Abstract
A potential fungal strain producing extracellular β-glucosidase enzyme was isolated from sea water and identified as Aspergillus sydowii BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of Aspergillus sydowii in the GenBank. A sequential optimization strategy was used to enhance the production of β-glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence β-glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for β-glucosidase production. The enzyme was purified by (NH4)2SO4 precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ∼95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50°C. It showed high affinity towards pNPG and enzyme has a K m and V max of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a K i of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of Saccharomyces cerevisiae in presence of cellulase and the purified β-glucosidase of Aspergilus sydowii BTMFS 55.
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Sandgrena, M., J. Stahlbergb, and C. Mitchinson (2005) Structural and biochemical studies of GH family 12 cellulases: improved thermal stability and ligand complexes. Prog. Biophy. Mol. Biol. 89: 246–291.
Riou, C., J. M. Salmon, M. J. Vallier, Z. Gunata, and P. Barre (1998) Purification, characterization, and substrate specificity of a novel highly glucose-tolerant β-Gluco sidase from Aspergillus oryzae. Appl. Environ. Microbol. 64: 3607–3614.
Jorgensen, H., A. Morkeberg, K. B. R. Krogh, and L. Olsson (2005) Production of cellulases and hemicellulases by three Penicillium species: effect of substrate and evaluation of cellulase adsorption by capillary electrophoresis. Enz. Microb. Technol. 36: 42–48.
Chandrasekaran, M (1996) Harnessing marine microorganisms through solid state fermentation. J. Scientific. Ind. Res. 55: 468–471.
Park, T. H., K. W. Choi, C. S. Park, S. B. Lee, H. Y. Kang, K. J. Shon, J. S. Park, and J. Cha (2005) Substrate specificity and transglycosylation catalyzed by a thermostable β-glucosidase from marine hyperthermophile Thermotoga neapolitana. Appl. Microbiol. Biotechnol. 4: 411–422.
Fischer, L., R. Bromann, S. W. M. Kengen, W. M. dVos, and F. Wagner (1996) Catalytical potency of β-Glucosidase from the extremophile Pyrococcus furiosus in glucoconjugate synthesis. Biotechnology 14: 88–91.
Swiatek, G., J. D. Bok, and D. A. Yernool (1996) Cloning and sequence analysis of a novel β-glucan-glucohydrolase: A gene from Thermotoga neapolitana. Abstracts of the 96th General Meeting of the American Society for Microbiology. May 19–23. Louisiana, USA.
O’Donnell, K. (1993) Fusarium and its near relatives. pp. 225–233. In: D. R. Reynolds and J. W. Taylor (eds.). The fungal holomorph: mitotics, meiotic, and pleomorphic speciation in fungal systamatics. CAB International, Oxon, UK.
Endo, K., Y. Hakamada, S. Takizawa, H. Kubota, N. Sumitomo, T. Kobayashi, and S. Ito (2001) A novel alkaline endoglucanase from an alkaliphilic Bacillus isolate: enzymatic properties, and nucleotide and deduced amino acid sequences. Appl. Microbiol. Biotechnol. 57: 109–116.
Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265–275.
Plackett, R. L. and J. P. Burman (1946) The design of optimum multifactorial experiments. Biometrica 33: 305–325.
Englard, S. and S. Seifter (1990) Precipitation techniques. Meth. Enzymol. 182: 285–300.
Laemmlli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Parry, N. J., D. E. Beever, E. Owen, I. Vandenberghe, J. Van Beeumen, and M. K. Bhat (2001) Biochemical characterization and mechanism of action of a thermostable β-glucosidase purified from Thermoascus aurantiacus. Biochem. J. 353: 117–127.
Lineweaver, H. and D. Burk (1934) The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56: 658–666.
Dixon, M. (1953) The determination of enzyme inhibitor constants. Biochem. J. 55: 170–171.
Miller, G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426–428.
Sugita, T. and A. Nishikawa (2003) Fungal identification method based on DNA sequence analysis: reassesment of the methods of the pharmaceutical society of japan and the japanese pharmacopoeia. J. Health Sci. 49: 531–533.
Xu, C. P. and J. W. Yun (2003) Optimization of submerged culture conditions for mycelial growth and exobiopolymer production by Auricularia polytricha (wood ears fungus) using the methods of uniform design and regression analysis. Biotechnol. Appl. Biochem. 38: 193–199.
Haaland, P. D. (1989) Experimental design in biotechnology. pp. 61–83. Marcel Dekker, NY, USA.
Woodward, J. and A. Wiseman (1982) Fungal and other β-glucosidases-their properties and applications. Enz. Microb. Technol. 4: 73–79.
Witte, K. and A. Wartenberg (1989) Purification and properties of two β-glucosidases isolated from Aspergillus niger. Acta. Biotechnol. 9: 179–190.
Galas, E. and I. Romanowska (1997) Purification and some properties of β-glucosidase from Aspergillus niger IBT-90. Acta. Microbiol. Pol. 46: 241–252.
Yan, T. R. and C. L. Lin (1997) Purification and characterization of a glucose-tolerant β-glucosidase from Aspergillus niger CCRC 31494. Biosci. Biotechnol. Biochem. 61: 965–970.
Kwon, K. S., H. G. Kang, and Y. C. Hah (1992) Purification and characterization of two extracellular β-glucosidases from Aspergillus nidulans. FEMS Microbiol. Lett. 97: 149–153.
Saha, B. C., and R. J. Bothast (1996) Production, purification, and characterization of a highly glucose-tolerant novel β-glucosidase from Candida peltata. Appl. Environ. Biotechnol. 62: 3165–3170.
