High-level expression of a novel thermostable and mannose-tolerant β-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli

BMC Biotechnology

Research Article

Xun Li¹  Yingjuan Huang¹  Wenqian Li¹  Fei Wang¹  Hao Shi¹  Yu Zhang¹ 

[1] College of Chemical Engineering, Nanjing Forestry University, 210037, Nanjing, China;Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, 210037, Nanjing, China;

关键词: Thermotoga thermarum;    β-mannosidase;    Mannose-tolerant;    Mannan;    Thermostability;    Mannooligosaccharides;   

DOI  :  10.1186/1472-6750-13-83

 received in 2013-07-09, accepted in 2013-10-04,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundMannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. β-Mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. Therefore, the mannose-tolerant β-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above.ResultsA β-mannosidase gene (Tth man5) of 1,827 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 608 amino acid residues, and was over-expressed in Escherichia coli BL21 (DE3). The results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the Tth Man5 is a novel β-mannosidase of glycoside hydrolase family 5. The optimal activity of the Tth Man5 β-mannosidase was obtained at pH 5.5 and 85°C and was stable over a pH range of 5.0 to 8.5 and exhibited 2 h half-life at 90°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 4.36±0.5 mM and 227.27±1.59 μmol min-1 mg-1, 58.34±1.75 mg mL-1 and 285.71±10.86 μmol min-1 mg-1, respectively. The kcat/Km values for p-nitrophenyl-β-D-mannopyranoside and 1,4-β-D-mannan were 441.35±0.04 mM-1 s-1 and 41.47±1.58 s-1 mg-1 mL, respectively. It displayed high tolerance to mannose, with a Ki value of approximately 900 mM.ConclusionsThis work provides a novel and useful β-mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes.

【 授权许可】

Unknown   
© Shi et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.