Characterization of a Thermophilic Monosaccharide Stimulated β-Glucosidase from Acidothermus cellulolyticus

Abstract

The gene(ABK51908) from Acidothermus cellulolyticus encodes a mature protein of 484 residues with a calculated molecular mass of 53.0 kDa. Sequence analysis revealed that the protein had 59% identity to the β-glucosidases CAA82733, which belongs to glycoside hydrolase family 1(GH1). We cloned and expressed the gene in Escherichia coli BL21-Gold(DE3). The recombinant protein(AcBg) had an optimal pH and temperature of 7.0 and 70 °C, respectively. The specific activities of AcBg under optimal conditions were 290 and 33 U/mg for p-nitrophenyl-β-D-glucopyranoside(pNPG) and cellobiose, respectively. AcBg hydrolyzed the oligosaccharide sequentially from the non-reducing end to produce glucose units according to the results of HPLC analysis. AcBg showed high salt tolerance and monosaccharide-stimulation properties. Its activity rose more than 2-fold when 5 mol/L NaCl/KCl were added. The activity of the β-glucosidase was remarkably enhanced in the presence of 0.2 mol/L D-glucose(increased more than 1.9-fold), 0.1 mol/L α-methyl-D-glucose(increased more than 1.4-fold) and 1.0 mol/L D-xylose(increased more than 1.9-fold). The catalysis kinetics and structural changes in various concentrations of glucose were determined. The results indicate that glucose reduces substrate affinity and causes conformational changes.

Keywords

β-Glucosidase / Monosaccharide-stimulation / Salt tolerance / Thermophilic enzyme

References

[1] Zhang Y. H. P., Himmel M. E., Mielenz J. R. Biotechnol. Adv., 2006, 24: 452.

[2] Gao S., Gao R. Chem. Res. Chinese Universities, 2017, 33(1): 1.

[3] Beguin P. Annu. Rev. Microbiol., 1990, 44: 219.

[4] Vinzant T., Adney W., Decker S., Baker J., Kinter M., Sherman N., Fox J., Himmel M. Appl. Biochem. Biotechnol., 2001, 91: 99.

[5] Pei J., Pang Q., Zhao L., Fan S., Shi H. Biotechnol. Biofuels, 2012, 5: 1.

[6] Chen M., Zhao J., Xia L. Carbohyd. Polym., 2008, 71: 411.

[7] Brimer L., Cicalini A. R., Federici F., Petruccioli M. Arch. Micro-biol., 1998, 169: 106.

[8] Pal S., Banik S. P., Ghorai S., Chowdhury S., Khowala S. Bioresour. Technol., 2010, 101: 2412.

[9] Bhatia Y., Mishra S., Bisaria V. Crit. Rev. Biotechnol., 2002, 22: 375.

[10] Choi I. S., Wi S. G., Jung S. R., Patel D. H., Bae H. J. J. Wood. Sci., 2009, 55: 329.

[11] Souza F. H. M., Meleiro L. P., Machado C. B., Zimbardi A. L. R. L., Maldonado R. F., Souza T. A. C. B., Masui D. C., Murakami M. T., Jorge J. A., Ward R. J. J. Mol. Catal. B: Enzym., 2014, 106: 1.

[12] Zhou C., Qian L., Ma H., Yu X., Zhang Y., Qu W., Zhang X., Xia W. Carbohyd. Polym., 2012, 90: 516.

[13] Kong F., Yang J., Zhen Z. Chem. Res. Chinese Universities, 2015, 31(5): 774.

[14] Uchima C. A., Tokuda G., Watanabe H., Kitamoto K., Arioka M. Appl. Environ. Microbiol., 2012, 78: 4288.

[15] Zhou J., Zhang R., Shi P., Huang H., Meng K., Yuan T., Yang P., Yao B. Appl. Microbiol. Biotechnol., 2011, 92: 305.

[16] Zhao L., Xie J., Zhang X., Cao F., Pei J. J. Mol. Cat. B: Enzym., 2013, 95: 62.

[17] Li Y. K., Chang L. F., Shu H. H., Chir J. J. Chin. Chem. Soc., 1997, 44: 81.

[18] Bhalla A., Bansal N., Kumar S., Bischoff K. M., Sani R. K. Bioresour. Technol., 2013, 128: 751.

[19] Gumerov V. M., Rakitin A. L., Mardanov A. V., Ravin N. V. Archaea, 2015, 2015: 978632.

[20] De C. P. J., Leite R. S., Prado H. F. A., Bocchini M. D. A., Gomes E., Silva R. Appl. Biochem. Biotechnol., 2014, 175: 723.

[21] Chen L., Li N., Zong M. H. Biochem., 2012, 47: 127.

[22] Krogh K. B., Harris P. V., Olsen C. L., Johansen K. S., Hojer Peder-sen J., Borjesson J., Olsson L. Appl. Microbiol. Biotechnol., 2010, 86: 143.

