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

高等学校化学研究 ›› 2015, Vol. 31 ›› Issue (5): 774-780.doi: 10.1007/s40242-015-5179-y

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

收稿日期:2015-05-05 修回日期:2015-07-14 出版日期:2015-10-01 发布日期:2015-08-24
通讯作者: XIE Guiqiu, E-mail: jiegq@jlu.edu.cn E-mail:jiegq@jlu.edu.cn
基金资助:
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).

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).

摘要/Abstract

摘要：

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.

关键词: β-Glucosidase, Galacto-oligosaccharide, Alkyl galactopyranoside, Thermostablility, Thermotoga naphthophila, Transglycosylation

Abstract:

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]. 高等学校化学研究, 2015, 31(5): 774-780.

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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

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

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