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Biotransformation of dl -lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166
Process Biochemistry 45 (2010) 1632–1637
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Biotransformation of dl-lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166夽 Zhi-Qiang Liu, Li-Zhuang Jia, Yu-Guo Zheng ∗ Institute of Bioengineering, Zhejiang University of Technology, No. 18, Chaowang Road, Hangzhou 310014, Zhejiang Province, People’s Republic of China
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Article history: Received 24 February 2010 Received in revised form 4 June 2010 Accepted 17 June 2010 Keywords: Serratia marcescens dl-Lactate Pyruvate Lactate dehydrogenase Identification Biotransformation
a b s t r a c t The production of pyruvate using biotransformation from dl-lactate has been recently drawn more and more attentions due to the wide applications of pyruvate in chemicals, drugs, and agrochemicals industries. In the current study, a strain ZJB-07166, which was capable of converting dl-lactate to pyruvate, was newly isolated and characterized and later identified as Serratia marcescens based on the morphology, physiological tests, ATB system and its 16S rDNA sequence. The strain S. marcescens ZJB-07166 was applied in biotransformation of dl-lactate to pyruvate and the detailed time courses for cultivation and biotransformation were investigated. The optimum nitrogen source and carbon source in the microorganism culture for production of lactate dehydrogenase were NH4 Cl and dl-lactate, respectively. The optimum substrate concentration for biotransformation was around 40 mM and EDTA had an obvious stabilizing effect on pyruvate in biotransformation process. The pyruvate production concentration of 210 mM was achieved under the optimum conditions. These results demonstrated that the newly isolated S. marcescens ZJB-07166 was a promising strain for pyruvate production in industrial scale. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction The commercial demand for pyruvate is expanding since it has been widely used in chemicals, drugs, and agrochemicals industries [1]. It is commonly known that pyruvate is regarded as a valuable drug due to its applications in weight reduction and cardioprotection [2–4] and a relatively new supplement to the athletic and sports nutrition. In addition, pyruvate is a useful precursor for synthesis of various chemicals [1]. As an important substrate, it has been widely used in enzymatic production of amino acids such as l-tyrosine, l-dihydroxyphenylalanine, and l-tryptophan [5,6]. Pyruvate is currently prepared by chemical synthesis, fermentation [7], and biocatalysis [8–13]. Due to the increasing concerns on environment-friendly processes, there is growing interest in the pyruvate production by biotechnological methods [14]. However, the difficulty of separating pyruvate from the fermentation broths results in a high-cost pyruvate production [13]. Recently, biocatalysis for pyruvate production has been attracted a considerable interest due to its advantages including improvement the conversion rate, the mild reaction conditions and easier separa-
夽 Note: Nucleotide sequence data reported in this study are available in the GenBank databases under the accession number EU031439. ∗ Corresponding author. Tel.: +86 571 88320614, fax: +86 571 88320630. E-mail addresses: [email protected], [email protected] (Y.-G. Zheng). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.06.012
tion process of product from reaction broth, as well as possible low cost for commercial production. dl-Lactate is a suitable substrate for producing pyruvate in the process of biocatalysis [8–12]. Biocatalysis for production of pyruvate is competitive and attractive for its low-cost and feasible applications in industrial scale, which is regarded as the most promising process for pyruvate production [12]. It is reported that lactate can be converted to pyruvate by lactate dehydrogenase [10], lactate oxidase [12], and glycolate oxidase [8]. Lactate dehydrogenase is classified into two categories: NAD-linked lactate dehydrogenase and NAD-independent lactate dehydrogenase. NAD-linked lactate dehydrogenase converts lactate to pyruvate requiring NAD+ as coenzyme; while NADindependent lactate dehydrogenase also catalyzes the reaction, but in most cases, the natural hydrogen acceptor is unknown [10]. Lactate oxidase and glycolate oxidase catalyze the transformation from lactate to pyruvate requiring FAD/FMN as coenzyme and produce H2 O2 [10]. By far, several strains were isolated and reported for preparing pyruvate from lactate. For instances, Edwardsiella tarda and Pseudomonas putida SM-6 could oxidize dl-lactate to prepare pyruvate using lactate oxidase [12,15]. Acinetobacter sp. was reported to produce pyruvate from dl-lactate without producing H2 O2 [1]. Hansenula polymorpha and Pichia pastoris also can express glycolate oxidase for converting l-lactate to pyruvate [8]. Recently, Pseudomonas stutzeri was reported to prepare pyruvate with both NAD-linked lactate dehydrogenase and NAD-independent lactate dehydrogenase [16]. However, there are no reports related to the
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stains that have been applied into industrial-scale production of pyruvate. Thus it is of vital necessity to screen new promising strains with industrially potential for biotransformation of lactate to produce pyruvate. In this present study, we investigated the morphology and biochemical characteristics of a new strain for the production of pyruvate using dl-lactate as substrate. This strain, later named Serratia sp. ZJB-07166, was isolated from soil samples and its 16S ribosomal DNA (rDNA) was sequenced for classification. Cultivation conditions and biotransformation conditions for the pyruvate production were optimized as well. In addition, the type of the lactate dehydrogenase from S. marcescens ZJB-07166 were studied and confirmed.
