Abstract
An open reading frame (ORF) encoding the enzyme β-glucosidase from the extremely thermophilic bacterium Fervidobacterium islandicum has been identified, cloned and sequenced. The bgl1A gene was cloned in a pET-Blue1 vector and transformed in Escherichia coli, resulting in high-level expression of β-glucosidase FiBgl1A that was purified to homogeneity in a two-step purification. FiBgl1A is composed of 459 amino acid residues and showed high homology to glycoside hydrolase family 1 proteins. It exhibited highest activity towards p-nitrophenyl-β-d-glucopyranoside with an optimum activity at pH 6.0 and 7.0 and at 90 °C. The enzyme is resistant to glucose inhibition. Furthermore, it did not require divalent cations for activity, nor was it affected by the addition of p-chloromercuribenzoate (10 mM), EDTA (10 mM), urea (10 mM) or dithiothreitol (10 mM). Addition of surfactants (with the exception of SDS) and a number of solvents enhanced the activity of FiBgl1A. It also displayed remarkable activity across a broad temperature range (80–100 °C). The thermoactivity and thermostability of FiBgl1A and its resistance to denaturing and reducing agents make this enzyme a potential candidate for industrial applications.
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References
Ait N, Creuzet N, Cattaneo J (1979) Characterization and purification of a thermostable β-glucosidase from Clostridium thermocellum. Biochem Biophys Res Commun 90:537–546
Altschul S, Gish W, Miller W, Myers E, Lipman D (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL Workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201
Barrett T, Suresh CG, Tolley SP, Dodson EJ, Hughes MA (1995) The crystal structure of a cyanogenic β-glucosidase from white clover, a family 1 glycosyl hydrolase. Structure 3:951–960
Béguin P (1990) Molecular biology of cellulose degradation. Annu Rev Microbiol 44:219–248
Bowers E, Ragland L, Byers L (2007) Salt effects on β-glucosidase: pH-profile narrowing. Biochim Biophys Acta 1774:1500–1507
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
Branden C (1991) The TIM barrel—the most frequently occurring folding motif in proteins. Curr Opin Struct Biol 1:978–983
Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971
Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker J (ed) The proteomics protocols handbook. Humana, Totowa
Gloster TM, Roberts S, Ducros VM, Perugino G, Rossi M, Hoos R, Moracci M, Vasella A, Davies GJ (2004) Structural studies of the beta-glycosidase from Sulfolobus solfataricus in complex with covalently and noncovalently bound inhibitors. Biochemistry 43:6101–6109
Gödde C, Sahm K, Brouns S, Kluskens L, van der Oost J, de Vos W, Antranikian G (2005) Cloning and expression of Islandin, a new thermostable subtilisin from Fervidobacterium islandicum, in Escherichia coli. Appl Environ Microbiol 71:3951–3958
Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon J, Davies G (1995) Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci USA 92:7090–7094
Hong J, Ladisch M, Gong C, Wankat P, Tsao G (1981) Combined product and substrate inhibition equation for cellobiase. Biotechnol Bioeng 23:2779–2788
Huber R, Woese C, Langworthy T, Kristjansson J, Stetter K (1990) Fervidobacterium islandicum sp. nov., a new extremely thermophilic eubacterium belonging to the "Thermotogales". Arch Microbiol 154:105–111
Jenkins J, Lo Leggio L, Harris G, Pickersgill R (1995) β-Glucosidase, β-galactosidase, family A cellulases, family F xylanases and two barley glycanases from a superfamily of enzymes with 8-fold β/α architecture and with two conserved glutamates near the carboxy-terminal ends of β-strands four and seven. FEBS Lett 362:281–285
Jones D (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202
Kengen S, Luesink E, Stams A, Zehnder A (1993) Purification and characterization of an extremely thermostable β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur J Biochem 213:305–312
Klippel B, and Antranikian G (2011) Lignocellulose converting enzymes from thermophiles. In Horikoshi K (ed) Extremophiles handbook. Springer, New York, pp 444–474
Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinform 23:2947–2948
Li X, Bhaskar R, Yang H, Wang D, Miao Y (2009) Screening and identification of new isolate: thermostable Escherichia coli with novel thermoalkalotolerant cellulases. Curr Microbiol 59:393–399
