About the Authors:
Chao Gao
Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, People’s Republic of China
Jianhua Qiu
Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, People’s Republic of China
Cuiqing Ma
* E-mail: [email protected]
Affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan, People’s Republic of China
Ping Xu
Affiliations State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, People’s Republic of China, State Key Laboratory of Microbial Technology, Shandong University, Jinan, People’s Republic of China
Introduction
Lactic acid, the most important hydroxycarboxylic acid, is currently commercially produced by the fermentation of sugars present in biomass [1]–[3]. In addition to its use in the synthesis of biodegradable polymers [4], lactic acid can also be regarded as a feedstock for the green chemistry of the future [1]. For example, pyruvate, another important platform chemical, can be produced from lactate through direct oxidation [5]–[7]. Considering the significant difference in their prices, the production of pyruvate from lactate by catalysis is a valuable process.
[Figure omitted. See PDF.]
Figure 1. Involvement of cytochrome c in lactate oxidation of P. stutzeri SDM.
(a) Native-PAGE of cytochrome c in P. stutzeri SDM. Lane M: molecular mass standards in kilodaltons (kDa) (GE Healthcare); lane 1: cell extract of P. stutzeri SDM; lane 2: membrane fractions of P. stutzeri SDM; lane 3: pooled fraction containing cytochrome c after DEAE-Sepharose; lanes 4 and 5: pooled fraction containing cytochrome c after DEAE-A25. (b) The absorption spectrum of cytochrome c in P. stutzeri SDM. Black line: reaction mixtures consisting of cytochrome c, crude extract of P. stutzeri SDM and dl-lactate. Red line: reaction mixtures consisting of cytochrome c and crude extract of P. stutzeri SDM.
https://doi.org/10.1371/journal.pone.0040755.g001
[Figure omitted. See PDF.]
Table 1. Effects of different respiratory chain depressors on the lactate oxidation activities.
https://doi.org/10.1371/journal.pone.0040755.t001
Most of the reported chemical catalysts convert a major part of lactate to acetaldehyde and CO2 rather than pyruvate [7]. Indeed, there have been very few attempts to bring about the oxidative dehydrogenation of lactate through chemical catalysis [5], [6]. Biocatalysts could catalyze lactate to pyruvate under relatively mild conditions [1], [6]. Different enzymes, such as NAD-dependent lactate dehydrogenases (nLDHs) and lactate oxidase, have been employed in the biotechnological production of pyruvate from lactate. However, the costliness of cofactor NAD or the production of the byproduct hydrogen peroxide restricted the industrial application of nLDHs and lactate oxidase, respectively [1], [7].
[Figure omitted. See PDF.]
Figure 2. Time course of P. stutzeri SDM growth in the media with different dissolved oxygen.
(a) 5%; (b) 15%; (c) 30%. (▪) OD620nm; (•) Biotransformation activity; (▴) dl-Lactate. Data are the average ± SD of three separate experiments.
https://doi.org/10.1371/journal.pone.0040755.g002
In a previous report, Pseudomonas stutzeri SDM was reported to have the ability to produce pyruvate from lactate with oxygen as the terminal electron acceptor [8]. No hydrogen peroxide was produced during P. stutzeri SDM catalyzed lactate oxidation, which made the strain a promising biocatalyst for the commercial pyruvate production. NAD-independent lactate dehydrogenases (iLDHs) were reported to be involved in the lactate oxidation process [8]–[12]. However, iLDHs could not directly use the oxygen as the electron acceptor, which made the lactate oxidation process in strain SDM rather confusing. To further explore the strain with regard to pyruvate production, the aim of the present study is to determine how iLDHs are involved in the oxidation of lactate. After illustrating the pyruvate-producing mechanism, optimal conditions for the production of pyruvate from a cheap substrate, dl-lactate, by the lactate-utilizing strain SDM were also developed.
