High-level expression of a novel thermostable and mannose-tolerant β-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli
Open Access
- 8 October 2013
- journal article
- Published by Springer Science and Business Media LLC in BMC Biotechnology
- Vol. 13 (1), 83
- https://doi.org/10.1186/1472-6750-13-83
Abstract
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. 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. 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.Keywords
This publication has 26 references indexed in Scilit:
- Biochemical properties of a novel thermostable and highly xylose-tolerant β-xylosidase/α-arabinosidase from Thermotoga thermarumBiotechnology for Biofuels, 2013
- A novel highly thermostable xylanase stimulated by Ca2+ from Thermotoga thermarum: cloning, expression and characterizationBiotechnology for Biofuels, 2013
- Bacillus subtilis: from soil bacterium to super-secreting cell factoryMicrobial Cell Factories, 2013
- Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-β-mannosidase from Bacillus licheniformis in Escherichia coliMicrobial Cell Factories, 2010
- Alanine-shaving Mutagenesis to Determine Key Interfacial Residues Governing the Assembly of a Nano-cage Maxi-ferritinPublished by Elsevier BV ,2010
- A MANBAmutation resulting in residual beta-mannosidase activity associated with severe leukoencephalopathy: a possible pseudodeficiency variantBMC Medical Genetics, 2009
- Clustal W and Clustal X version 2.0Bioinformatics, 2007
- Potential and utilization of thermophiles and thermostable enzymes in biorefiningMicrobial Cell Factories, 2007
- Structure of a Full Length Psychrophilic Cellulase from Pseudoalteromonas haloplanktis revealed by X-ray Diffraction and Small Angle X-ray ScatteringJournal of Molecular Biology, 2005
- Use of Dinitrosalicylic Acid Reagent for Determination of Reducing SugarAnalytical Chemistry, 1959