Bioresource Technology 102 (2011) 4595–4599

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Bioresource Technology

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Kinetic resolution of 2-hydroxybutanoate racemic mixtures by NAD-independentL-lactate dehydrogenase

Chao Gao a,b, Wen Zhang a, Cuiqing Ma a,⇑, Peng Liu a, Ping Xu a,b,⇑a State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of Chinab MOE Key Laboratory of Microbial Metabolism and School of Life Sciences & 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 5 November 2010Received in revised form 4 January 2011Accepted 4 January 2011Available online 7 January 2011

Keywords:D-2-Hydroxybutanoate2-OxobutanoateNAD-Independent L-lactate dehydrogenaseKinetic resolutionIon exchange

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.01.003

⇑ Corresponding authors. Address: School of LifeShanghai Jiao Tong University, Shanghai 200240, PeopTel.: +86 21 34206647; fax: +86 21 34206723 (P. Xu),+86 531 88369463 (C. Ma).

E-mail addresses: macq@sdu.edu.cn (C. Ma), pingx

a b s t r a c t

Optically active D-2-hydroxybutanoate is an important building block intermediate for medicines andbiodegradable poly(2-hydroxybutanoate). Kinetic resolution of racemic 2-hydroxybutanoate may be agreen and desirable alternative for D-2-hydroxybutanoate production. In this work, D-2-hydroxybutano-ate at a high concentration (0.197 M) and a high enantiomeric excess (99.1%) was produced by anNAD-independent L-lactate dehydrogenase (L-iLDH) containing biocatalyst. 2-Oxobutanoate, anotherimportant intermediate, was co-produced at a high concentration (0.193 M). Using a simple ion exchangeprocess with the macroporous anion exchange resin D301, D-2-hydroxybutanoate was separated fromthe biotransformation system with a high recovery of 84.7%.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction electron donor NADH and thus is difficult to undertake on an indus-

2-Hydroxybutanoate is an important raw material for produc-tion of isoleucine, 2-oxobutanoate, and some kinds of medicines(Gao et al., 2010; Nakajima et al., 1994; Scheer et al., 1987,1988). In particular, highly optically pure 2-hydroxybutanoatecan be used as a feedstock monomer for production of poly(2-hydroxybutanoate), a polymer which is susceptible to hydrolyticdegradation and thus can be utilized as a biodegradable materialfor biomedical, pharmaceutical, and environmental applications(Tsuji and Okumura, 2009). As for D-2-hydroxybutanoate, it couldalso be used to produce azinothricin family of antitumour antibiot-ics (Karl et al., 1995; Nakagawa et al., 2007). Therefore, develop-ment of practicable techniques for production of optically activeD-2-hydroxybutanoate is desirable.

Chemical processes for 2-hydroxybutanoate production result ina racemic mixture of both stereospecific forms. Utilization of chem-ical catalysts has also made the green production of 2-hydroxybut-anoate difficult to accomplish. Several methods using biocatalystshave been reported for formation of optically active D-2-hydroxy-butanoate. For example, 2-oxobutanoate, the oxidation product of2-hydroxybutanoate, could be reduced to D-2-hydroxybutanoateby an NAD-dependent D-lactate dehydrogenase (D-nLDH) (Simonet al., 1989). This process depends on the addition of expensive

ll rights reserved.

Sciences and Biotechnology,le’s Republic of China (P. Xu).tel.: +86 531 88364003; fax:

u@sjtu.edu.cn (P. Xu).

trial scale. Racemic 2-hydroxybutanoate, which is much cheaperthan 2-oxobutanoate (Gao et al., 2010), can replace 2-oxobutanoatefor D-2-hydroxybutanoate production through kinetic resolution.The resolution can be performed through lipase-catalyzed enantio-selective acetylation or glycolate oxidase-catalyzed enantioselec-tive oxidation (Adam et al., 1997, 1998a, 1998b). Althoughoxidative resolution of the racemate produces low concentrationsof D-2-hydroxybutanoate, this process is promising because of itslow substrate price and exclusion of cosubstrate addition.

NAD-independent L-lactate dehydrogenase (L-iLDH), whichcould catalyze the oxidation of L-2-hydroxybutanoate with a fla-vin-dependent mechanism, has been documented in previousworks (Futai and Kimura, 1977; Gao et al., 2010; Jasso-Chávezet al., 2001; Ma et al., 2007). This enzyme might also be used inthe kinetic resolution of racemic 2-hydroxybutanoate into D-2-hydroxybutanoate. To identify the application potential of L-iLDHin D-2-hydroxybutanoate production, kinetic resolution of racemic2-hydroxybutanoate by L-iLDH containing biocatalyst was con-ducted in this work. The separation between D-2-hydroxybutano-ate and 2-oxobutanoate, another valuable product of the kineticresolution process, was also studied.