Results
Pyruvate-producing Mechanism in P. stutzeri SDM
P. stutzeri SDM possesses 2 inducible iLDHs that made the strain a good biocatalyst for the 2-oxo-carboxylate production [9], [13]–[15]. Unlike lactate oxidase, iLDHs could not oxidize lactate with oxygen as the directly electron acceptor. There should be an electron transfer system between iLDHs and oxygen in the lactate-utilizing strain SDM. To elucidate the electron transfer system in this case, the effects of different electron transfer inhibitors such as diphenylamine, antimycin A, NaN3, and salicylhydroxamic acid on the pyruvate-producing activity of the P. stutzeri SDM crude extract were studied. As shown in Table 1, the cytochrome c reductase inhibitor antimycin A distinctly inhibited the pyruvate-producing activity. This implied that iLDHs in P. stutzeri SDM might use quinone as the natural electron acceptor. The electron acquired by quinone would be transferred to cytochrome (by cytochrome c reductase) and then terminally transferred to oxygen (by cytochrome oxidase). In addition to the traditionally cytochrome oxidase, Pseudonomas strains also have some alternative cytochrome oxidase, such as cytochrome cbb3 oxidase [16]. Although the cytochrome oxidase inhibitors (diphenylamine, NaN3, and salicylhydroxamic acid) exhibited different inhibition effects on the l- and d-lactate oxidation activities of P. stutzeri SDM due to the complexity of the cytochrome oxidase system in Pseudonomas strains, those results implied the roles of cytochrome in the lactate oxidation process.
[Figure omitted. See PDF.]
Figure 3. Optimization of biocatalysis conditions.
(a) Effect of pH on pyruvate production; (b) Effect of lactate concentrations on pyruvate production.
https://doi.org/10.1371/journal.pone.0040755.g003
Next, a cytochrome c fraction was purified from P. stutzeri SDM (Figure 1a). After oxidizing cytochrome c with ferricyanide, a crude extract of P. stutzeri SDM and dl-lactate were added. As shown in Figure 1b, the addition of dl-lactate to the reaction system produced a characteristic of reduced cytochrome c. The reduction of cytochrome c by dl-lactate further supported the participation of cytochrome in the lactate oxidation process. On the basis of the results mentioned above, it is concluded that iLDHs and the electron transport chain account for the observed oxidation of lactate in P. stutzeri SDM.
[Figure omitted. See PDF.]
Figure 4. Effect of temperature on the production of pyruvate from dl-lactate.
https://doi.org/10.1371/journal.pone.0040755.g004
Effect of Dissolved Oxygen on Biotransformation Activity
In the case of the lactate oxidation mechanism in P. stutzeri SDM, the components of the respiratory chain may influence biotransformation activity distinctly. Because the components of the respiratory chain are different under various dissolved oxygen (DO) concentrations, the effect of DO on the biotransformation activity of P. stutzeri SDM was studied in a 5-l reactor. As shown in Figure 2, when 15% DO was employed in the culture, the highest biotransformation activity was obtained.
[Figure omitted. See PDF.]
Figure 5. Effect of concentration of P. stutzeri SDM on the production of pyruvate from dl-lactate.
BTA: biotransformation activity.
https://doi.org/10.1371/journal.pone.0040755.g005
d-Lactate or l-lactate has been employed in pyruvate production [1], [5], [7]. Because of the low price and large sources of racemic lactate compared to optical lactate, dl-lactate was used as the substrate for pyruvate production in this work.
[Figure omitted. See PDF.]
Table 2. Effect of DO on the pyruvate production.
https://doi.org/10.1371/journal.pone.0040755.t002
Optimal Range of pH
Influence of reaction pH on pyruvate production was determined in 100 mM phosphate buffer containing 6 g dry cell weight (DCW) l−1 of cell biomass of P. stutzeri SDM and 0.4 M dl-lactate at 30°C. As shown in Figure 3a, after biocatalysis for 24 h, the highest pyruvate production was detected at pH 8.0.
[Figure omitted. See PDF.]
Figure 6. Time course of pyruvate production by P. stutzeri SDM.
10 mM EDTA was added in the biocatalysis system to inhibit pyruvate degradation. (▪) Pyruvate; (•) dl-lactate. Data are the average ± SD of three separate experiments.
https://doi.org/10.1371/journal.pone.0040755.g006
Effect of dl-lactate Concentration on Biocatalysis
Substrate concentration was also important for the conversion of DL-lactate to pyruvate. Effect of the concentration of dl-lactate on biocatalysis was investigated to determine its optimal range. The dl-lactate concentration was varied from 0.15 to 0.75 M. As shown in Figure 3b, after 2 h of biocatalysis, the highest pyruvate production was detected when the concentration of dl-lactate was 0.45 M. High DL-lactate concentration (more than 0.6 M) would lead to substrate inhibition and resulted in lower pyruvate concentrations.
