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

Shi H, Huang Y, Zhang Y, Li W, Li X, Wang F - BMC Biotechnol. (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China. hgwf@njfu.edu.cn.

ABSTRACT

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.

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SDS-PAGE analysis of recombinant Tth Man5 β-mannosidase in E. coli BL21 (DE3). Lane M: protein marker, lane 1: cell-free extract of E. coli BL21 (DE3) harboring pET-20b plasmids, lane 2: the purified Tth Man5 β-mannosidase eluted with 0.2 M 1 mL imidazole buffer, lane 3,4: the purified Tth Man5 β-mannosidase eluted with 0.4 M 1 mL imidazole buffer (lane 3: first tube collection, lane 4: second tube collection).
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Figure 2: SDS-PAGE analysis of recombinant Tth Man5 β-mannosidase in E. coli BL21 (DE3). Lane M: protein marker, lane 1: cell-free extract of E. coli BL21 (DE3) harboring pET-20b plasmids, lane 2: the purified Tth Man5 β-mannosidase eluted with 0.2 M 1 mL imidazole buffer, lane 3,4: the purified Tth Man5 β-mannosidase eluted with 0.4 M 1 mL imidazole buffer (lane 3: first tube collection, lane 4: second tube collection).

Mentions: When using native gene from T. thermarum for expression, the protein production was very difficult to detected (data not shown). Thus, in order to increase the expression level of Tth Man5 β-mannosidase in Escherichia coli, rare codons were replaced by optimal codons without change of amino acid sequence (data not shown). The mature protein without the signal peptide, allowing the insertion of a His6-tag at the C-terminus, was successfully expressed in E. coli BL21 (DE3), after induction with IPTG for 5 h at 37°C. The recombinant protein in the cell-free extract was purified by a heat treatment followed by a nickel affinity column (Table 1). Finally, the purified recombinant enzyme displayed a single band on SDS-PAGE with an estimated molecular weight (MW) of 70 kDa (Figure 2), which was consistent with the predicted MW of monomer (71, 725 Da). Size exclusion chromatography was also carried out using the AKTAFPLC™ system to compute the oligomerization state of the target protein. It was deduced that the native protein formed 7-mer in solution with a calculated MW 508,019 Da according to the calibration curve of the gel filtration column.


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

Shi H, Huang Y, Zhang Y, Li W, Li X, Wang F - BMC Biotechnol. (2013)

SDS-PAGE analysis of recombinant Tth Man5 β-mannosidase in E. coli BL21 (DE3). Lane M: protein marker, lane 1: cell-free extract of E. coli BL21 (DE3) harboring pET-20b plasmids, lane 2: the purified Tth Man5 β-mannosidase eluted with 0.2 M 1 mL imidazole buffer, lane 3,4: the purified Tth Man5 β-mannosidase eluted with 0.4 M 1 mL imidazole buffer (lane 3: first tube collection, lane 4: second tube collection).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852774&req=5

Figure 2: SDS-PAGE analysis of recombinant Tth Man5 β-mannosidase in E. coli BL21 (DE3). Lane M: protein marker, lane 1: cell-free extract of E. coli BL21 (DE3) harboring pET-20b plasmids, lane 2: the purified Tth Man5 β-mannosidase eluted with 0.2 M 1 mL imidazole buffer, lane 3,4: the purified Tth Man5 β-mannosidase eluted with 0.4 M 1 mL imidazole buffer (lane 3: first tube collection, lane 4: second tube collection).
Mentions: When using native gene from T. thermarum for expression, the protein production was very difficult to detected (data not shown). Thus, in order to increase the expression level of Tth Man5 β-mannosidase in Escherichia coli, rare codons were replaced by optimal codons without change of amino acid sequence (data not shown). The mature protein without the signal peptide, allowing the insertion of a His6-tag at the C-terminus, was successfully expressed in E. coli BL21 (DE3), after induction with IPTG for 5 h at 37°C. The recombinant protein in the cell-free extract was purified by a heat treatment followed by a nickel affinity column (Table 1). Finally, the purified recombinant enzyme displayed a single band on SDS-PAGE with an estimated molecular weight (MW) of 70 kDa (Figure 2), which was consistent with the predicted MW of monomer (71, 725 Da). Size exclusion chromatography was also carried out using the AKTAFPLC™ system to compute the oligomerization state of the target protein. It was deduced that the native protein formed 7-mer in solution with a calculated MW 508,019 Da according to the calibration curve of the gel filtration column.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China. hgwf@njfu.edu.cn.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus