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Process Biochemistry 45 (2010) 1912–1915

Contents lists available at ScienceDirect

Process Biochemistry

journa l homepage: www.e lsev ier .com/ locate /procbio

yruvate producing biocatalyst with constitutive NAD-independent lactateehydrogenases

hao Gaoa, Xiaoman Xua, Chunhui Hua, Wen Zhanga, Ye Zhanga, Cuiqing Maa,∗, Ping Xub

State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People’s Republic of ChinaMOE Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 14 November 2009eceived in revised form 19 May 2010ccepted 24 May 2010

a b s t r a c t

Production of pyruvate from lactate through biocatalysis is a valuable process for its simple composi-tion of reaction system and convenience of recovery. Biocatalyst with lactate-induced NAD-independentlactate dehydrogenases (iLDHs) can effectively catalyze lactate into pyruvate. To reduce the cost of bio-

eywords:iocatalystAD-Independent lactate dehydrogenaseonstitutive expressionyruvate

catalyst preparation caused by indispensable lactate addition, the mutants with constitutive iLDH ofPseudomonas sp. XP-M2 were screened. Mutant XP-LM exhibited high iLDHs activities in minimal saltmedium with cheap substrate glucose as the carbon source. The biocatalyst (8.2 g dry cell weight l−1)containing 169.8 U l−1 l-iLDH was prepared with 20 g 1−1 glucose. The cost-effective biocatalyst preparedfrom the mutant XP-LM could efficiently catalyze lactate into pyruvate with high yield (0.961 mol mol−1).Based on the different thermostability of d-iLDH and l-iLDH in the biocatalyst, whole cells of the strain

ntial i

actate might also have the pote

. Introduction

Pyruvate is an important platform chemical widely applied inhemical, pharmaceutical, and agrochemical industries [11,15,21].t can be produced by chemical or biotechnological processes. Withegards to the issues of environmental protection, the pyruvateroduction by biotechnology methods has been a research focus11,21].

The biotechnological production of pyruvate could be per-ormed through direct fermentative or biocatalysis processes7,14,15,19]. Although the fermentation process currently playsdominant role in biotechnological production of pyruvate, bio-

atalysis processes are also desirable, for the simple compositionf reaction mixture and the high conversion rate of substrate, asell as the convenience of recovery of pyruvate [11,21].

Among the substrates reported for pyruvate production, lac-ate, which is now commercially produced from sugars presentedn biomass, has directed researchers’ attention [3,10,16,17,21].ifferent enzymes such as glycolate oxidase, NAD-dependent lac-

ate dehydrogenases, NAD-independent lactate dehydrogenases

iLDHs) and lactate oxidase might be employed in the enzymaticynthesis of pyruvate from lactate [7,12,21]. Compared with othernzymes, the iLDHs catalyzed lactate oxidation avoiding the hydro-en peroxide formation and the cofactor regeneration processes

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

359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2010.05.029

n production of pyruvate and d-lactate from racemic lactate.© 2010 Elsevier Ltd. All rights reserved.

[12,21]. Hence, the whole cells containing iLDHs might have poten-tial for a commercial production of pyruvate. However, inducementof iLDHs in different strains needed the addition of lactate, whichmade the biocatalyst preparation cost high [1,8,9]. With the targetof low cost biocatalyst preparation, the mutants with constitutiveiLDH of Pseudomonas sp. XP-M2 for the biocatalysis were screenedin this study. After preparation of the biocatalyst from the cheapersubstrate glucose, the feasibility of pyruvate production using amutant as biocatalyst was also investigated.

2. Materials and methods

2.1. Bacterial strains, media, and growth conditions

Pseudomonas sp. XP-M2 was used as the wild type [20]. For growth exper-iments and determination of the iLDHs activities, strain XP-M2 was cultured inLuria–Bertani (LB) medium and then inoculated into a 500-ml baffled shake flaskcontaining LB medium or minimal salt medium (MSM) supplemented with l-lactate,glucose or pyruvate as the sole carbon source at 37 ◦C and 120 rpm. The MSM con-tained 1.0 g NH4Cl, 1.1 g KH2PO3·3H2O, 0.9 g K2HPO4, 0.5 g MgSO4·7H2O, and 1.2 mltrace elements solution in 1 l of distilled water. The trace elements solution consistedof 50.0 g Na2EDTA, 5.5 g CaCl2, and 5.0 g FeSO4·7H2O in 1 l of distilled water.

2.2. Isolation of the mutants with constitutive iLDH

After being cultured in LB medium at 37 ◦C for 10 h, the cells of Pseudomonassp. XP-M2 were collected and suspended in sterilized physiological salt solution

and then implanted with ion beam. The energy used was 30 keV and the dosagesof N+ implantation were 1 × 1014 ions/cm2, 5 × 1014 ions/cm2, 1 × 1015 ions/cm2,5 × 1015 ions/cm2, and 1 × 1016 ions/cm2. The mutants with constitutive iLDH wereobtained on the lactate/glycollate agar plate according to the previous report [2].Portions (0.1 ml) were spread on MSM containing 5.0 g 1−1 l-lactate and 5.0 g 1−1

glycollate and incubated at 37 ◦C. Mutants picked at random were purified by

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espreading on the isolation medium and iLDHs activities of the mutants in LBedium were examined.

.3. Analytical procedure

The cells of strain were harvested, washed, resuspended in 67 mM phos-hate buffer (pH 7.4) containing 10 mM dithiothreitol (DTT), 5 mM MgSO4, andmM ethylenediaminetetraacetic acid (EDTA) and disrupted by sonication (Son-

cs 500 W/20 kHz, USA). The disrupted cells were centrifuged for 5 min at 8000 × g,nd the supernatant was used as the crude cell extract. The activities of iLDHsere determined at 30 ◦C in 1 ml of 50 mM Tris–HCl, pH 7.5, 0.2 mM 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and the crude cellxtract or whole cells. The reaction was started by addition of 50 mM l-lactate ormM d-lactate, and the rate of MTT reduction was determined by measuring thebsorbance changes at 578 nm. One unit of iLDH activity was defined as the amountf reducing 1.0 �mol of MTT per minute under the test conditions. The specific activ-ties of iLDH were defined on the basis of milligram crude proteins in the cell extractsr gram dry cell weight (DCW) of whole cells.

Accurate concentrations of pyruvate, lactate and glucose were determinted byigh-performance liquid chromatography (HPLC) (Agilent 1100 series, Hewlett-ackard, USA) at 210 nm [13]. The HPLC was fitted with a reverse-phase C18 column4.6 mm × 150 mm; Hewlett-Packard). The mobile phase consisted of sulfuric acid1 mM) and methanol (96:4) pumped at 0.4 ml min−1.

. Results and discussion

.1. Screening of the mutants with constitutive iLDH

Some Pseudomonas spp. utilized lactate for growth through theactate-induced iLDH. Glycollate inhibited the induction of iLDHsxpression by lactate in the Pseudomonas strains [2]. Therefore, theild strain XP-M2 could not grow on lactate/glycollate medium.nly the mutants whose iLDHs were constitutive could utilize the

actate for growth on lactate/glycollate medium. About 100 iso-ates were obtained from lactate/glycollate agar plate in this work.mong them, a mutant, designated strain XP-LM showed the high-st iLDHs activities in LB medium. Fig. 1 showed iLDHs activities ofhe mutant XP-LM grown in different carbon sources. In absence ofactate, mutant XP-LM exhibited much higher iLDH activities thanhe parent strain.

Regulation of iLDHs expression has been studied in Corynebac-erium glutamicum and Escherichia coli [4,6]. Binding of LldR, autative regulator of the lactate utilization operon to the pro-oter region of the operon has been demonstrated experimentally

n C. glutamicum [6]. Although the regulation of iLDHs expres-ion in Pseudomonas has never been studied, we speculate thathere might have a regulator involved in the iLDHs expressionrocess. The constitutive expression of iLDHs activities in Pseu-omonas sp. XP-LM might be caused by the mutant of putativeegulator.