[Figure omitted. See PDF.]
Table 3. Overview of literature on the enzymatic synthesis of pyruvate from lactate.
https://doi.org/10.1371/journal.pone.0040755.t003
Optimal Reaction Temperature
To investigate the influence of reaction temperature on pyruvate production, the reaction was carried out at different temperatures at pH 8.0. As shown in Figure 4, the highest pyruvate production was detected at 30°C after 24 h. Although high pyruvate production could be detected at 42°C after 2 h, because of biocatalyst instability under high temperature, the optimal reaction temperature was determined to be 30°C.
Optimal Range of Cell Biomass
Biocatalysis was carried out with 2–16 g DCW l–1 of P. stutzeri SDM as the biocatalyst. As shown in Figure 5, 10 g DCW l–1 of P. stutzeri SDM was optimal and produced the highest pyruvate concentration and relatively high specific biotransformation activity.
Optimal Range of DO
The biocatalysis production of pyruvate from lactate was a bio-oxidation process. Oxygen would be the terminal electron acceptor. Thus, oxygen was also the substrate of the biocatalysis process and the effect of DO on pyruvate production should be investigated. As shown in Table 2, 0.39 M pyruvate was produced after 39 h with a low amount of DO (5%). Pyruvate production was improved by increasing the DO content to 15%. However, with higher DO (30%), the lactate oxidation decreased, and only 0.19 M pyruvate was produced. This might be due to the substrate inhibition effect of oxygen. The optimal DO content was then determined to be 15%. This result was consistent with our previous report which related to the production of pyruvate from l-lactate [17].
Combining these results, an optimal biotransformation system for the production of pyruvate from dl-lactate was developed. The biocatalysis was conducted at 30°C in distilled water (pH was adjusted to 8.0) with 10 g DCW l−1 of P. stutzeri SDM as the biocatalyst. The DO saturation was controlled at 15%. The dl-lactate concentration in the 5-l reactor was about 0.45 M. As shown in Figure 6, 0.44 M pyruvate was obtained from 0.45 M dl-lactate after 29 h of biotransformation.
Discussion
Pyruvate is an important starting material widely applied in chemical, pharmaceutical, and agrochemical industries [6], [18]. Commercial pyruvate is produced by dehydration and decarboxylation of tartaric acid. This classical chemical route is energy-intensive and controversial with issues of environmental protection and sustainable process development [6], [7]. Novel systems which produce pyruvate by biotechnological methods have been the research focus [19]–[22]. Microbial fermentation currently plays a dominant role in biotechnological production of pyruvate [23]–[26]. High concentration (135 g l−1) and high volumetric productivity (6 g l−1 h−1) of pyruvate have been obtained through fermentation [25], [26]. However, biocatalysis processes, due to their simple composition of reaction mixture, high conversion rate of substrate, and convenience of recovery, are also promising in the biotechnological production of pyruvate [6].
Lactate, which can be easily produced from biomass, is the most promising substrate in the biocatalysis production of pyruvate [1], [7]. Of the enzymes employed in the lactate-based pyruvate production, lactate oxidase has been studied extensively [1], [7]. Lactate oxidase catalyzes pyruvate formation from l-lactate with oxygen as the second substrate, giving hydrogen peroxide as a byproduct. Hydrogen peroxide decomposes pyruvate to acetate, carbon dioxide, and water, lowering the production yield. If the problem of further oxidation of pyruvate by hydrogen peroxide can be solved, the biocatalysis production of pyruvate from lactate has the potential to be commercialized because of the low price of lactate [1], [6], [7]. P. stutzeri SDM was confirmed to have the ability to produce pyruvate from lactate with the merit of no hydrogen peroxide production. iLDHs in P. stutzeri SDM firstly acquired the electron from the lactate and then the electron would be terminally transferred to oxygen. However, the electron transfer process has never been clarified in previous works.
