato, T., Yoshioka, S., Koshijima, T., & Kuwahara, M. (1992). Purification and properties of Aspergillus niger β-glucosidase. European Journal of Biochemistry, 209, 651–659.
Riou, C., Salmon, J. M., Vallier, M. J., Gunata, Z., & Barre, P. (1998). Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae. Applied and Environmental Microbiology, 64, 3607–3614.
Yan, T. R., & Lin, C. L. (1997). Purification and characterization of a glucose tolerant β-glucosidase from Aspergillus niger CCRC 31494. Bioscience, Biotechnology, and Biochemistry, 61, 965–970.
Saha, B. C., & Bothast, R. J. (1996). Production, purification, and characterization of a highly glucose-tolerant novel β-glucosidase from Candida peltata. Applied and Environmental Microbioly, 62, 3165–3170.
Deshpande, V., Eriksson, K. E., & Pettersson, B. (1978). Production, purification and partial characterization of 1,4-β-glucosidase enzymes from Sporotrichum pulverulentum. European Journal of Biochemistry, 90, 191–198.
Smith, M. H., & Gold, M. H. (1979). Phanerochaete chrysosporium β-glucosidase: induction, cellular localization, and physical characterization. Applied and Environmental Microbiology, 37, 938–942.
Tsukada, T., Igarashi, K., Yoshida, M., & Samejima, M. (2006). Molecular cloning and characterization of two intracellular β-glucosidases belonging to glycoside hydrolase family 1 from the basidiomycete Phanerochaete chrysosporium. Applied Microbiology and Biotechnology, 73, 807–814.
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.
Hrmova, M., De Gori, R., Smith, B. J., Fairweather, J. K., Driguez, H., Varghese, J. N., et al. (2002). Structural basis for broad substrate specificity in higher plant β-d-glucan glucohydrolases. Plant Cell, 14, 1033–1052.
Hrmova, M., Streltsov, V. A., Smith, B. J., Vasella, A., Varghese, J. N., & Fincher, G. B. (2005). Structural rationale for low-nanomolar binding of transition state mimics to a family GH3 β-D-glucan glucohydrolase from barley. Biochemistry, 44, 16529–16539.
Bamford, V. A., Kolade, O. O., Osbourn, A. E., & Hemmings, A. M. (2004). Purification, crystallization and preliminary X-ray diffraction analysis of a fungal saponin-detoxifying enzyme. Acta Crystallographica Section D, 60, 1331–1333.
Yoshida, E., Hidaka, M., Fushinobu, S., Koyanagi, T., Minami, H., Tamaki, H., et al. (2010). Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus. Biochemical Journal, 431, 39–49.
Ingham, K. C., Brew, S. A., Broekelmann, T. J., & McDonald, J. A. (1984). Thermal stability of human plasma fibronectin and its constituent domains. The Journal of Biological Chemistry, 259(19), 11901–11907.
Acknowledgments
Fatma Abdeljalil is thanked for reviewing English language. Lamia Jmal-Hammami and Mosbeh Dardouri are thanked for their technical help. We thank Professors Raja Mokdad-Gargouri and Hafedh Belghith for fruitful discussion of scientific interpretations. This work was supported by Grants from the Ministry of Higher Education and Scientific Research, Tunisia.
Author information
Authors and Affiliations
Corresponding author
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Abdeljalil, S., Trigui-Lahiani, H., Lazzez, H. et al. Cloning, Molecular Characterization, and mRNA Expression of the Thermostable Family 3 β-Glucosidase from the Rare Fungus Stachybotrys microspora . Mol Biotechnol 54, 842–852 (2013). https://doi.org/10.1007/s12033-012-9633-5
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-012-9633-5