[23] Jabbour D., Klippel B., Antranikian G. Appl. Microbiol. Biotechnol., 2012, 93: 1947.

[24] Giuseppe P. O., Souza F. H. M., Zanphorlin L. M., Machado C. B., Ward R. J., Jorge J. A., Furriel R. P. M., Murakami M. T. Acta Crystallogr. Sect. D: Biol. Crystallogr., 2014, 70: 1631.

[25] Yang Y., Zhang X., Yin Q., Fang W., Fang Z., Wang X., Zhang X., Xiao Y. Sci. Rep., 2015, 5: 17296.

[26] Barabote R. D., Xie G., Leu D. H., Normand P., Necsulea A., Daubin V., Médigue C., Adney W. S., Xu X. C., Lapidus A. Geno. Res., 2009, 19: 1033.

[27] Alahuhta M., Adney W. S., Himmel M. E., Lunin V. V. Acta Crystallogr. Sect. F: Struct. Biol. Cryst. Commun., 2013, 69: 1335.

[28] Wang J., Gao G., Li Y., Yang L., Liang Y., Jin H., Han W., Feng Y., Zhang Z. Int. J. Mol. Sci., 2015, 16: 25080.

[29] Zhang Q., Zhang W., Lin C., Xu X., Shen Z. Prot. Exp. Purif., 2012, 82: 279.

[30] Zhang Y. H. P., Cui J., Lynd L. R., Kuang L. R. Biomacromol., 2006, 7: 644.

[31] Mamiatis T., Fritsch E., Sambrook J., Engel J. Molecular Cloning—A Laboratory Manual, 1985, New York: Cold Spring Harbor Laboratory, Wiley Online Library.

[32] Chen Y., Sun D., Zhou Y., Liu L., Han W., Zheng B., Wang Z., Zhang Z. Int. J. Mol. Sci., 2014, 15: 5717.

[33] Bradford M. M. Plant, 1976, 168: 214.

[34] Rauscher E., Neumann U., Schaich E., Von Bülow S., Wahlefeld A. Clin. Chem., 1985, 31: 14.

[35] Lineweaver H., Burk D. J. Amer. Chem. Soc., 1934, 56: 658.

[36] Miller G. L. Anal. Chem., 1959, 31: 426.

[37] Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Nucl. Acid. Res., 1997, 25: 3389.

[38] Badieyan S., Bevan D. R., Zhang C. Biochem., 2012, 51: 8907.

[39] Miao L. L., Hou Y. J., Fan H. X., Qu J., Qi C., Liu Y., Li D. F., Liu Z. P. Appl. Environ. Microbiol., 2016, 82: 2021.

[40] Mai Z., Yang J., Tian X., Li J., Zhang S. Appl. Biochem. Biotechnol., 2013, 169: 1512.

[41] Voorhorst W., Eggen R., Luesink E. J., De Vos W. J. Bacteriol., 1995, 177: 7105.

[42] Lee J. M., Kim Y. R., Kim J. K., Jeong G. T., Ha J. C., Kong I. S. Biopro. Biosys. Eng., 2015, 38: 1335.

[43] Bowers E. M., Ragland L. O., Byers L. D. Biochim. Biophy. Acta, 2007, 1774: 1500.

[44] Chamoli S., Kumar P., Navani N. K., Verma A. K. Int. J. Biol. Macromol., 2016, 85: 425.

[45] Ramachandran P., Tiwari M. K., Singh R. K., Haw J. R., Jeya M., Lee J. K. Biochem., 2012, 47: 99.

[46] Shin K. C., Oh D. K. J. Biotechnol., 2014, 172: 30.

[47] Pérez-Pons J. A., Rebordosa X., Querol E. Biochim. Biophy. Acta, 1995, 1251: 145.

[48] Uchiyama T., Yaoi K., Miyazaki K. Front. Microbiol., 2015, 6: 548.

[49] Lu J., Du L., Wei Y., Hu Y., Huang R. Acta. Biochim. Biophy. Sin., 2013, 45: 664.

[50] Saha B. C., Bothast R. J. Appl. Environ. Microbiol., 1996, 62: 3165.

[51] Riou C., Salmon J. M., Vallier M. J., Günata Z., Barre P. Appl. En-viron. Microbiol., 1998, 64: 3607.

[52] Rajasree K. P., Mathew G. M., Pandey A., Sukumaran R. K. J. Ind. Microbiol. Biotechnol., 2013, 40: 967.

[53] Yang F., Yang X., Li Z., Du C., Wang J., Li S. Appl. Microbiol. Bio-technol., 2015, 99: 8903.

[54] Yan T. R., Lin C. L. Biosci. Biotechnol. Biochem., 1997, 61: 965.