Pyruvate production by (2R)-hydroxycarboxylate-viologen-oxidoreductase (HVOR) in Proteus vulgaris or Proteus mirabilis with the addition of artificial redox mediators was studied in previous reports [27], [28]. The regeneration of artificial redox mediators was achieved by chemical or electrochemical methods. However, in the case of P. stutzeri SDM, pyruvate was produced without the addition of an artificial redox mediator. The reduction of cytochrome c in P. stutzeri SDM by dl-lactate implied that the electron transfer components of respiratory chain might play an important role in lactate oxidation. Effects of different respiratory chain inhibitors on the pyruvate production further identified the involvement of the electron transfer chain in the lactate oxidation process. Unlike Proteus strains, the cofactor regeneration of iLDHs in P. stutzeri SDM may employ the inherent electron transfer system of the strain. This process, which excludes the expensive cofactor regeneration step, makes P. stutzeri SDM a rather practicable alternative for the biocatalytic production of pyruvate. Under the optimal conditions, with biocatalyst prepared from 10 g DCW l−1 of P. stutzeri SDM, 0.44 M pyruvate was obtained after 29 h.
As shown in Table 3, pyruvate production from d-lactate, l-lactate, and dl-lactate has been studied in previous works [29]–[32]. Genetically modified Hansenula polymorpha and Pichia pastoris cells expressing glycolate oxidase could catalyze l-lactate into pyruvate with rather high yield [29]. For the decomposition of the byproduct hydrogen peroxide, co-expression of catalase with glycolate oxidase in genetically modified yeasts was needed [1], [7]. Whole cells of P. stutzeri SDM catalyzed lactate oxidation without the production of hydrogen peroxide. On the other hand, the glycolate oxidase could only utilize l-lactate as the substrate but a large amount of lactate produced today is a racemic mixture of both stereospecific forms. P. stutzeri SDM possesses 2 inducible iLDHs which give the strain the ability to use dl-lactate, a much cheaper substrate than the optical lactate, as the substrate to produce pyruvate.
Recently, Serratia marcescens ZJB-07166 has been applied in the biotransformation of dl-lactate to pyruvate. The pyruvate concentration of 0.21 M was achieved under an optimum condition [32]. The newly isolated S. marcescens ZJB-07166 was regarded as a promising strain for pyruvate production at an industrial scale [32]. Under the conditions optimized in the present study, a much higher concentration of pyruvate (0.44 M) was obtained from the cheap substrate dl-lactate than in previous reports.
In conclusion, the lactate-utilizing P. stutzeri strain SDM was confirmed to catalyze lactate oxidation through iLDHs and the inherent electron transfer chain. Preparation of pyruvate from dl-lactate was carried out under the following optimal conditions: DCW, 10 g l−1; pH 8.0; temperature, 30°C; DO saturation, 15%; and dl-lactate concentration, 0.45 M. After 29 h of biotransformation, pyruvate was obtained at a high concentration (48.4 g l−1) and a high yield (98%). The biocatalysis process introduced in this study provides not only a rather feasible method for pyruvate production but also a green pathway for the utilization of lactate produced from biomass.
Materials and Methods
Chemicals
l-Lactate and bovine serum albumin were purchased from Sigma. d-Lactate was purchased from Fluka. dl-Lactate was purchased from Wujiang Ciyun Flavor & Fragrance Co. Ltd. (P. R. China). Diphenylamine, antimycin A, and salicylhydroxamic acid were purchased from Sigma. All other chemicals were of reagent grade.
Microorganism and Biocatalyst Preparation
P. stutzeri SDM (China Center for Type Culture Collection No. M 206010) isolated from soil was used [9]. The minimal salt medium (MSM) supplemented with 10.0 g 1−1 dl-lactate was used as the fermentation medium [9]. For biocatalyst preparation, cells of the SDM strain cultivated at 30°C were harvested from MSM by centrifugation, washed twice with 0.85% (w/v) sterile salt water, and then resuspended in various concentrations with distilled water.
Preparation of Crude Cell Extract
Cells of the SDM strain grown in MSM containing dl-lactate as the sole carbon source were resuspended in 50 mM Tris-HCl (pH 8.0) and disrupted by sonication (Sonics 500 W/20 KHz, USA) in an ice bath. The disrupted cells were subjected to centrifugation for 20 min at 12,000 g, and the supernatant was used as crude cell extract.