.2. Selection of nitrogen sources

The mutant XP-LM could grow with glucose and exhibit high

LDHs activities, which made the preparation of iLDHs contain-ng biocatalyst from cheap substrate glucose rather feasible. Theiocatalyst preparation from strain XP-LM was carried out withlucose and seven different nitrogen sources, including ammo-ium chloride, ammonium sulfate, ammonium nitrate, ammonium

able 1ffect of nitrogen sources on the activities of iLDHs in Pseudomonas sp. XP-LM.

Nitrogen source l-iLDH (U g−1) d-iL

Diammonium hydrogen phosphate 33.84 ± 4.08 13.8Ammonium sulfate 21.66 ± 2.89 4.7Ammonium nitrate 39.96 ± 2.88 20.9Ammonium acetate 31.96 ± 1.81 14.9Ammonium chloride 18.14 ± 2.70 13.9Ammonium bicarbonate 20.40 ± 0.46 14.3Sodium nitrate 36.26 ± 0.33 12.0

Fig. 1. Effects of carbon sources on the l- and d-iLDH activities in Pseudomonas sp.XP-LM (left sparse) and Pseudomonas sp. XP-M2 (right sparse). (a) l-iLDH activitiesand (b) d-iLDH activities.

acetate, ammonium bicarbonate, sodium nitrate and diammoniumhydrogen phosphate. MSM medium at pH 7.0 containing 1 g l−1 ofnitrogen source and 5 g l−1 of glucose was used; and the culturewas incubated at 37 ◦C for 12 h. As shown in Table 1, biocatalystprepared with ammonium nitrate as the nitrogen source had thehighest specific iLDHs activities. Therefore, the ammonium nitratewas selected as nitrogen source.

3.3. Selection of nitrogen and carbon source concentrations

The effects of concentrations of glucose and ammonium nitrateon the specific growth rate of the mutant XP-LM were investi-

gated. The ammonium nitrate concentration was firstly varied from0.25 g l−1 to 2.0 g l−1, while the glucose concentration was fixedat 5 g l−1. As shown in Fig. 2a, the maximum value of the specificgrowth rate was 0.68 h−1 with 0.75 g l−1 of ammonium nitrate. Thenthe ammonium nitrate concentration was fixed at 0.75 g l−1, while

DH (U g−1) Cell density (g DCW l−1) pH

0 ± 2.65 1.19 ± 0.09 3.64 ± 0.076 ± 0.23 1.29 ± 0.06 3.54 ± 0.018 ± 2.90 1.78 ± 0.12 3.56 ± 0.023 ± 0.79 0.33 ± 0.01 4.3 ± 0.126 ± 0.82 1.28 ± 0.12 3.67 ± 0.147 ± 0.62 1.84 ± 0.15 3.57 ± 0.053 ± 0.20 1.53 ± 0.16 6.67 ± 0.19

1914 C. Gao et al. / Process Biochemistry 45 (2010) 1912–1915

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ig. 2. Effects of the culture conditions on the specific growth rate of Pseudomonalucose concentrations on cell growth; (c) effect of pH on cell growth; and (d) effehe initial rate data.

he glucose concentration was varied from 2.5 g l−1 to 50 g l−1. Ashown in Fig. 2b, the maximum value of the specific growth rateccurred at 15–20 g l−1 glucose.

.4. Selection of pH and temperature

For the selection of pH, cell cultivation was carried out in MSMedium containing 20 g l−1 of glucose and 0.75 g l−1 of ammonium

itrate, and the pH was adjusted from 3.0 to 10.0. As shown in

ig. 2c, the optimal value of pH for growth of the mutant XP-LMas found to be 5.0–7.0. The effect of the temperature on specific

rowth rate of the mutant XP-LM was investigated in the MSMedium at pH 7.0. As shown in Fig. 2d, the maximum value of the

pecific growth rate occurred at 37 ◦C.

ig. 3. (a) Time-course of batch culture of Pseudomonas sp. XP-LM. (�) l-iLDH activity; (�

p. XP-LM. 10 mM EDTA was added in the biocatalysis system to inhibit the pyruvate deg

P-LM: (a) effect of ammonium nitrate concentrations on cell growth; (b) effect ofmperature on cell growth. The specific growth rate was calculated on the basis of

3.5. Biocatalyst preparation under optimal conditions

Batch fermentation was conducted in a 5-l B. Braun reactor, with4 l of the MSM medium containing 20 g l−1 glucose and 0.75 g l−1

ammonium nitrate at 37 ◦C. The pH was maintained at pH 7.0 bysupplementing the 5 M sodium hydroxide. The DO saturation wascontrolled as 15%. As shown in Fig. 3a, after 13 h culture, 8.2 g drycell weight (DCW) l−1 of strain XP-LM containing 169.8 U l−1 l-iLDHwas prepared from 20 g l−1 glucose.

3.6. Production of pyruvate from lactate by biocatalysis

The preparation of pyruvate from lactate in a total reactionvolume of 20 ml in 100-ml flask was carried out under the

) cell density; (�) glucose; (b) time-course of pyruvate production by Pseudomonasradation. (�) Pyruvate; (�) lactate.

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ig. 4. The effect of temperature on the L- and d-iLDH activities in whole cells ofseudomonas sp. XP-LM. (�) l-iLDH; (�) d-iLDH.

onditions: cell concentration, 4 g DCW l−1; pH 7.0; 100 mMotassium phosphate; temperature, 30 ◦C; shaking speed,00 rpm. EDTA (10 mM) which could inhibit the degradationf pyruvate was used in the reaction. As shown in Fig. 3b,ith 4 g DCW l−1 whole cells of strain XP-LM as biocatalyst,

.198 M pyruvate was obtained from 0.206 M l-lactate over8 h.

The degradation of pyruvate by the biocatalyst will decreasehe yield and the by-products could greatly influence the down-tream process [13]. Although the catalysis efficiency of strainP-LM prepared from glucose is lower than other studies, the yiledf pyuvate is higher (0.961 mol mol−1). The result suggests thathe biocatalyst prepared in this work might have the potential inyruvate production. For the efficient production of pyruvate usinghe mutant introduced in this work, the high-cell-density culti-ation and biocatalysis conditions will be optimized in successiveork.

.7. Effects of temperature on the stability of d- and l-iLDHctivities

Highly optically pure d-lactic acid has great potential in theiodegradable polylactic acid polymer production and asymmet-ic synthesis [5,18]. Co-production of d-lactate and pyruvate fromacemic lactate by a biocatalyst which exhibited only l-lactatexidation activity has been studied in previous report [5]. Wholeells of strain XP-LM were preincubated at different tempera-ures for 10 min, and then the iLDH activities were measured.s shown in Fig. 4, the d- and l-iLDH activities in strain XP-M were affected differently by heat treatment. Both of d-iLDHnd l-iLDH activities were fairly stable at 30 ◦C. Although the-iLDH was inactivated completely at temperature higher than0 ◦C, the l-iLDH was abolished partly which might decreasehe l-lactate conversion ratio. After 10 min exposure at 65 ◦C,

ost of the d-iLDH activities were inactivated, while majority of

he l-iLDH activities reserved. Taking advantage of the greaterhermostability of the l-iLDH, the whole cells of strain XP-LM prein-ubated at 65 ◦C for 10 min might also have the potential in thenantioselective oxidation of racemic lactate to d-lactate and pyru-ate.

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4. Conclusions

Production of pyruvate from lactate with iLDHs containing bio-catalyst is a valuable process. A mutant with constitutive iLDH ofPseudomonas sp. XP-M2 was screened. The iLDHs containing bio-catalyst was prepared using the cheaper substrate glucose as thecarbon source from the mutant. The cost-effective biocatalyst pre-pared from glucose exhibited high yield of pyruvate from lactate(0.961 mol mol−1). Based on the greater thermostability of the l-iLDH in strain XP-LM, the biocatalyst might also be used in theco-production of pyruvate and d-lactate from racemic lactate.

Acknowledgements

The work was supported by the Research Fund for the DoctoralProgram of Higher Education of China (Grant no. 20090131110036),and the Chinese National Programs for High Technology Researchand Development (2006AA020100 and 2007AA10Z360).

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