MacLellan J (2010) Strategies to enhance enzymatic hydrolysis of cellulose in lignocellulosic biomass. MMG 445 Basic Biotech 6:31–35
Moracci M, Capalbo L, Ciaramella M, Rossi M (1996) Identification of two glutamic acid residues essential for catalysis in the β-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus. Protein Eng 9:1191–1195
Noh K, Oh DK (2009) Production of the rare ginsenosides compound K, compound Y and compound Mc by a thermostable β-glycosidase from Sulfolobus acidocaldarius. Biol Pharm Bull 32:1830–1835
Ozaki H, Yamada K (1991) Isolation of Streptomyces sp. producing glucose-tolerant beta-glucosidases and properties of the enzymes. Agric Biol Chem 55:979–987
Patchett M, Daniel R, Morgan H (1987) Purification and properties of a stable β-glucosidase from an extremely thermophilic anaerobic bacterium. Biochem J 243:779–787
Perez-Pons JA, Robordosa X, Querol E (1995) Properties of a novel glucose enhanced β-glucosidase purified from Streptomyces sp. ATCC11238. Biochim Biophys Acta 1251:145–153
Riou C, Salmon J, Vallierl M, 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
Romaniec M, Huskisson N, Barker P, Demain A (1993) Purification and properties of the Clostridium thermocellum bgl gene product expressed in Escherichia coli. Enzyme Microb Technol 15:393–400
Ryu D, Mandels M (1980) Cellulases: biosynthesis and applications. Enzyme Microb Technol 2:91–102
Saha B, Bothast R (1996) Production, purification and characterization of a highly glucose-tolerant novel β-glucosidase from Candida peltata. Appl Environ Microbiol 62:3165–3170
Singh A, Hayashi K (1995) Construction of chimeric β-glucosidases with improved enzymatic properties. J Biol Chem 270:21928–21933
Trimbur D, Warren R, Withers S (1992) Region-directed mutagenesis of residues surrounding the active site nucleophile in β-glucosidase from Agrobacterium faecalis. J Biol Chem 267:10248–10251
Tull D, Withers S, Gilkes N, Kilburn D, Warren R, Aebersold R (1991) Glutamic acid 274 is the nucleophile in the active site of a “retaining” exoglucanase from Cellulomonas fimi. J Biol Chem 266:15621–15625
Tykarska E, Lebioda L, Marchut E, Steczko J, Stec B (1990) Crystallization of alcohol oxidase from Pichia pastoris. Secondary structure predictions indicate a domain with the eightfold beta/alpha-barrel fold. J Protein Chem 9:83–86
Vallmitjana M, Ferrer-Navarro M, Planell R, Abel M, Ausin C, Querol E, Planas A, Pérez-Pons JA (2001) Mechanism of the family 1 β-glucosidase from Streptomyces sp: catalytic residues and kinetic studies. Biochem 40:5975–5982
Voorhorst W, Eggen R, Luesink E, De Vos W (1995) Characterization of the celB gene coding for β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli. J Bacteriol 177:7105–7111
Wang Q, Trimbur D, Graham R, Warren R, Withers S (1995) Identification of the acid/base catalyst in Agrobacterium faecalis beta-glucosidase by kinetic analysis of mutants. Biochem 34:14554–14562
Wierenga R (2001) The TIM-barrel fold: a versatile framework for efficient enzymes. FEBS Lett 492:193–198
Wood T (1985) Properties of cellulolytic enzyme systems. Biochem Soc Trans 13:407–410
Wood T, McCrae S, Bhat K (1989) The mechanism of fungal cellulase action: synergism between components of Penicillum pinophilum cellulase in solubilizing hydrogen bond ordered cellulose. Biochem J 260:37–43
Wright R, Yablonsky M, Shalita Z, Goyal A, Eveleigh D (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
Xin Z, Yinbo Q, Peiji G (1993) Acceleration of ethanol production from paper mill waster fiber by supplementation with β-glucosidases. Enzyme Microb Technol 15:62–65
Yan T, Lin C (1997) Purification and characterization of a glucose-tolerant β-glucosidase from Aspergillus niger CCRC 31494. Biosci Biotechnol Biochem 61:965–970
Zheng Y, Pan Z, Zhang R, Wang D, Jenkins B (2008) Non-ionic surfactants and non-catalytic protein treatment on enzymatic hydrolysis of pretreated creeping wild ryegrass. Appl Biochem Biotechnol 146:231–248
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D. J. received a scholarship from DAAD (Deutscher Akademischer Austausch Dienst).
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Jabbour, D., Klippel, B. & Antranikian, G. A novel thermostable and glucose-tolerant β-glucosidase from Fervidobacterium islandicum . Appl Microbiol Biotechnol 93, 1947–1956 (2012). https://doi.org/10.1007/s00253-011-3406-0
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DOI: https://doi.org/10.1007/s00253-011-3406-0