Purification of Cytochrome in P. stutzeri SDM
The membrane fraction of P. stutzeri SDM was prepared from the crude cell extract by centrifugation at 147,000 g for 180 min. Triton X-100 (10%, w/v) was added to the membrane fraction to a final concentration of 1 mg mg−1 of protein. The supernatant (detergent extract) was applied to a column of DEAE Sepharose Fast Flow equilibrated with Buffer A: 50 mM Tris-HCl (pH 8.0) containing 1 mM dithiothreitol (DTT), 5 mM MgSO4, 0.1% Triton X-100, and 1 mM EDTA. The column was washed with Buffer B: 50 mM Tris-HCl (pH 8.0) containing 1 mM DTT, 200 mM KCl, 5 mM MgSO4, 0.1% Triton X-100, and 1 mM EDTA at a flow rate of 5 ml min−1. Cytochrome from column effluents was monitored by measuring the absorbance at 410 nm. The fractions containing cytochrome were concentrated by ultrafiltration and desalted with gel G-25. The cytochrome pool after desalting was then applied to a column of DEAE A-25 pre-equilibrated with Buffer A. The column was washed with a linear gradient of 0–100% Buffer B at a flow rate of 0.5 ml min−1. The fractions containing cytochrome were concentrated and stored at 0 to 4°C. Native polyacrylamide gel electrophoresis (native-PAGE) was performed on a 5–12% native polyacrylamide gradient gel with a Mini-Protean III system (Bio-Rad) according to the protocol by Davis [33]. After electrophoresis, the gel was stained for the protein with Coomassie Brilliant Blue R-250.
Pyruvate-producing Activity Assay
Pyruvate-producing activity was determined in 1 ml of 67 mM phosphate buffer (pH 7.4) containing 20 mM l-lactate or d-lactate. The reaction was initiated by the addition of whole cells or crude cell extract of SDM at 37°C for 10 min. The reaction was stopped by adding 0.05 ml of 1 M NaOH. The mixture was subjected to centrifugation for 1 min at 16,000 g, and a 0.5-ml portion of the supernatant was used for pyruvate detection. One unit was defined as the amount of enzyme that converted lactate to 1.0 µM pyruvate per minute under the test conditions.
Analytical Methods
Accurate concentrations of lactate and pyruvate were analyzed by HPLC (Agilent 1100 series, Hewlett-Packard, USA) using an Aminex HPX-87H column (Bio-Rad), which was running at 0.4 ml min−1 with 10 mM H2SO4 as eluent at 55°C [34]. UV and visible absorption spectra of the cytochrome were measured by an Ultrospec™ 2100 pro UV/visible spectrophotometer (GE Healthcare). Protein was determined by the Markwell variation of the Lowry method, with bovine serum albumin as the standard [35].
Author Contributions
Conceived and designed the experiments: PX CM. Performed the experiments: CG JQ. Analyzed the data: CG JQ. Contributed reagents/materials/analysis tools: PX CM. Wrote the paper: CG CM PX.
Citation: Gao C, Qiu J, Ma C, Xu P (2012) Efficient Production of Pyruvate from DL-Lactate by the Lactate-Utilizing Strain Pseudomonas stutzeri SDM. PLoS ONE 7(7): e40755. https://doi.org/10.1371/journal.pone.0040755
1. Gao C, Ma C, Xu P (2011) Biotechnological routes based on lactic acid production from biomass. Biotechnol Adv 29: 930–939.C. GaoC. MaP. Xu2011Biotechnological routes based on lactic acid production from biomass.Biotechnol Adv29930939
2. John RP, Nampoothiri KM, Pandey A (2007) Fermentative production of lactic acid from biomass: an overview on process developments and future perspectives. Appl Microbiol Biotechnol 74: 524–534.RP JohnKM NampoothiriA. Pandey2007Fermentative production of lactic acid from biomass: an overview on process developments and future perspectives.Appl Microbiol Biotechnol74524534
3. Qin J, Wang X, Zheng Z, Ma C, Tang H, et al. (2010) Production of l-lactic acid by a thermophilic Bacillus mutant using sodium hydroxide as neutralizing agent. Bioresour Technol 101: 7570–7576.J. QinX. WangZ. ZhengC. MaH. Tang2010Production of l-lactic acid by a thermophilic Bacillus mutant using sodium hydroxide as neutralizing agent.Bioresour Technol10175707576
