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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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Scheme for kinetic resolution of 2-hydroxycarboxylic acids. R is CH3: lactic acid [13]; C2H5: 2-hydroxybutyric acid [14]; C6H5: mandelic acid.
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Figure 1: Scheme for kinetic resolution of 2-hydroxycarboxylic acids. R is CH3: lactic acid [13]; C2H5: 2-hydroxybutyric acid [14]; C6H5: mandelic acid.

Mentions: The NAD-independent l-lactate dehydrogenase (l-iLDH) of Pseudomonas stutzeri SDM is located on the cell membrane, and quinine, as its electron acceptor, could be directly regenerated by the membrane electron transport chain [11]. So it may exhibit higher catalytic efficiency than the soluble FMN-dependent α-hydroxyacid dehydrogenases. Previous report showed that it exhibits high catalytic efficiency and enantioselectivity toward small aliphatic 2-hydroxycarboxylic acids such as l-lactate and l-2-hydroxybutanoate [12]. Cells of P. stutzeri SDM have been used in the kinetic resolution of lactate and 2-hydroxybutanoate racemic mixtures to produce d-lactate and d-2-hydroxybutanoate [13,14]. Considering the similar structures of lactic acid and mandelic acid, l-iLDH might also be able to catalyze the kinetic resolution of racemic mandelic acid (Figure 1). l-iLDH from P. stutzeri SDM was purified, and then it was characterized further [12]. It showed rather low activity towards l-mandelate [12], which restricts its potential application for the kinetic resolution of racemic 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)

Scheme for kinetic resolution of 2-hydroxycarboxylic acids. R is CH3: lactic acid [13]; C2H5: 2-hydroxybutyric acid [14]; C6H5: mandelic acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Scheme for kinetic resolution of 2-hydroxycarboxylic acids. R is CH3: lactic acid [13]; C2H5: 2-hydroxybutyric acid [14]; C6H5: mandelic acid.
Mentions: The NAD-independent l-lactate dehydrogenase (l-iLDH) of Pseudomonas stutzeri SDM is located on the cell membrane, and quinine, as its electron acceptor, could be directly regenerated by the membrane electron transport chain [11]. So it may exhibit higher catalytic efficiency than the soluble FMN-dependent α-hydroxyacid dehydrogenases. Previous report showed that it exhibits high catalytic efficiency and enantioselectivity toward small aliphatic 2-hydroxycarboxylic acids such as l-lactate and l-2-hydroxybutanoate [12]. Cells of P. stutzeri SDM have been used in the kinetic resolution of lactate and 2-hydroxybutanoate racemic mixtures to produce d-lactate and d-2-hydroxybutanoate [13,14]. Considering the similar structures of lactic acid and mandelic acid, l-iLDH might also be able to catalyze the kinetic resolution of racemic mandelic acid (Figure 1). l-iLDH from P. stutzeri SDM was purified, and then it was characterized further [12]. It showed rather low activity towards l-mandelate [12], which restricts its potential application for the kinetic resolution of racemic 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