Amouri, B. and A. Gargouri (2006) Characterization of a novel β-glucosidase from a Stachybotrys strain. Biochem. Eng. J. 32: 191–197.
Li, X., J. Pei, G. Wu, and W. Shao (2005) Expression, purification, and characterization of a recombinant β-glucosidase from Volvariella volvacea. Biotech. Lett. 27: 1369–1373.
Cascalheira, J. F. and J. A. Queiroz (1999) Kinetic study of the cellobiase activity of Trichoderma reesei cellulase complex at high substrate concentrations. Biotech. Lett. 21: 651–655.
Zhang, C., D. Li, H. Yu, B. Zhang, and F. Jin (2007) Purification and characterization of piceid-β-D-glucosidase from Aspergillus oryzae. Proc. Biochem. 42: 83–88.
Yazdi, M. T., A. A. Khosravi, M. Nemati, and N. D. V. Motlagh (2003) Purification and characterization of two intracellular β-glucosidases from the Neurospora crassa mutant cell-1. W. J. Microbiol. Biotechnol. 19: 79–84.
Ximenes, E. A., C. R. Felix, and C. J. Ulhoa (1996) Production of cellulases by Aspergillus fumigatus and characterization of one β-glucosidase. Curr. Microbiol. 32: 119–123.
Decker, C. H., J. Visser, and P. Schreier (2001) β-Glucosidase multiplicity from Aspergillus tubingensis CBS 643.92: purification and characterization of four β-glucosidases and their differentiation with respect to substrate specificity, glucose inhibition, and acid tolerance. Appl. Microbiol. Biotechnol. 55: 157–163
Christakopoulos, P., P. W. Goodenough, D. Kekos, B. J. Macris, M. Claeyssens, and M. K. Bhat (1994) Purification and characterization of an extracellular β-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum. Eur. J. Biochem. 224: 379–385.
Plant, A. R., J. E. Oliver, M. L. Patchett, R. M. Daniel, and H. W. Morgan (1988) Stability and substrate specificity of a β-glucosidase from the thermophilic bacterium Tp8 cloned into Escherichia coli. Arch. Biochem. Biophys. 262: 181–188.
Nakkharat, P. and D. Haltrich (2006) Purification and characterisation of an intracellular enzyme with β-glucosidase and β-galactosidase activity from the thermophilic fungus Talaromyces thermophilus CBS 236.58. J. Biotech. 123: 304–313
Gueguen, Y., P. Chemardin, A. Arnaud, and P. Galzy (1995) Purification and characterization of an intracellular β-glucosidases from Botrytis cinerea. Enz. Microb. Technol. 17: 900–906.
Pitson, S. M., R. J. Seviour, and B. M. McDougall (1997) Purification and characterization of an extracellular β-glucosidase from the filamentous fungus Acremonium persicinum and its probable role in β-glucan degradation. Enz. Microb. Technol. 21: 182–190
Lin, J., B. Pillay, and S. Singh (1999) Purification and biochemical characteristics of β-D-glucosidase from a thermophilic fungus, Thermomyces lanuginosus-SSBP. Biotechnol. Appl. Biochem. 30: 81–87.
Lymer, E. S. and V. Renganathan (1995) Purification and characterization of a cellulose-binding β-gluco-sidase from cellulose-degrading cultures of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 61: 2976–2980.
Ozaki, H. and K. Yamada (1992) Isolation of Streptomyces sp. producing glucose-tolerant β-glucosidases and properties of the enzymes. Agric. Biol. Chem. 55: 979–987.
Kengen, S. W. M., E. J. Luesink, A. J. M. Stams, and A. J. B. Zehnder (1993) Purification and characterization of an extremely thermostable β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur. J. Biochem. 213: 305–312.
Saha, B. C., L. B. Iten, M. A. Cotta, and Y. V. Wu (2005) Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol. Proc. Biochem. 40: 3693–3700.
Tewari, H. K., S. S. Marwaha, J. F. Kennedy, and L. Singh (1987) Acid and enzymatic saccharification of agricultural mixed polymers for alcohol production. British Polym. J. 19: 425–428.
Ballesteros, M., J. M. Oliva, M. J. Negro, P. Manzanares, and I. Ballesteros (2004) Ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (SFS) with Kluyveromyces marxianus CECT 10875. Proc. Biochem. 39: 1843–1848.
Sharma, S. K., K. L. Kalra, and G. S. Kocher (2004) Fermentation of enzymatic hydrolysate of sunflower hulls for ethanol production and its scale-up. Biomass Bioenerg. 27: 399–402.
Wang, L. S., X. Y. Ge, and W. G. Zhang (2007) Improvement of ethanol yield from raw corn flour by Rhizopus sp. W. J. Microbiol. Biotechnol. 23: 461–465.
Hari Krishna, S., K. Prasanthi, G. V. Chowdary, and C. Ayyanna (1998) Simultaneous saccharification fermentation of pretreated sugar cane leaves to ethanol. Proc. Biochem. 33: 825–830.
Adrados, B. P., M. Galbe, and G. Zacchi (2005) Pretreatment of barley husk for bioethanol production. J. Chem. Technol. Biotechnol. 80: 85–91.
Nigam, J. N. (2007) Bioconversion of water-hyacinth hemicellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting yeast. J. Biotech. 97: 107–116.
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Madhu, K.M., Beena, P.S. & Chandrasekaran, M. Extracellular β-glucosidase production by a marine Aspergillus sydowii BTMFS 55 under solid state fermentation using statistical experimental design. Biotechnol Bioproc E 14, 457–466 (2009). https://doi.org/10.1007/s12257-008-0116-2
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DOI: https://doi.org/10.1007/s12257-008-0116-2