Bioresource Technology 100 (2009) 1878–1880

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Bioresource Technology

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Short Communication

Enantioselective oxidation of racemic lactic acid to D-lactic acid and pyruvic acid byPseudomonas stutzeri SDM

Chao Gao a,1, Jianhua Qiu a,1, Jingchen Li a, Cuiqing Ma a,*, Hongzhi Tang b, Ping Xu b

a State Key Laboratory of Microbial Technology, Shandong University, Shandananlu 27, Jinan 250100, People’s Republic of Chinab Key Laboratory of Microbial Metabolism, Ministry of Education, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China

a r t i c l e i n f o

Article history:Received 31 July 2008Received in revised form 25 September2008Accepted 26 September 2008Available online 11 November 2008

Keywords:D-Lactic acidPyruvic acidLactate oxidase componentsEnantioselective oxidationSeparation

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.09.053

* Corresponding author. Tel.: +86 531 88369463; faE-mail address: macq@sdu.edu.cn (C. Ma).

1 These authors contributed equally to this work.

a b s t r a c t

D-Lactic acid and pyruvic acid are two important building block intermediates. Production of D-lactic acidand pyruvic acid from racemic lactic acid by biotransformation is economically interesting. Biocatalystprepared from 9 g dry cell wt l�1 of Pseudomonas stutzeri SDM could catalyze 45.00 g l�1

DL-lactic acid into25.23 g l�1

D-lactic acid and 19.70 g l�1 pyruvic acid in 10 h. Using a simple ion exchange process, D-lacticacid and pyruvic acid were effectively separated from the biotransformation system. Co-production of D-lactic acid and pyruvic acid by enantioselective oxidation of racemic lactic acid is technically feasible.

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1. Introduction

Highly optically pure lactic acid is used as the feedstock mono-mer for biodegradable polylactic acid polymer production (Dattaand Tsai, 1995). Stereocomplex of poly L-lactic acid and poly D-lac-tic acid is characterized by its high melting temperature which af-fects the physical properties of the plastic (Tsuji, 2005). D-Lacticacid is also used as a chiral building block for the asymmetric syn-thesis (Su et al., 2004). These applications make the production ofD-lactic acid more important than before. However, D-lactic acid ismuch more difficult to produce than the L-enantiomer and produc-tion of optically pure lactic acid is dominated by L-lactic acid (Johnet al., 2007; Okino et al., 2008).

Pyruvic acid is also an important starting material widely ap-plied in the chemical, pharmaceutical, and agrochemical industries(Xu et al., 2008). Reflecting the variety of its application fields, thecommercial demand for pyruvic acid has been increasing. Co-pro-duction of D-lactic acid and pyruvic acid from the cheap substrateracemic lactic acid by biocatalysis would be a valuable process. Inthis report, racemic lactic acid was enantioselective oxidated toD-lactic acid and pyruvic acid by the L-lactate oxidase component(L-LOC) of Pseudomonas stutzeri SDM. After biotransformation,

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D-lactic acid and pyruvic acid could be separated by ion exchangechromatography.

2. Methods

2.1. Chemicals

L-Lactate was purchased from Sigma. D-Lactate was purchasedfrom Fluka. SD-4 anion exchange resin was purchased from Shan-dong Lukang Pharmaceutical Co., Ltd. DL-lactate which contained44% L-lactic acid and 56% D-lactic acid was purchased from WujiangCiyun Flavor and Fragrance Co., Ltd. All other chemicals were of re-agent grade.

2.2. Biocatalyst preparation

Crude extract of strain SDM was prepared according to the pre-vious report (Ma et al., 2007). After preincubated at different tem-peratures for different time, activities of the D- and L-LOC in thecrude extract were determined in 67 mM phosphate buffer (pH7.4) containing 20 mM L- or D-lactate. Reaction was initiated bythe addition of crude extract at 37 �C and then incubated for10 min. After stopping the reaction by 1 M HCl, the concentrationof pyruvic acid was determined. The preincubated crude extractwhich only exhibited the L-LOC activity was used as biocatalystin this report.

C. Gao et al. / Bioresource Technology 100 (2009) 1878–1880 1879

2.3. Optimization of biotransformation conditions

To optimize the biotransformation conditions, 50 ml samples ofthe reaction mixture in 500 ml flask was used with variations asfollows. The biocatalysts were prepared from 1.2 to 12 g dry cellwt l�1 [g (DCW) l�1] of P. stutzeri SDM. The ethylenediaminetetra-acetic acid (EDTA) concentrations were 2.5 to 15 mM. The DL-lacticacid concentrations were 15.00 to 75.00 g l�1. Biotransformationwas carried out at 30 �C and pH 7.0 for 2–30 h.

