Gene Cloning and Molecular Characterization of a β-Glucosidase from Thermotoga Naphthophila RUK-10: an Effective Tool for Synthesis of Galacto-oligosaccharide and Alkyl Galactopyranosides

doi:10.1007/s40242-015-5179-y

Chemical Research in Chinese Universities ›› 2015, Vol. 31 ›› Issue (5): 774-780.doi: 10.1007/s40242-015-5179-y

• Articles • Previous Articles Next Articles

Gene Cloning and Molecular Characterization of a β-Glucosidase from Thermotoga Naphthophila RUK-10: an Effective Tool for Synthesis of Galacto-oligosaccharide and Alkyl Galactopyranosides

KONG Fansi¹, YANG Jingwen¹, ZHEN Zhen², LIANG Tingting², ZHU Dongliang², GAO Renjun¹, XIE Guiqiu²

1. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, School of Life Science, Jilin University, Changchun 130012, P. R. China;
2. College of Pharmacy, Jilin University, Changchun 130021, P. R. China

Received:2015-05-05 Revised:2015-07-14 Online:2015-10-01 Published:2015-08-24
Contact: XIE Guiqiu, E-mail: jiegq@jlu.edu.cn E-mail:jiegq@jlu.edu.cn
Supported by:
Supported by the National High Technology Research and Development Program of China(No.2013AA102104) and the National Natural Science Foundation of China(Nos.20772046, 21072075).

Abstract

Abstract:

A novel thermostable β-glucosidase(Tnap0602) with β-galactosidase activity was cloned from Thermotoga naphthophila RUK-10 and overexpressed in Escherichia coli BL21(DE3) with the aid of pET28b(+) vector. The recombinant β-glucosidase was purified to homogeneity by heat precipitation and Ni²⁺-affinity chromatography. The molecular weight of the recombinant enzyme was estimated to be 51 kDa by SDS-PAGE analysis. The optimum temperature for the hydrolyses of p-nitrophenyl-β-D-glucopyranoside and o-nitrophenyl-β-D-galactopyranoside by the recombinant β-glucosidase were both above 95℃, and the corresponding optimum pH value was found to be the same as 7.0. Thermostability studies show that the half-lives of the recombinant enzyme at 75, 80, 85 and 90℃ are respectively 84, 32, 14, and 3 h, and it is quite stable in a pH range of 5.0-10.0. The K_m and V_max values of the recombinant β-glucosidase for the hydrolysis of pNPGlu at 80℃ are 0.127 mmol/L and 18389.1 μmol·min^-1·mg^-1, the corresponding values are 0.625 mmol/L and 6250 μmol·min^-1·mg^-1 for the hydrolysis of oNPGal, respectively. The enzyme also display the hydrolysis activity for lactose and cellobiose. Galacto-oligosaccharide and alkyl galactopyranosides could be synthesized from Tnap0602 when lactose was used as the transglycosylation substrate, indicating that the thermostable β-glucosidase could be a candidate for industrial application.

Key words: β-Glucosidase, Galacto-oligosaccharide, Alkyl galactopyranoside, Thermostablility, Thermotoga naphthophila, Transglycosylation

KONG Fansi, YANG Jingwen, ZHEN Zhen, LIANG Tingting, ZHU Dongliang, GAO Renjun, XIE Guiqiu. Gene Cloning and Molecular Characterization of a β-Glucosidase from Thermotoga Naphthophila RUK-10: an Effective Tool for Synthesis of Galacto-oligosaccharide and Alkyl Galactopyranosides[J]. Chemical Research in Chinese Universities, 2015, 31(5): 774-780.

