Rationally re-designed mutation of NAD-independent l-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli
© Jiang et al.; licensee BioMed Central Ltd. 2012
Received: 18 June 2012
Accepted: 4 October 2012
Published: 23 November 2012
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.
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.
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.
KeywordsNAD-independent l-lactate dehydrogenase Site-directed mutagenesis Optical resolution d-mandelic acid
d-Mandelic acid, an aromatic 2-hydroxycarboxylic acid, is a valuable chiral building block for the synthesis of various pharmaceuticals, such as anti-obesity agents, antitumor agents, penicillins, and semisynthetic cephalosporins [1–3]. Chemical processes for mandelic acid production result in the racemic mixture of both stereospecific forms. Several biocatalytic methods, including lipase catalyzed enantioselective esterification , oxidoreductase catalyzed enantioselective oxidation, and microbial mediated enantioselective degradation [5–10], have been developed to prepare d-mandelic acid from racemic mandelic acid. Among these routes, oxidative resolution of racemic mandelic acid is much more promising because of its easy manipulation, exclusion of co-substrate addition, and high yield.
In recent years, rational re-design mutagenesis has emerged as a practical technique for the construction of biocatalysts with high catalytic efficiencies toward unnatural substrates, like the re-design of phenylalanine dehydrogenase [15–19], NAD-dependent l-lactate dehydrogenase , and d-amino-acid oxidase . In this study, l-iLDH from P. stutzeri SDM was rationally re-designed on the basis of the sequence alignment and active site structure of its homologous enzyme flavocytochrome b2. The mutant enzyme was expressed in E. coli, purified, and characterized. l-Mandelate dehydrogenation activity of the mutant enzyme was successfully enhanced. Whole cells of E. coli expressing the mutant l-iLDH were then used to perform the kinetic resolution of racemic mandelic acid.
Results and discussion
Rationally re-designed mutation
l-iLDH is a member of the l-α-hydroxyacid-oxidizing flavoprotein family, and is therefore related both structurally and by sequence to a number of other enzymes, including flavocytochrome b2 from Saccharomyces cerevisiae. The X-ray crystal structure of flavocytochrome b2 has been resolved (PDB ID code 1FCB), and the active site for 2-α-hydroxyacid dehydrogenation has been identified . Six amino acids which interact directly to the substrate have been pinpointed in the crystal structure , in which four amino acids are highly conserved in this protein family. However, the other two residues, Ala-198 and Leu-230 in flavocytochrome b2, which interact with the alkyl group of substrates, are not well conserved (Additional file 1 Figure S1). They are considered important for the substrate specificity of the enzyme . A s an aromatic 2-hydroxycarboxylic acid, l-mandelic acid is similar in structure with l-lactic acid except that phenyl group replaces alkyl group (Figure 1). Double mutation of the Ala-198 and Leu-230 to amino acids with smaller side chains (Gly and Ala, respectively) increased the ability of flavocytochrome b2 to utilize l-mandelate as a substrate . The altered substrate specificity may result from enlargement of the active site space to accommodate the phenyl group .
Kinetics of V108A l-iLDH
V108A l-iLDH was expressed in E. coli C43 (DE3), and it was purified using the protocol reported for the wild-type l-iLDH . The activity of wild-type l-iLDH for l-mandelate was below the detection limit for a reliable measurement with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) as the electron acceptor . However, when the reaction time was extended, the oxidation product benzoylformate could be detected by HPLC, suggesting weak activity of l-iLDH for l-mandelate. The more sensitive electron acceptor 2,6-dichloroindophenol (DCIP) was used to determine the accurate kinetic parameters of the enzyme.
Kinetics of wild-type (WT) and V108A l -iLDH
445 ± 44
0.18 ± 0.01
185 ± 36
0.8 ± 0.1
8.3 ± 1.3
6.8 ± 1.0
97 ± 13
1.6 ± 0.1
Feasibility in co-production of d-mandelic acid and benzoylformic acid
Optimization of biocatalysis conditions
To increase the efficiency of whole-cell biocatalysis in the kinetic resolution of dl-mandelic acid, the bioconversion conditions were optimized. Benzoylformic acid could be degraded by microbial cells as other 2-keto-acids (Figure 3B and Additional file 6 Figure S6). The degradation decreased the bioconversion ratio and produced some by-products. EDTA was added at a concentration of 20 mM in the reaction system, which removed bivalent ions necessary for 2-keto-acid decarboxylase-catalyzed reactions  and then prevents the degradation of benzoylformic acid (Additional file 6 Figure S6).
Effect of dl -mandelic acid concentration on the biotransformation
dl-Mandelic acid concentration (g·L-1)
Reaction time (h)
Benzoylformic acid concentration (g·L-1)
Biotransformation efficiency (g·L-1·h-1)
Residual l-mandelic acid (g·L-1)
Residual d-mandelic acid (g·L-1)
Enantiomeric excess (%)
In this work, l-iLDH was rationally re-designed on the basis of sequence alignment and the active site structure of a homologous enzyme; a new biocatalyst with high catalytic efficiency toward an unnatural substrate was successfully constructed. A one-pot biotransformation system producing 2 building block intermediates was established using the biocatalyst. Under optimal conditions, a concentration of 20.1 g·L-1 of d-mandelic acid with high enantiomeric excess (> 99.5%) and 19.3 g·L-1 of benzoylformic acid was obtained from 40 g·L-1 of dl-mandelic acid.
Materials and methods
Enzymes and chemicals
Restriction enzymes were purchased from TaKaRa Bio Inc (China). FastPfu DNA polymerase and T4 DNA ligase were purchased from Transgen Biotech (China) and MBI (USA), respectively. d-mandelic acid, l-mandelic acid, racemic mandelic acid, benzoylformic acid, and 2,6-dichloroindophenol (DCIP) were all purchased from Sigma-Aldrich (USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG), dithiothreitol (DTT), and phenylmethanesulfonyl fluoride (PMSF) were obtained from Merck (Germany). All other chemicals were of reagent grade.