2. Methods

2.1. Chemicals

D-2-Hydroxybutanoate, L-2-hydroxybutanoate, racemic 2-hydroxybutanoate and 2-oxobutanoate were all purchased from

Table 1Substrate specificity of L-iLDH activity in P. stutzeri SDM.

Substrate Km (mM) Vmax (lmol min�1 mg�1)

L-Lactate 22.9 ± 1.1 13.2 ± 0.3

L-2-Hydroxybutanoate 62.7 ± 3.4 15.1 ± 0.2

Values are the average ± SD of three separate determinations.

4596 C. Gao et al. / Bioresource Technology 102 (2011) 4595–4599

Sigma. The adsorbents were provided by Shandong Lukang Phar-maceutical Co. Ltd. (PR China). All other chemicals were of reagentgrade.

2.2. Microorganism and growth conditions

Pseudomonas stutzeri SDM (China Center for Type Culture Col-lection No. M206010) isolated from soil was used in this study(Ma et al., 2007). The minimal salt medium (MSM) was supple-mented with 5.0 g 1�1

DL-lactate as the seed medium and with10.0 g 1�1

DL-lactate as the fermentation medium (Ma et al.,2007). P. stutzeri SDM cells were inoculated into 50 ml seed med-ium in a 300 ml flask and incubated with shaking at 30 �C for10 h. Then they were inoculated into a 5 l flask with 1 l fermenta-tion medium and cultivated at 30 �C for 16 h.

2.3. Biocatalyst preparation

The cells of strain SDM grown in MSM containing DL-lactate asthe carbon source were harvested at the late log phase. The har-vested cells were then washed and resuspended with distilledwater (pH 7.4). After preincubated at 55 �C for 15 min, the wholecells were used as the biocatalyst for kinetic resolution of DL-2-hydroxybutanoate.

2.4. Kinetic resolution of DL-2-hydroxybutanoate

For kinetic resolution of racemic 2-hydroxybutanoate, a 50 mlreaction mixture containing 6 g dry cell weight l�1 preincubatedP. stutzeri SDM and 0.4 M DL-2-hydroxybutanoate was incubatedin a 500 ml flask. Reactions were carried out at 30 �C and150 rpm on a reciprocating shaker. After stopping the reaction byadding of 1 M HCl, the final concentrations of 2-oxobutanoateand 2-hydroxybutanoate were determined.

2.5. Adsorbents tests

Adsorption was carried out with 20 ml reaction mixture at25 �C. The fresh resin was treated with 10 times the volume of1.0 M NaOH, water, 1.0 M HCl, and water in turn. Calculated capac-ities are based on the dry weights calculated from dried samples.

2.6. Adsorption characteristics of resin

A column (20 cm � 16 mm) packed with 15 ml wet resin wasused for column separation at room temperature. The columnwas washed with water after the resin was regenerated with1.0 M HCl. Different pH values of reaction mixture were chosento determine the effect of pH on D-2-hydroxybutanoate and 2-oxo-butanoate adsorption. The initial pH value of the solution was ad-justed by 1.0 M HCl.

2.7. Analytical procedures

The activity of L-iLDH was determined at 30 �C in 1 ml of 50 mMTris–HCl, pH 7.5, 0.2 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT), and 0.02–0.05 dry cell weight l�1

whole cells of P. stutzeri SDM. The reaction was started by additionof 10 mM L-lactate or L-2-hydroxybutanoate, and the rate of MTTreduction was determined by measuring the absorbance changesat 578 nm in 1 minute (Ma et al., 2007). One unit of enzyme activ-ity is defined as the amount of iLDH required to catalyze 1 lmol ofsubstrate oxidation per minute.

