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

BMC Biotechnology

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

[1] Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, China

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

Others  :  835316
DOI  :  10.1186/1472-6750-13-83

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

【 摘 要 】

Background

Mannan 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.

Results

A 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 K_m and V_max 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 k_cat/K_m 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 K_i value of approximately 900 mM.

Conclusions

This 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】

   
2013 Shi et al.; licensee BioMed Central Ltd.

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