4. Distel KA, Zhu G, Wang P (2005) Biocatalysis using an organic-soluble enzyme for the preparation of poly(lactic acid) in organic solvents. Bioresour Technol 96: 617–623.KA DistelG. ZhuP. Wang2005Biocatalysis using an organic-soluble enzyme for the preparation of poly(lactic acid) in organic solvents.Bioresour Technol96617623
5. Ai M, Ohdan K (1997) Oxidative dehydrogenation of lactic acid to pyruvic acid over iron phosphate catalyst. Appl Catal A: Gen 150: 13–20.M. AiK. Ohdan1997Oxidative dehydrogenation of lactic acid to pyruvic acid over iron phosphate catalyst.Appl Catal A: Gen1501320
6. Li Y, Chen J, Lun SY (2001) Biotechnological production of pyruvic acid. Appl Microbiol Biotechnol 57: 451–459.Y. LiJ. ChenSY Lun2001Biotechnological production of pyruvic acid.Appl Microbiol Biotechnol57451459
7. Xu P, Qiu JH, Gao C, Ma CQ (2008) Biotechnological routes to pyruvate production. J Biosci Bioeng 105: 169–175.P. XuJH QiuC. GaoCQ Ma2008Biotechnological routes to pyruvate production.J Biosci Bioeng105169175
8. Hao J, Ma C, Gao C, Qiu J, Wang M, et al. (2007) Pseudomonas stutzeri as a novel biocatalyst for pyruvate production from dl-lactate, Biotechnol Lett 29: 105–110.J. HaoC. MaC. GaoJ. QiuM. Wang2007Pseudomonas stutzeri as a novel biocatalyst for pyruvate production from dl-lactate, Biotechnol Lett29105110
9. Ma C, Gao C, Qiu J, Hao J, Liu W, et al. (2007) Membrane-bound l- and d-lactate dehydrogenase activities of a newly isolated Pseudomonas stutzeri strain. Appl Microbiol Biotechnol 77: 91–98.C. MaC. GaoJ. QiuJ. HaoW. Liu2007Membrane-bound l- and d-lactate dehydrogenase activities of a newly isolated Pseudomonas stutzeri strain.Appl Microbiol Biotechnol779198
10. Gao C, Hu CH, Zheng ZJ, Ma CQ, Jiang TY, et al. (2012) Lactate utilization is regulated by the FadR-type regulator LldR in Pseudomonas aeruginosa. J Bacteriol 194: 2687–2692.C. GaoCH HuZJ ZhengCQ MaTY Jiang2012Lactate utilization is regulated by the FadR-type regulator LldR in Pseudomonas aeruginosa.J Bacteriol19426872692
11. Gao C, Jiang TY, Dou PP, Ma CQ, Li LX, et al. (2012) NAD-Independent l-lactate dehydrogenase is required for l-lactate utilization in Pseudomonas stutzeri SDM PLoS ONE 7: e36519.C. GaoTY JiangPP DouCQ MaLX Li2012NAD-Independent l-lactate dehydrogenase is required for l-lactate utilization in Pseudomonas stutzeri SDM PLoS ONE7e36519
12. Jiang TY, Gao C, Su F, Zhang W, Hu CH, et al. (2012) Genome sequence of Pseudomonas stutzeri SDM-LAC, a typical strain for studying the molecular mechanism of lactate utilization. J Bacteriol 194: 894–895.TY JiangC. GaoF. SuW. ZhangCH Hu2012Genome sequence of Pseudomonas stutzeri SDM-LAC, a typical strain for studying the molecular mechanism of lactate utilization.J Bacteriol194894895
13. Gao C, Qiu JH, Li JC, Ma CQ, Tang HZ, et al. (2009) Enantioselective oxidation of racemic lactic acid to d-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM. Bioresour. Technol 100: 1878–1880.C. GaoJH QiuJC LiCQ MaHZ Tang2009Enantioselective oxidation of racemic lactic acid to d-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM. Bioresour.Technol10018781880
14. Gao C, Zhang W, Lv CJ, Li LX, Ma CQ, et al. (2010) Efficient production of 2-oxobutyrate from 2-hydroxybutyrate by using whole cells of Pseudomonas stutzeri strain SDM. Appl Environ Microbiol 76: 1679–1682.C. GaoW. ZhangCJ LvLX LiCQ Ma2010Efficient production of 2-oxobutyrate from 2-hydroxybutyrate by using whole cells of Pseudomonas stutzeri strain SDM.Appl Environ Microbiol7616791682
15. Gao C, Zhang W, Ma CQ, Liu P, Xu P (2011) Kinetic resolution of 2-hydroxybutanoate racemic mixtures by NAD-independent l-lactate dehydrogenase. Bioresour Technol 102: 4595–4599.C. GaoW. ZhangCQ MaP. LiuP. Xu2011Kinetic resolution of 2-hydroxybutanoate racemic mixtures by NAD-independent l-lactate dehydrogenase.Bioresour Technol10245954599