Accurate concentrations of 2-oxobutanoate and 2-hydroxybut-anoate were analyzed by high-performance liquid chromatography(HPLC, Agilent 1100 series, Hewlett–Packard, USA) using an

Aminex HPX-87H column (Bio-Rad) and the eluent using 10 mMH2SO4 solution at 1 ml min�1 flow-rate (Hao et al., 2007).Stereoselective assays of D-2-hydroxybutanoate and L-2-hydroxy-butanoate were performed by HPLC analysis using a chiral column(MCI GEL CRS10 W, Japan) and a tunable UV detector at 254 nm(Gao et al., 2009). The mobile phase was a mixture of water andacetonitrile (90:10), containing 2 mM copper sulphate. The opticalpurity of D-2-hydroxybutanoate was expressed as enantiomericexcess which was defined as the ratio of

ðD� 2� hydroxybutanoateÞ � ðL� 2� hydroxybutanoateÞðD� 2� hydroxybutanoateÞ þ ðL� 2� hydroxybutanoateÞ � 100

3. Results and discussion

3.1. Identification of D-2-hydroxybutanoate production feasibility

Addition of DL-lactate in culture medium induced the expressionof L-iLDH and NAD-independent D-lactate dehydrogenase (D-iLDH)in P. stutzeri SDM (Ma et al., 2007). Different thermostabilities ofL-iLDH and D-iLDH have been documented in previous work. L-iLDHis more thermostable than D-iLDH (Ma et al., 2007). Thus, the bio-catalyst with only L-iLDH activity, could be prepared through pre-incubation of P. stutzeri SDM under proper temperature (seeSupplementary Material).

Lactate is taken up into the Escherichia coli cell either by theL-lactate permease or by the glycolate permease in previousreports (Núñez et al., 2002). Because of the similar structure of2-hydroxybutanoate and lactate, we expected that the 2-hydroxy-butanoate might also be transported into P. stutzeri SDM by thosetransporters. On the other hand, iLDHs often play their essentialrole in the aerobic utilization of lactate through electron transportchain (Castro-Guerrero et al., 2005; Moreno-Sánchez et al., 2000;Philippe et al., 2004). The L-lactate oxidation activity with oxygenas the electron acceptor (including the L-iLDH and some compo-nents of electron transport chain) is also not affected by heat pre-incubation under proper temperature (see SupplementaryMaterial). As shown in Table 1, L-iLDH in P. stutzeri SDM alsoexhibited stereospecific NAD-independent L-2-hydroxybutanoatedehydrogenase (L-iBDH) activity. Thus, the preincubated wholecells of P. stutzeri SDM might have the ability to enantioselectivelyoxidize racemic 2-hydroxybutanoate into D-2-hydroxybutanoateand 2-oxobutanoate.

Kinetic resolution of racemic 2-hydroxybutanoate was con-ducted with preincubated whole cells of P. stutzeri SDM as the bio-catalyst. As shown in Fig. 1, D-2-hydroxybutanoate, which was notoxidized in the biocatalytic process, accumulated with a high con-centration (0.197 M) and a high enantiomeric excess (99.1%) (seeSupplementary Material). Reaction with higher concentrations ofracemic 2-hydroxybutanoate would result in a much lower enan-tiomeric excess of D-2-hydroxybutanoate (see supplementarymaterial).

As shown in Table 2, different biocatalysts such as lipase, glyco-late oxidase, 4-chloro-3-hydroxybutanoate hydrolase, and horse-radish peroxidase have been used in production of opticallyactive D-2-hydroxybutanoate (Adam et al., 1995, 1997, 1998a ,

0 2 4 6 8 10 12

0.00

0.05

0.10

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0.20

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Fig. 1. Time course of kinetic resolution of racemic 2-hydroxybutanoate bypreincubated whole cells of P. stutzeri SDM. (h) L-2-hydroxybutanoate; (d)2-oxobutanoate; (j) D-2-hydroxybutanoate. Accurate concentrations of 2-oxobut-anoate and 2-hydroxybutanoate in the reaction mixture were analyzed by HPLC atthe indicated times. Values are the average ± SD of three separate determinations.

Table 3Screening of adsorbents for D-2-hydroxybutanoic acid and 2-oxobutanoic acidseparation.

Adsorbent Type Adsorption capacity (mmol g�1)

D-2-hydroxybutanoic acid 2-oxobutanoic acid

D315 Acrylic 0.247 ± 0.006 0.321 ± 0.009D301 Styrene 0.528 ± 0.019 0.711 ± 0.015D345 Phenolic 0.354 ± 0.009 0.404 ± 0.012335 Epoxy 0.351 ± 0.014 0.433 ± 0.013318 Acrylic 0.274 ± 0.009 0.379 ± 0.007330 Epoxy 0.364 ± 0.013 0.476 ± 0.010D941 Acrylic 0.259 ± 0.011 0.341 ± 0.008CAD45 Styrene 0.041 ± 0.002 0.034 ± 0.002CAD40 Styrene 0.042 ± 0.001 0.064 ± 0.003D311 Acrylic 0.159 ± 0.007 0.100 ± 0.005

Adsorption capacities of 10 sorts of adsorbents were studied in reaction mixture(pH 7.0) at 25 �C. The ratio of the solution volume to the adsorbent was 20 ml to 5 g.Values are the average ± SD of three separate determination.