2.4. Separation of D-lactic acid and pyruvic acid

A column (1.6 � 20 cm) packed with 30 ml wet SD-4 anion ex-change resin was used for column separation (Ma et al., 2005).Thirty milliliters of biotransformational solutions was applied tothe column at a flow rate of 0.75 ml min�1. Then the column waswashed with 45 ml of water at a flow rate of 0.5 ml min�1 to eluteD-lactic acid. Loaded pyruvic acid was eluted by 2.0 M HCl at a flowrate of 0.75 ml min�1. The concentrations of D-lactic acid and pyru-vic acid in the eluate were determined by HPLC (Hao et al., 2007).

2.5. Stereoselective assays of D-lactic and L-lactic acid

Stereoselective assays of D-lactic and L-lactic acid were per-formed by HPLC analysis using a chiral column (MCI GEL CRS10 W,Japan) and a tunable UV detector at 254 nm. The mobile phase con-sisted of 2 mM copper sulfate pumped at 0.4 ml min�1 (25 �C). Theoptical purity of D-lactic acid was expressed as enantiomericexcess (ee value) which was defined as the ratio ofðd-lactic acidÞ � ðl-lactic acidÞðd-lactic acidÞ þ ðl-lactic acidÞ � 100%.

3. Results and discussion

3.1. Optimization of biotransformation by the biocatalyst from P.stutzeri SDM

Pseudomonas stutzeri SDM exhibited stereospecific D- and L-LOCactivities (Hao et al., 2007). For the stereospecific oxidation of L-lactic acid into pyruvic acid and preservation of D-lactic acid, theD-LOC in strain SDM needs to be inactivated while the L-LOC is pre-served. D- and L-LOC activities were affected differently by heattreatment. Complete inactivation of D-LOC activity occurred after5 min exposure at 55 �C, without affecting the L-LOC activity. Tak-ing advantage of the greater thermostability of the L-LOC activity,the crude extract of strain SDM incubated at 55 �C for 5 min wasused as the biocatalyst in the subsequent experiments.

Biotransformation was carried out with biocatalyst preparedfrom P. stutzeri SDM at different concentrations. Table 1 showsthe results of biotransformation with biocatalyst prepared from1.2 to 12 g (DCW) l�1 of P. stutzeri SDM. The biocatalyst preparedfrom 3 g (DCW) l�1 of P. stutzeri SDM was insufficient to carry the

Table 1Effects of Pseudomonas stutzeri SDM concentrations on the biotransformation.

Pseudomonas stutzeri SDMconcentration (g (DCW) l�1)

1.2 3 6 9 12

Reaction time (h) 12 12 10 4 4Pyruvic acid concentration (g l�1) 1.02 1.90 13.65 13.59 13.76Productivity (g pyruvic acid h�1 g�1 dry

weight)0.09 0.06 0.28 0.46 0.35

Residual L-lactic acid (g l�1) 12.38 11.69 <0.005Residual D-lactic acid (g l�1) 16.25 16.75 16.24 16.50 16.22Enantiomeric excess (%) 13.49 17.80 >99.5

Values are the average of three separate determinations.

reaction to completion. However, biocatalyst prepared from9 g (DCW) l�1 of P. stutzeri SDM had the highest specific biotrans-formation rate and the reaction was complete in 4 h.

The effect of the concentrations of DL-lactic acid on biotransfor-mation was investigated to determine its optimal range. As shownin Table 2, the biotransformation efficiency decreased as the DL-lac-tic acid concentration increased. However, the reaction with45.00 g l�1

DL-lactic acid as the substrate exhibited relatively highbiotransformation efficiency, high concentration of D-lactic acidand pyruvic acid, and high optical purity of D-lactic acid.

The degradation of pyruvic acid decreased the bioconversion ra-tio and the by-products could greatly influence the downstreamproces. Addition of EDTA was needed for stabilizing the pyruvicacid in other reports (Hao et al., 2007). In this work, the effect ofEDTA on the biotransformation was also studied. However, no dif-ference in biotransformation efficiency, pyruvic acid yield or ee ofD-lactic acid was detected when different EDTA concentrationswere used. It is possible that the pyruvate-metabolizing enzymesmight be inactivated by the heat preincubation, allowing the L-lac-tic acid to be transformed into pyruvic acid with high yield and noproduction of other organic acid was detected. Elimination of EDTAaddition would simplify the biotransformation process and prod-uct separation.

