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

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.