C. Gao et al. / Bioresource Technology 102 (2011) 4595–4599 4597

1998b; Nakagawa et al., 2007). The process utilizing 4-chloro-3-hydroxybutanoate hydrolase resulted in the highest product(D-2-hydroxybutanoate ester) concentration and has been used inpractical production of D-2-hydroxybutanoate (Nakagawa et al.,2007). The kinetic resolution catalyzed by L-iLDH also producedhigh concentration of D-2-hydroxybutanoate at a high enantio-meric excess. This route excluded the hydrolysis of D-2-hydroxy-butanoate ester for preparation of D-2-hydroxybutanoate. Thus,practical production of D-2-hydroxybutanoate might be also per-formed using L-iLDH catalyzed kinetic resolution.

On the other hand, the kinetic resolution process also produced0.193 M 2-oxobutanoate, which was an important raw material insynthesis of chiral 2-aminobutyric acid, isoleucine, and some kindsof medicines (Eggeling et al., 1987; Furuyoshi et al., 1991; Gaoet al., 2010 ). This process used the cheap starting material racemic2-hydroxybutanoate and produced two important block interme-diates with high concentrations. Thus, it is a promising alternativefor the biotechnological production of D-2-hydroxybutanoate and2-oxobutanoate.

3.2. Screening for adsorbents

In order to simplify the isolation of D-2-hydroxybutanoate and2-oxobutanoate, a distilled water system was used in the kineticresolution of DL-2-hydroxybutanoate. Due to the simple composi-tion of the resultant reaction mixture, D-2-hydroxybutanoate and2-oxobutanoate were the main components needing separation(see Supplementary Material). In this work, ion exchange, a

Table 2Comparison of the processes for D-2-hydroxybutanoate production.

Enzyme Substrate

Horseradish peroxidase Racemic 2-hydroperoxy esters4-Chloro-3-hydroxybutyrate hydrolase Racemic 2-hydroxybutanoate esterGlycolate oxidase Racemic 2-hydroxybutanoateGlycolate oxidase and D-nLDH Racemic 2-hydroxybutanoateLipase Racemic 2-hydroxybutanoatel-iLDH Racemic 2-hydroxybutanoate

practical method in industry because of its economy and facility,was utilized in the D-2-hydroxybutanoate recovery process.

As shown in Table 3, the macroporous anion exchange resinD301 resin had higher adsorption capacity than other adsorbents.Its capacities were 0.528 mmol g�1 for D-2-hydroxybutanoate and0.711 mmol g�1 for 2-oxobutanoate, respectively.

3.3. Adsorption characteristics of D301 under different pHs

Both D-2-hydroxybutanoic acid and 2-oxobutanoic acid areweak acids and have different pKa. The dissociation of D-2-hydroxybutyric acid and 2-oxobutyric acid can be shifted underdifferent pHs. The pH value of reaction mixture would have signif-icant effect on the adsorption property of ion exchange adsorbent.A column (20 cm � 16 mm) packed with 15 ml wet resin was usedfor separation of D-2-hydroxybutyric acid and 2-oxobutyric acid.

The breakthrough curves of D-2-hydroxybutyric acid and 2-oxobutyric acid at pH 2.4, 3.5, and 4.6 were shown in Fig. 2.Selectivity is the critical factor in the separation process of D-2-hydroxybutyric acid and 2-oxobutyric acid. Compared with the re-sults at pH 2.4 and 4.6 (Figs. 2a and c), the operation at pH 3.5(Fig. 2b) got the best separation of D-2-hydroxybutyric acid and2-oxobutyric acid. Thus, the following work was conducted at pH3.5.

3.4. Separation of D-2-hydroxybutyric acid and 2-oxobutyric acid

Reaction mixtures (40 ml, pH 3.5) were applied on the D301 ex-change resin column at a flow rate of 0.5 ml min�1. Then the col-umn was washed with 60 ml of water at a flow rate of0.5 ml min�1 for elution of D-2-hydroxybutyric acid. Loaded 2-oxobutyric acid was eluted by 1.0 M HCl at a flow rate of0.5 ml min�1.