Site-directed mutagenesis and enzyme expression
To construct the mutant enzyme with valine-108 changed to alanine (V108A l-iLDH), site-directed mutagenesis was performed using the MutanBEST Kit (TaKaRa). The lldD gene coding for the wild-type l-iLDH was subcloned into the pMDTM18-T vector (TaKaRa) to construct the pMDTM18-T-lldD vector. The mutation was introduced using the following primers: 5′TTCACCCTTTCCACCGCG TCGGTCT3′ and 5′GGGAATCCCTTTCTTGTCTGCCGC3′ to amplify the entire sequence of pMDTM18-T-lldD. The linear PCR products were then cyclized using the ligase from the MutanBEST Kit. The mutant lldD gene (confirmed by automated DNA sequencing) was subcloned into the Hind III and Xho I restriction sites of the pETDuet-1 expression vector with a T7 promoter. Plasmid purification, DNA manipulation, and transformation were performed by the standard methods described by Sambrook et al. . E. coli DH5α and C43 (DE3) were used for general cloning and expression procedures, respectively. Lysogenic broth (LB) medium was used for E. coli cultivations. Ampicillin was used at a concentration of 100 μg· mL-1.
The expression and purification procedure of wild-type l-iLDH from recombinant E. coli has been described previously . The same procedure was used to express and purify the V108A mutant of l-iLDH. The purified enzymes were concentrated by ultrafiltration, desalted with Sephadex G-25, and then stored in 100 mM sodium phosphate buffer (pH 8.0, containing 0.1% Triton X-100) at −20°C. The expressed and purified enzyme was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The activities of wild-type and mutant l-iLDH were determined at 30°C in 1 mL of 50 mM Tris–HCl, pH 7.5, 0.0625 mM DCIP, and the enzyme. Protein amounts of wild-type enzyme and V108A l-iLDH mutant used to assay the kinetic parameters towards l-lactate were 0.05 μg and 0.2 μg, repectively; 4.0 μg and 1.0 μg proteins of wild-type and V108A l-iLDH were used to assay the kinetic parameters towards l-mandelate, respectively. The reaction was started by the addition of l-lactate or l-mandelate, and the rates of DCIP reduction were determined by measuring the absorbance change at 600 nm . To study effects of temperature and pH on enzyme stability, the enzyme was incubated at different temperature for 0.5 h or at different pH for 2 h, and then assayed with 1.25 mM l-lactate as substrate. MTT was used at a concentration of 0.2 mM instead of 0.0625 mM DCIP as electron acceptor in the assay of pH stability, since the molar extinction coefficient of DCIP changes with different pH. The rate of MTT reduction was determined by measuring changes in absorbance at 578 nm . One unit of l-iLDH activity was defined as the amount reducing 1.0 μmol of electron acceptor per minute under the test conditions. Protein concentration was determined by the Lowry method with BSA as a standard .
Recombinant E. coli C43 (DE3) cells were grown at 37°C on a rotary shaker (180 rpm) in LB medium containing ampicillin (100 μg·mL-1) to an OD620 of 0.6. Expression of the recombinant gene was induced by adding 1 mM IPTG at the same temperature for 6 h. After induction, the cells were harvested by centrifugation at 6,000 × g for 10 min at 4°C and then washed twice with 0.85% NaCl. The whole cells resuspended in ddH2O were used as the biocatalyst for the kinetic resolution of dl-mandelic acid.
Optimization of biotransformation conditions
To optimize the biotransformation conditions, 20-mL samples of the reaction mixture in a 100-mL flask were used. Ethylenediaminetetraacetic acid (EDTA) was added to a concentration of 20 mM in the reaction system. The biocatalysts were prepared from 12.5 g (DCW) L-1 of E. coli C43 (DE3) expressing the V108A mutant l-iLDH for the optimization of pH and temperature. The pH was adjusted from 4.0 to 10.0. The temperatures ranges were from 16°C to 58°C. The activity of whole-cell biocatalyst was judged by the concentration of benzoylformic acid produced. The dl-mandelic acid concentrations were 10.0–50.0 g·L-1, and the biotransformation was performed at 42°C at pH 7.0 for 12–54 h with 25 g (DCW) L-1 of biocatalyst.
Accurate concentrations of mandelic acid and benzoylformic acid were analyzed by high-performance liquid chromatography (HPLC, Agilent 1100 series, USA) using an Aminex HPX-87H column (Bio-Rad, USA) and the eluent using 10 mM H2SO4 solution at a flow rate of 0.4 mL·min-1. Samples from reaction systems during biocatalysis were centrifuged at 140,000 × g for 5 min to remove cells, and the supernatant was filtered by 0.22 μm pore size membrane filter for HPLC analysis.
Stereoselective assays of d-mandelic acid and l-mandelic acid were performed by HPLC analysis using a chiral column (DAICEL CHIRALCEL OJ-RH, Japan) and a tunable UV detector at 205 nm. The mobile phase consisted of 90% H2O (with 0.1% acetic acid added) and 10% acetonitrile (v/v) pumped at 0.4 mL·min-1 (15°C). Samples were prepared by the same procedure as using Aminex HPX-87H column. The optical purity of d-mandelate was expressed as enantiomeric excess (ee value), which was defined as the ratio of .
The work was supported by National Natural Science Foundation of China (31170052) and Chinese National Program for High Technology Research and Development (2011AA02A207).
The authors thank Professor Jan-Willem de Gier for providing E. coli C43 (DE3) for protein expression.
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