Combining the results above, an optimal biotransformation sys-tem was developed for enantioselective oxidation of racemate lac-tic acid. Biocatalyst prepared from 9 g (DCW) l�1 of P. stutzeri SDMcould catalyze 45.00 g l�1

DL-lactic acid into 25.23 g l�1D-lactic acid

and 19.70 g l�1 pyruvic acid in 10 h. The biotransformation pro-duced high enantiomeric excess (>99.5%) of D-lactic acid at highD-lactic acid concentrations which were much higher than otherbiocatalysis process (Motosugi et al., 1984; Su et al., 2004).Although genetically modified Escherichia coli TG113 (Grabaret al., 2006) and Corynebacterium glutamicum DldhA/pCRB204(Okino et al., 2008) have been previously used to produce high con-centrations of D-lactate than biocatalysis, the simple active conver-sion system in this report is a promisingly alternative because ituses the cheaper starting material DL-lactic acid and brings highyields of two building block intermediates (D-lactic acid and pyru-vic acid) by a one-pot biotransformation method.

3.2. Separation of D-lactic acid and pyruvic acid

Recently, separation methods of lactic acid (Cao et al., 2002;Thang and Novalin, 2008) and pyruvic acid (Zelic et al., 2004; Maet al., 2006) have received increasing attention. Because of the sim-ple composition of the reaction mixture in the biotransformationsystem recovery of the D-lactic acid and pyruvic acid is convenientto perform. Separation of L-lactic acid from pyruvic acid in bio-transformation solutions by ion exchange chromatography wasperformed in our previous work (Ma et al., 2005). The SD-4 anionexchange resin was also used to separate D-lactic acid from pyruvicacid in this study. Column separation curves of D-lactic acid andpyruvic acid at pH 2.0 are shown in Fig. 1. The D-lactic acid concen-tration in the loading stage was very low. The elution of D-lactic

Table 2Effects of DL-lactic acid concentration on the biotranformation.

DL-lactic acid concentration (g l�1)

15 30 45 60 75

Reaction time (h) 2 4 10 20 30Pyruvic acid concentration (g l�1) 5.98 13.91 19.70 24.96 21.99Biotransformation efficiency (g l�1 h�1) 3.19 3.38 2.12 1.42 0.94Residual L-lactic acid (g l�1) <0.005 11.73Residual D-lactic acid (g l�1) 7.80 16.53 25.23 34.63 43.05Enantiomeric excess (%) >99.5 59.83

Values are the average of three separate determinations.

0 10 20 30 40 50 60 70 80 90 100 110 120 130

0

10

20

30

40

D-l

actic

aci

d &

pyr

uvic

aci

d (g

l-1)

Volume (ml)

Fig. 1. Column separation of D-lactic and pyruvic acid on SD-4 anion exhange resin.(j) D-lactic acid; (d) pyruvic acid.

1880 C. Gao et al. / Bioresource Technology 100 (2009) 1878–1880

acid from the SD-4 anion exchange resin could be easily performedusing water as eluant. Washing the column with water resulted ina high D-lactic acid recovery of 91.2%. After washing with water,the pyruvic acid was eluted from the resin by 1 M HCl with a yieldof 87.1%. The D-lactic acid and pyruvic acid were perfectly sepa-rated by this simple ion exchange process. This separation processis feasible and might be coupled with the biotransformation.

4. Conclusions

In conclusion, we introduced an enantioselective oxidation pro-cedure to produce D-lactic acid and pyruvic acid from cheap race-mic lactic acid. Using the ion exchange method, recovery ofD-lactic acid and pyruvic acid from the simple reaction liquid isrelatively easy and inexpensive to perform. The enantioselectiveoxidation of racemic lactic acid into higher-value D-lactic acidand pyruvic acid is a potentially valuable process.

Acknowledgements

The work was supported by the National Natural Science Foun-dation of China (Grant No. 20676072), and by the grants from the

Ministry of Science and Technology of China, National Basic Re-search Program of China (2007CB707803) and Chinese NationalPrograms for High Technology Research and Development(2006AA020100 and 2006AA02Z244). The authors thank Dr. Pam-ela Holt for editing the manuscript.

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