Abstract
A β-glucosidase gene bglI from Aspergillus niger NL-1 was cloned and expressed in Pichia pastoris. The bglI gene consists of a 2583 bp open reading frame encoding 861 amino acids; the enzyme was classified into glycoside hydrolases 3. To improve the expression level of recombinant BGL in P. pastoris, fermentation conditions were optimized by the single-factor experiments. The optimal fermentation conditions were obtained: initial pH 5.0, methanol concentration 0.5% added into the culture every 24 h, and initial cell density (OD600) of 10 for induction. The activity of BGL was increased from 4 U/mL to 45 U/mL in optimal conditions. The BGL was purified by ultrafiltration and (NH4)2SO4 precipitation showing a single band on SDS-PAGE. The optimal activity was at pH 4.0 and 60°C. The recombinant enzyme was stable over a pH range of 3.0–7.0 and retained more than 85% activity after incubation at 60°C for 30 min. The kinetic experiments revealed K m and V max for p-nitrophenyl-β-D-glucoside of 0.64 mM and 370 U/mg, for cellobiose 8.59 mM and 1480 U/mg. The activity of BGL was not or only a little affected by many metal ions and EDTA and was enhanced by methanol or n-butyl alcohol. The BGL had a K i of 48 mM for glucose and retained 76% activity in the presence of 50 mM glucose. The favorable properties of BGL offer the potential for industrial application.
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References
Bhat, M.K. and Bhat, S., Cellulose degrading enzyme and their potential industrial applications, Biotechnol. Adv., 1997, vol. 15, pp. 583–620.
Sun, Y. and Cheng, J., Hydrolysis of lignocellulosic materials for ethanol production: a review, Biores. Technol., 2002, vol. 83, pp. 1–11.
Neil, A.H., Charles, R.W., and Harvey, W.B., Enhanced cellulase production in fed-batch culture of Trichoderma reesei C30, Enzyme Microb. Technol., 1984, vol. 6, pp. 73–77.
Shin, H.J. and Yang, J.W., Galactooligosaccharide synthesis from lactose by Penicillium funiculosum cellulase, Biotechnol. Lett., 1996, vol. 18, pp. 143–144.
Sternberg, D., β-Glucosidase of Trichoderma: its biosynthesis and role in saccharification of cellulose, Appl. Environ. Microbiol., 1976, vol. 31, pp. 164–178.
Bayonove, C.L., Delcroix, A., Günata, Z., Sapis, J.C., and Salmon, J.M., Glycosidase activities of three enological yeast strains during wine making: effect on the terpenol content of muscat wine, Am. J. Enol. Vitic., 1994, vol. 45, pp. 291–296.
Engel, K.H. and Tressl, R., Formation of aroma components from nonvolatile precursors in passion fruit, J. Agric. Food Chem., 1983, vol. 31, pp. 998–1002.
Gueguen, Y., Chemardin, P., Janbon, G., Arnoud, A., and Galzy, P., A very efficient β-glucosidase catalyst for the hydrolysis of flavor precursors of wines and fruit juices, J. Agric. Food Chem., 1996, vol. 44, pp. 2336–2340.
Gunata, Y.Z., Bayonove, C.L., Baumes, R.L., and Cordonnier, R.E., The extraction and determination of free and glycosidically bound fractions of some grape aroma substances, J. Chromatogr., 1985, vol. 331. pp. 83–90.
Williams, P.J., Christopher, R.S., Bevan, W., and Massy-Westropp, R.A., Studies on the hydrolysis of vitis vinifera monoterpenne precursor compounds and model monoterpene β-D-glucosides rationalizing the monoterpene composition of grapes, J. Agric. Food Chem., 1982, vol. 30, pp. 1219–1223.
Wen, Z., Liao, W., and Chen, S., Production of cellulose/β-glucosidase by the mixed fungi culture and on dairy manure, Process Biochem., 2005, vol. 40, pp. 3087–3094.