16. Buschmann S, Warkentin E, Xie H, Langer JD, Ermler U, et al. (2010) The structure of cbb3 cytochrome oxidase provides insights into proton pumping. Science 329: 327–330.S. BuschmannE. WarkentinH. XieJD LangerU. Ermler2010The structure of cbb3 cytochrome oxidase provides insights into proton pumping.Science329327330
17. Ma CQ, Xu P, Qiu JH, Zhang ZJ, Wang KW, et al. (2004) An enzymatic route to produce pyruvate from lactate. Appl Microbiol Biotechnol 66: 34–39.CQ MaP. XuJH QiuZJ ZhangKW Wang2004An enzymatic route to produce pyruvate from lactate.Appl Microbiol Biotechnol663439
18. Stottmeister U, Aurich A, Wilde H, Andersch J, Schmidt S, et al. (2005) White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses. J Ind Microbiol Biotechnol 32: 651–664.U. StottmeisterA. AurichH. WildeJ. AnderschS. Schmidt2005White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses.J Ind Microbiol Biotechnol32651664
19. Miyazaki M, Shibue M, Ogino K, Nakamura H, Maeda H (2001) Enzymatic synthesis of pyruvic acid from acetaldehyde and carbon dioxide. Chem Commun 18: 1800–1801.M. MiyazakiM. ShibueK. OginoH. NakamuraH. Maeda2001Enzymatic synthesis of pyruvic acid from acetaldehyde and carbon dioxide.Chem Commun1818001801
20. Ogawa J, Soong CL, Masashi I, Shimizu S (2001) Enzymatic production of pyruvate from fumarate–an application of microbial cyclic–imide transforming pathway. J Mol Catal B: Enzym 11: 355–359.J. OgawaCL SoongI. MasashiS. Shimizu2001Enzymatic production of pyruvate from fumarate–an application of microbial cyclic–imide transforming pathway.J Mol Catal B: Enzym11355359
21. Buto S, Pollegioni L, Angiurl L, Pilone MS (1994) Evaluation of d-amino-acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids. Biotechnol Bioeng 44: 1288–1294.S. ButoL. PollegioniL. AngiurlMS Pilone1994Evaluation of d-amino-acid oxidase from Rhodotorula gracilis for the production of alpha-keto acids.Biotechnol Bioeng4412881294
22. Dixon NM, James EW, Lovitt RW, Kell DB (1989) Electromicrobial transformation using the pyruvate synthase system of Clostridium sporagenes. Bioelectrochem Bioenerg 21: 245–259.NM DixonEW JamesRW LovittDB Kell1989Electromicrobial transformation using the pyruvate synthase system of Clostridium sporagenes.Bioelectrochem Bioenerg21245259
23. Causey TB, Shanmugam KT, Yomano LP, Ingram LO (2003) Engineering Escherichia coli for efficient conversion of glucose to pyruvate. Proc Nat Acad Sci USA 101: 2235–2240.TB CauseyKT ShanmugamLP YomanoLO Ingram2003Engineering Escherichia coli for efficient conversion of glucose to pyruvate.Proc Nat Acad Sci USA10122352240
24. Li Y, Hugenholtz J, Chen J, Lun SY (2002) Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy. Appl Microbiol Biotechnol 60: 101–106.Y. LiJ. HugenholtzJ. ChenSY Lun2002Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy.Appl Microbiol Biotechnol60101106
25. van Maris AJ, Geertman JM, Vermeulen A, Groothuizen MK, Winkler AA, et al. (2004) Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast. Appl Environ Microbiol 70: 159–166.AJ van MarisJM GeertmanA. VermeulenMK GroothuizenAA Winkler2004Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast.Appl Environ Microbiol70159166
26. Zelić B, Gostovic S, Vuorilehto K, Vasić-Racki D, Takors R (2004) Process strategies to enhance pyruvate production with recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis. Biotechnol Bioeng 85: 638–646.B. ZelićS. GostovicK. VuorilehtoD. Vasić-RackiR. Takors2004Process strategies to enhance pyruvate production with recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis.Biotechnol Bioeng85638646
