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Rationally re-designed mutation of NAD-independent L-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli.

Jiang T, Gao C, Dou P, Ma C, Kong J, Xu P - Microb. Cell Fact. (2012)

Bottom Line: The L-iLDH mutant exhibited much higher activity than wide-type L-iLDH towards L-mandelate, an aromatic 2-hydroxycarboxylic acid.Using the engineered Escherichia coli expressing the mutant L-iLDH as a biocatalyst, 40 g·L(-1) of DL-mandelic acid was converted to 20.1 g·L(-1) of D-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L(-1) of benzoylformic acid.Two building block intermediates (optically pure D-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.

ABSTRACT

Background: NAD-independent L-lactate dehydrogenase (L-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids.

Results: Val-108 of L-iLDH was changed to Ala by rationally site-directed mutagenesis. The L-iLDH mutant exhibited much higher activity than wide-type L-iLDH towards L-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant L-iLDH as a biocatalyst, 40 g·L(-1) of DL-mandelic acid was converted to 20.1 g·L(-1) of D-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L(-1) of benzoylformic acid.

Conclusions: A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure D-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system.

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Optimization of pH and temperature for biocatalysis. (A) Optimization of pH. (B) Optimization of temperature. Unshaded bars represent the production of benzoylformic acid in 4 h. Shaded bars represent the production of benzoylformic acid in 10 h. Values are the mean ± SD of 3 separate determinations.
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Figure 4: Optimization of pH and temperature for biocatalysis. (A) Optimization of pH. (B) Optimization of temperature. Unshaded bars represent the production of benzoylformic acid in 4 h. Shaded bars represent the production of benzoylformic acid in 10 h. Values are the mean ± SD of 3 separate determinations.

Mentions: Since pH and temperature are parameters that often limit enzyme activity and stability in technical applications, studies addressing the effects of temperature and pH on whole-cell catalysis were performed. The optimal pH was found to be 7.0 after adjusting the pH of the reaction system from 4.0 to 10.0 (Figure 4A). The effect of the reaction temperature was examined in the range of 16°C to 58°C after 4 h and 10 h of reaction. As shown in Figure 4B, with increasing temperature, higher activity of the biocatalyst was observed over the short term, as shown by the benzoylformic acid production after 4 h of reaction. However, the activity decreased more rapidly at higher temperatures, as shown by the benzoylformic acid production after 10 h of reaction. The optimal reaction temperature was chosen to be 42°C, which optimized enzyme activity and stability. Either the lower activity at lower temperature or the lower stability at higher temperature may cause a decrease of overall l-mandelic acid degrading capacity of the biocatalyst, which will then decrease the production capacity of d-mandelic acid.


Rationally re-designed mutation of NAD-independent L-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli.

Jiang T, Gao C, Dou P, Ma C, Kong J, Xu P - Microb. Cell Fact. (2012)

Optimization of pH and temperature for biocatalysis. (A) Optimization of pH. (B) Optimization of temperature. Unshaded bars represent the production of benzoylformic acid in 4 h. Shaded bars represent the production of benzoylformic acid in 10 h. Values are the mean ± SD of 3 separate determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526519&req=5

Figure 4: Optimization of pH and temperature for biocatalysis. (A) Optimization of pH. (B) Optimization of temperature. Unshaded bars represent the production of benzoylformic acid in 4 h. Shaded bars represent the production of benzoylformic acid in 10 h. Values are the mean ± SD of 3 separate determinations.
Mentions: Since pH and temperature are parameters that often limit enzyme activity and stability in technical applications, studies addressing the effects of temperature and pH on whole-cell catalysis were performed. The optimal pH was found to be 7.0 after adjusting the pH of the reaction system from 4.0 to 10.0 (Figure 4A). The effect of the reaction temperature was examined in the range of 16°C to 58°C after 4 h and 10 h of reaction. As shown in Figure 4B, with increasing temperature, higher activity of the biocatalyst was observed over the short term, as shown by the benzoylformic acid production after 4 h of reaction. However, the activity decreased more rapidly at higher temperatures, as shown by the benzoylformic acid production after 10 h of reaction. The optimal reaction temperature was chosen to be 42°C, which optimized enzyme activity and stability. Either the lower activity at lower temperature or the lower stability at higher temperature may cause a decrease of overall l-mandelic acid degrading capacity of the biocatalyst, which will then decrease the production capacity of d-mandelic acid.

Bottom Line: The L-iLDH mutant exhibited much higher activity than wide-type L-iLDH towards L-mandelate, an aromatic 2-hydroxycarboxylic acid.Using the engineered Escherichia coli expressing the mutant L-iLDH as a biocatalyst, 40 g·L(-1) of DL-mandelic acid was converted to 20.1 g·L(-1) of D-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L(-1) of benzoylformic acid.Two building block intermediates (optically pure D-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.

ABSTRACT

Background: NAD-independent L-lactate dehydrogenase (L-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids.

Results: Val-108 of L-iLDH was changed to Ala by rationally site-directed mutagenesis. The L-iLDH mutant exhibited much higher activity than wide-type L-iLDH towards L-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant L-iLDH as a biocatalyst, 40 g·L(-1) of DL-mandelic acid was converted to 20.1 g·L(-1) of D-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L(-1) of benzoylformic acid.

Conclusions: A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure D-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system.

Show MeSH
Related in: MedlinePlus