Decker, C.H., Visser, J., and Schreier, P., 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., 2001, vol. 55, pp. 157–163.
Yan, T.R. and Liau, J.C., Purification and characterization of a glucose-tolerant β-glucosidase from Aspergillus niger CCRC 31494, Biosci. Biotech. Biochem., 1997, vol. 61, pp. 965–970.
Saha, B.C. and Bothast, R.J., Production, purification, and characterization of a highly glucose-tolerant novel β-glucosidase from Candida peltata, Appl. Environ. Microb., 1996, vol. 62, pp. 3165–3170.
Yan, T.R. and Liau, J.C., Synthesis of alkyl β-glucosidases from cellobiose with Aspergillus niger β-glucosidase II, Biotechnol. Lett., 1998, vol. 20, pp. 653–657.
Decker, C.H., Visser, J., and Schreier, P., β-Glucosidases from five black Aspergillus species: study of their physico-chemical and biocatalytic properties, J. Agric. Food Chem., 2000, vol. 48, pp. 4929–4936.
Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
Li, X., Pei, J., Wu, G., and Shao, W., Expression, purification and characterization of a recombinant glucosidase from Volvariella volvacea, Biotechnol. Lett., 2005, vol. 27, pp. 1369–1373.
Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, vol. 227, pp. 680–685.
Bhat, M.K., Cellulases and related enzymes in biotechnology, Biotechnol. Adv., 2000, vol. 18, pp. 355–383.
Brethauer, S. and Wyman, C.E., Review: continuous hydrolysis and fermentation for cellulosic ethanol production, Biores. Technol., 2010, vol. 101, pp. 4862–4874.
Domingues, F.C., Queiroz, J.A., Cabral, J.M.S., and Fonseca, L.P., Production of cellulases in batch culture using a mutant strain of Trichoderma reesei Growing on soluble carbon source, Biotechnol. Lett., 2001, vol. 23, pp. 771–775.
George, S.P., Ahmad, A., and Rao, M.B., Studies on carboxymethyl cellulose produced by an alkalothermophilic actinomycete, Biores. Technol., 2001, vol. 77, pp. 171–175.
Jan, H.D. and Chen, K.S., Production and characterization of thermostable cellulases from Streptomyces transformant T3-1, World J. Microbiol. Biotechnol., 2003 vol. 19, pp. 263–268.
Cregg, J.M., Cereghino, J.L., Shi, J.Y., and Higgins, D.R., Recombinant protein expression in Pichia pastoris, Mol. Biotechnol., 2000, vol. 16, pp. 23–52.
Fischer, R., Drossard, J., and Emans, N., Towards molecular farming in the future: Pichia pastoris based production of single-chain antibody fragments, Biotechnol. Appl. Biochem., 1999, vol. 30, pp. 117–120.
Pei, J.J., Pang, Q., Zhao, L.G., Fan, S., and Shi, H., Thermoanaerobacterium thermosaccharolyticum β-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose, Biotechnol. Biofuels, 2012, vol. 5, p. 31.
Belancic, A., Gunata, Z., Vallier, M.J., and Agosin, E., β-Glucosidase from the grape native yeast Debaryomyces vanrijiae: puification, characterization, and its effect on monoterpene content of a muscat grape juice, J. Agric. Food Chem., 2003, vol. 51, pp. 1453–1459.
Riou, C., Salmon, J.M., Vallier, M.J., Gunata, Z., and Barre, P., Purification, characterization, and substrate specificity of a novel highly glucose-tolerant glucosidase from Aspergillus oryzae, Appl. Environ. Microbiol., 1998, vol. 64, pp. 3607–3614.
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Zhao, L., Zhou, T., Li, X. et al. Expression and characterization of GH3 β-Glucosidase from Aspergillus niger NL-1 with high specific activity, glucose inhibition and solvent tolerance. Microbiology 82, 356–363 (2013). https://doi.org/10.1134/S0026261713030181
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DOI: https://doi.org/10.1134/S0026261713030181