27. Hekmat D, Danninger J, Simon H, Vortmeyer D (1999) Production of pyruvate from (R)-lactate in an enzyme-membrane reactor with coupled electrochemical regeneration of the artificial mediator anthraquinone-2,6-disulfonate. Enzyme Microb Technol 24: 471–479.D. HekmatJ. DanningerH. SimonD. Vortmeyer1999Production of pyruvate from (R)-lactate in an enzyme-membrane reactor with coupled electrochemical regeneration of the artificial mediator anthraquinone-2,6-disulfonate.Enzyme Microb Technol24471479
28. Schinschel C, Simon H (1993) Preparation of pyruvate from (R)-lactate with Proteus species. J Biotechnol 31: 191–203.C. SchinschelH. Simon1993Preparation of pyruvate from (R)-lactate with Proteus species.J Biotechnol31191203
29. Eisenberg A, Seip JE, Gavagan JE, Payne MS, Anton DL, et al. (1997) Pyruvic acid production using methylotrophic yeast transformants as catalyst. J Mol Catal B: Enzym 2: 223–232.A. EisenbergJE SeipJE GavaganMS PayneDL Anton1997Pyruvic acid production using methylotrophic yeast transformants as catalyst.J Mol Catal B: Enzym2223232
30. Cooper B (1989) Microbial manufacture of pyruvic acid from d-(–)-lactic acid. DE patent 3733157. B. Cooper1989Microbial manufacture of pyruvic acid from d-(–)-lactic acid.DE patent 3733157
31. Gu JS, Xu P, Qu YB (2002) A biocatalyst for pyruvate preparation from dl-lactate: lactate oxidase in a Pseudomonas sp. J Mol Catal B: Enzym 18: 299–305.JS GuP. XuYB Qu2002A biocatalyst for pyruvate preparation from dl-lactate: lactate oxidase in a Pseudomonas sp.J Mol Catal B: Enzym18299305
32. Liu ZQ, Jia LZ, Zheng YG (2010) Biotransformation of dl-lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166. Process Biochem 45: 1632–1637.ZQ LiuLZ JiaYG Zheng2010Biotransformation of dl-lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166.Process Biochem4516321637
33. Davis BJ (1964) Disc electrophoresis II. Method and application to human serum proteins. Ann NY Acad Sci 121: 404–427.BJ Davis1964Disc electrophoresis II. Method and application to human serum proteins.Ann NY Acad Sci121404427
34. Gao C, Xu XX, Hu CH, Zhang W, Zhang Y, et al. (2010) Pyruvate producing biocatalyst with constitutive NAD-independent lactate dehydrogenases. Process Biochem 45: 1912–1915.C. GaoXX XuCH HuW. ZhangY. Zhang2010Pyruvate producing biocatalyst with constitutive NAD-independent lactate dehydrogenases.Process Biochem4519121915
35. Markwell MAK, Haas SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87: 206–210.MAK MarkwellSM HaasLL BieberNE Tolbert1978A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal Biochem87206210
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
© 2012 Gao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.
Abstract
Background
The platform chemical lactate is currently produced mainly through the fermentation of sugars presented in biomass. Besides the synthesis of biodegradable polylactate, lactate is also viewed as a feedstock for the green chemistry of the future. Pyruvate, another important platform chemical, can be produced from lactate through biocatalysis.
Methodology/Principal Findings
It was established that whole cells of Pseudomonas stutzeri SDM catalyze lactate oxidation with lactate-induced NAD-independent lactate dehydrogenases (iLDHs) through the inherent electron transfer chain. Unlike the lactate oxidation processes observed in previous reports, the mechanism underlying lactate oxidation described in the present study excluded the costliness of the cofactor regeneration step and production of the byproduct hydrogen peroxide.
Conclusions/Significance
Biocatalysis conditions were optimized by using the cheap dl-lactate as the substrate and whole cells of the lactate-utilizing P. stutzeri SDM as catalyst. Under optimal conditions, the biocatalytic process produced pyruvate at a high concentration (48.4 g l−1) and a high yield (98%). The bioconversion system provides a promising alternative for the green production of pyruvate.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer