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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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Effects of pH and temperature on the activity and stability of the recombinant Tth Man5 β-mannosidase. a. Optimal pH of the Tth Man5 β-mannosidase. b. pH stability of the Tth Man5 β-mannosidase. c. Effect of temperature on Tth Man5 β-mannosidase activity. d. The thermostability of the Tth Man5β-mannosidase. The residual activity was monitored, and the maximum activity was defined as 100% (a, c) or initial activity was defined as 100% (b, d). Values shown were the mean of triplicate experiments, and the variation about the mean was below 5%.
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Figure 3: Effects of pH and temperature on the activity and stability of the recombinant Tth Man5 β-mannosidase. a. Optimal pH of the Tth Man5 β-mannosidase. b. pH stability of the Tth Man5 β-mannosidase. c. Effect of temperature on Tth Man5 β-mannosidase activity. d. The thermostability of the Tth Man5β-mannosidase. The residual activity was monitored, and the maximum activity was defined as 100% (a, c) or initial activity was defined as 100% (b, d). Values shown were the mean of triplicate experiments, and the variation about the mean was below 5%.

Mentions: The recombinant Tth Man5 β-mannosidase has a pH optimum of 5.5 as shown in Figure 3a. The enzyme was relatively stable at a pH range of 5.0 to 8.5 and most stable at pH 5.5 (Figure 3b). More than 70% of the initial enzyme activity remained at this range. The enzyme was most active at 85°C, and it retained approximately 50% of the maximum activity at 95°C (Figure 3c). The thermostability data showed that it remained above 56% of its initial activity after 2 h of pre-incubation at temperature ranging from 75°C to 90°C (Figure 3d).


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)

Effects of pH and temperature on the activity and stability of the recombinant Tth Man5 β-mannosidase. a. Optimal pH of the Tth Man5 β-mannosidase. b. pH stability of the Tth Man5 β-mannosidase. c. Effect of temperature on Tth Man5 β-mannosidase activity. d. The thermostability of the Tth Man5β-mannosidase. The residual activity was monitored, and the maximum activity was defined as 100% (a, c) or initial activity was defined as 100% (b, d). Values shown were the mean of triplicate experiments, and the variation about the mean was below 5%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effects of pH and temperature on the activity and stability of the recombinant Tth Man5 β-mannosidase. a. Optimal pH of the Tth Man5 β-mannosidase. b. pH stability of the Tth Man5 β-mannosidase. c. Effect of temperature on Tth Man5 β-mannosidase activity. d. The thermostability of the Tth Man5β-mannosidase. The residual activity was monitored, and the maximum activity was defined as 100% (a, c) or initial activity was defined as 100% (b, d). Values shown were the mean of triplicate experiments, and the variation about the mean was below 5%.
Mentions: The recombinant Tth Man5 β-mannosidase has a pH optimum of 5.5 as shown in Figure 3a. The enzyme was relatively stable at a pH range of 5.0 to 8.5 and most stable at pH 5.5 (Figure 3b). More than 70% of the initial enzyme activity remained at this range. The enzyme was most active at 85°C, and it retained approximately 50% of the maximum activity at 95°C (Figure 3c). The thermostability data showed that it remained above 56% of its initial activity after 2 h of pre-incubation at temperature ranging from 75°C to 90°C (Figure 3d).

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