D-2-Hydroxybutanoateconcentration (mM)

Enantiomericexcess (%)

Reference

1.5 97 Adam et al., 1995s 340 98.5 Nakagawa et al., 2007

0.025 >99 Adam et al., 19970.4 95 Adam et al. 1998a0.05 77 Adam et al., 1998b197 99.1 This study

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Fig. 2. Breakthrough curve of D-2-hydroxybutanoic acid and 2-oxobutanoic acid onmacroporous anion exchange resin D301. After the pH was adjusted to a desired pH,reaction mixtures containing 0.197 M D-2-hydroxybutanoic acid and 0.193 M2-oxobutanoic acid were applied on the column at a flow rate of 0.5 ml min�1.Effluent was collected and the D-2-hydroxybutanoic acid and 2-oxobutanoic acidconcentrations were measured by HPLC. (a) pH 2.4; (b) pH 3.5; (c) pH 4.6. (j) D-2-hydroxybutanoic acid; (d) 2-oxobutanoic acid. Values are the average ± SD of threeseparate determinations.

0 20 40 60 80 100 120 140 160 180

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Fig. 3. Column separation of D-2-hydroxybutanoic acid and 2-oxobutanoic acid onmacroporous anion exchange resin D301. Accurate concentrations of D-2-hydrox-ybutanoic acid and 2-oxobutanoic acid in the effluent were analyzed by HPLC. (j)D-2-Hydroxybutanoic acid; (d) 2-oxobutanoic acid. Values are the average ± SD ofthree separate determinations.

4598 C. Gao et al. / Bioresource Technology 102 (2011) 4595–4599

Column separation curves of D-2-hydroxybutyric acid and 2-oxobutyric acid are shown in Fig. 3. After the loading stage, wash-ing the column with water resulted in a D-2-hydroxybutyric acidrecovery of 84.7%. The 2-oxobutyric acid was eluted from the resinby 1 M HCl with yield of 95.7%. Although the purity of 2-oxobutyricacid was not high (81.5%), the ion exchange process got a good sep-aration of D-2-hydroxybutyric acid from the reaction mixture (withthe purity of 98.7%, see Supplementary Material).

2-Hydroxycarboxylic acids and 2-oxocarboxylic acids could beproduced by biotechnological routes (Adam et al., 1997, 1998a,1998b; Causey et al., 2003; Furuyoshi et al., 1991; Gao et al.,

2009; Gao et al., 2010; Qin et al., 2009; Qin et al., 2010; Wanget al., 2010; Zhao et al., 2010a,b). Separation methods such as reac-tive extraction, ion exchange, and electro-dialysis have been usedin recovery of those carboxylic acids (Cao et al., 2002; Ma et al.,2006; Thang and Novalin, 2008; Zelic et al., 2004). Most of thosemethods face the separation of the target product from the minor-ity impurities and might be used in recovery of 2-oxobutyric acidfrom the effluent gotten in this work. On the other hand, enantio-selective oxidation of racemic 2-hydroxycarboxylic acids has beenextensively studied for production of optically pure 2-hydroxy-carboxylic acids. (Adam et al., 1997, 1998a; Gao et al., 2009). Thoseprocesses would result in a mixture of 2-hydroxycarboxylic acidsand 2-oxocarboxylic acids with approximate concentrations. Theion exchange process using the resin D301 gave a good examplefor separation of optically pure 2-hydroxycarboxylic acids fromits relevant 2-oxocarboxylic acids. Thus, it might be coupled withother enantioselective oxidation processes.

4. Conclusions

A simple kinetic resolution process was utilized to produce D-2-hydroxybutanoate from racemic 2-hydroxybutanoate. Using bio-catalyst which exhibited L-iLDH activity, D-2-hydroxybutanoate ata high concentration (0.197 M) and a high enantiomeric excess(99.1%) was produced. The separation of D-2-hydroxybutanoatewith 2-oxobutanoate, another important intermediate, was real-ized by the ion exchange process using resin D301. The green con-version system should be a promising one for the practicalproduction of D-2-hydroxybutanoate, because it uses the cheaperstarting material DL-2-hydroxybutanoate and brings higher yieldsof two building block intermediates (D-2-hydroxybutanoate and2-oxobutanoate) by a one-pot biotransformation method.

Acknowledgements

The work was supported by National Natural Science Foundationof China (31000014, 31070062), Major State Basic ResearchDevelopment Program of China (2007CB714303), China Postdoc-toral Science Foundation (20100480600) and Research Fundfor the Doctoral Program of Higher Education of China(20090131110036).

C. Gao et al. / Bioresource Technology 102 (2011) 4595–4599 4599

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2011.01.003.

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