References

[1] Bhatia Y., Mishra S., Bisaria V. S., Crit. Rev. Biotechnol., 2002, 22, 375
[2] Harnpicharnchai P., Champreda V., Sornlake W., Eurwilaichitr L., Protein Expr. Purif., 2009, 67, 61
[3] Turner P., Svensson D., Adlercreutz P., Karlsson E. N., J. Biotechnol., 2007, 130, 67
[4] Krisch J., Bencsik O., Papp T., Vágvölgyi C., TakóM., Bioresour. Technol., 2012, 114, 555
[5] Sivakumar R., Divakar S., Enzyme Microb. Technol., 2009, 44, 33
[6] Mahoney R. R., Food Chem., 1998, 63, 147
[7] Urrutia P., Rodriguez-Colinas B., Fernandez-Arrojo L., Ballesteros A. O., Wilson L., Illanes A., Plou F. J., J. Agric. Food Chem., 2013, 61, 1081
[8] Macfarlane G. T., Steed H., MacFarlane S., J. Appl. Microbiol., 2008, 104, 305
[9] El-Sukkary M. M. A., Nagla A. S., Aiad I., El-Azab W. I. M., J. Surfact Deterg., 2008, 11, 129
[10] Andersson M., Adlercreutz P., Patrick A., Journal of Molecular Catalysis B:Enzymatic, 2001, 14, 69
[11] Daabrowski S., Sobiewska G., Maciunska J., Synowiecki J., Ku J., Protein Expression and Purification, 2000, 19, 107
[12] Kang S. K., Cho K. K., Ahn J. K., Bok J. D., Kang S. H., Woo J. H., Lee H. G., You S. K., Choi Y. J., J. Biotechnol., 2005, 116, 337
[13] Hong M. R., Kim Y. S., Park C. S., Lee J. K., Kim Y. S., Oh D. K., J. Biosci. Bioeng., 2009, 108, 36
[14] Nakkharat P., Haltrich D., J. Biotechnol., 2006, 123, 304
[15] Kengen S. W. M., Luesink E. J., Stams A. J. M., Zehnder A. J. B., Eur. J. Biochem., 1993, 213, 305
[16] Gabelsberger J., Liebl W., Schleifer K. H., Appl. Microbiol. Biotechnol., 1993, 40, 44
[17] Kim Y. S., Park C. S., Oh D. K., Enzyme Microb. Technol., 2006, 39, 903
[18] Onishi N., Tanaka T., Appl. Environ. Microbiol., 1995, 61, 4026
[19] D?browski S., Maciuńska J., Synowiecki J., Mol. Biotechnol., 1998, 10, 217
[20] Juajun O., Nguyen T. H., Maischberger T., Iqbal S., Haltrich D., Yamabhai M., Appl. Microbiol. Biotechnol., 2011, 89, 645
[21] Iqbal S., Nguyen T. H., Nguyen H. A., Nguyen T. T., Maischberger T., Kittl R., Haltrich D., J. Agric. Food Chem., 2011, 59, 3083
[22] Splechtna B., Nguyen T. H., Steinböck M., Kulbe K. D., Lorenz W., Haltrich D., J. Agric. Food Chem., 2006, 54, 4999
[23] Kobayashi T., Adachi S., Nakanishi K., Matsuno R., Journal of Molecular Catalysis B:Enzymatic, 2000, 11, 13

Gene Cloning and Molecular Characterization of a β-Glucosidase from Thermotoga Naphthophila RUK-10: an Effective Tool for Synthesis of Galacto-oligosaccharide and Alkyl Galactopyranosides

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 2

Recommended Articles

Metrics

Comments

[1]	LI Yuwei, BU Mingwei, CHEN Peng, LI Xiaohong, CHEN Changwu, GAO Gui, FENG Yan, HAN Weiwei, ZHANG Zuoming. Characterization of a Thermophilic Monosaccharide Stimulated β-Glucosidase from Acidothermus cellulolyticus [J]. Chemical Research in Chinese Universities, 2018, 34(2): 212-220.
[2]	YANG Jingwen, DI Xiangjun, WANG Man, GAO Renjun. Gene Clone and Characterization of a Novel Thermostable β-Galactosidase with Transglycosylation Activity from Thermotoga naphthophila RUK-10 [J]. Chemical Research in Chinese Universities, 2015, 31(4): 564-568.