(Takara, Japan) by using the T/A cloning procedure [20,21]. The constructed vector was transferred into the competent cell Escherichia coli JM109 according to the method of Chung et al. [22], and then spread on the LB plate containing the X-gal (5-bromo-4-chloro-3-indolyl--d-galactoside), IPTG (isopropyl-1-thio--dgalactoside) and ampicillin (50 g mL−1 ). Subsequently a positive clone, designated E. coli JM109/pMD18-T-ZJB-07166, was obtained. DNA was sequenced on both strands with an Applied Biosystems Model 377 Bautomatic DNA sequencer, and a dye-labeled terminator sequencing kit (Applied Biosystems, Foster, CA, USA). The sequences obtained were compiled and compared with sequences in the GenBank databases using BLAST program [23]. The sequences were aligned using multiple sequence alignment software, CLUSTAL W ver. 1.81 [24]. A phylogenetic tree was constructed with MegAlign software (DNASTAR Inc., Madison, WI, USA) based on the partial 16S rDNA sequences of 15 strains similar to strain ZJB-07166.
2. Materials and methods
To determine whether the lactate dehydrogenase produced in strain ZJB-07166 requires NAD+ as coenzyme when it functioned, the crude enzyme was dialyzed overnight to extract NAD+ or NADH, and the biotransformation ability of lactate dehydrogenase was compared before and after dialysis. H2 O2 in the biotransformation mixture was detected based on the color reaction of H2 O2 with KI in the presence of starch [25].
2.1. Materials dl-Lactate, pyruvate and 2,4-dinitrophenylhydrazine (DNPH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The soil samples used for isolation were collected from different regions in Hangzhou city of Zhejiang province, China. All the other chemicals were of reagent grade and commercially available. 2.2. Media and culture conditions The lactate inorganic salt medium used as selective enrichment medium for isolation was composed of the following components: dl-lactate 1%, NH4 Cl 0.3%, KH2 PO4 0.1% and K2 HPO4 0.1%. The initial pH of the medium was adjusted to 7.0 with 1 mM NaOH. The enrichment medium plates consisted of dl-lactate 1%, peptone 0.15%, yeast extract 0.15%, agar 2%, and pH was adjusted to 7.0. The enrichment liquid medium used for strain isolation consisted of dl-lactate 1%, NH4 Cl 0.3%, KH2 PO4 0.1%, K2 HPO4 0.1% and MgSO4 ·7H2 O 0.0003%. Nitrogen-free medium consisted of dl-lactate 1%, KH2 PO4 0.1%, K2 HPO4 0.1% and MgSO4 ·7H2 O 0.0003%. Carbon-free medium consisted of NH4 Cl 0.3%, KH2 PO4 0.1%, K2 HPO4 0.1% and MgSO4 ·7H2 O 0.0003%. Fermentation was carried out at 30 ◦ C for 25 h on a rotary shaker with a shaking speed of 200 rpm. 2.3. Screening and isolation of strains for production of pyruvate Soil samples (1 g) was inoculated into 49 mL of the lactate inorganic salt medium, and incubated at 30 ◦ C for 48 h. Then 1.0 mL of the resulting fermentation broth was transferred into the same isolation medium and incubated for 48 h under the same conditions. After enrichment, the fermentation broth was spread onto the enrichment medium plate, and incubated at 30 ◦ C for 24 h to get the single colonies. The rapid screening method was based on the detection of pyruvate amount by the DNPH method [17]. The single colonies were transferred to the lactate inorganic salt medium and incubated at 30 ◦ C for 36 h. The fermentation broth was then centrifuged at 10,000 × g for 10 min under 4 ◦ C, and 0.1 mL of supernatant was mixed with 1 mL DNPH solution (0.5 g L−1 DNPH in 1 M HCl). The reaction was conducted at 37 ◦ C for 10 min in a water bath, and 10 mL 0.4 M NaOH was added to stop the reaction. DNPH can react with keto acid and forms reddish brown 2,4dinitrophenylhydrazone and the production of pyruvate was judged by the color change. These strains with high yield of pyruvate were further studied for biotransformation of lactate to produce pyruvate. The lactate inorganic salt medium was also tested by the DNPH method, and there is no color change. 2.4. Phenotypic and biochemical characterization Morphology of strain ZJB-07166 was observed with a light microscope (Leica DM4000 B, Germany) and transmission electron microscope (JEM-1200EX, Japan). The oxidase activity was assayed with oxidase reagent (bioMerieux, MarcyI’Etoile, France). The carbon source utilization was examined by a standardized micromethod, with ID32 E (bioMerieux, arcy-I’Etoile, France). The results were obtained using an automated reader (bio-Merieux ATB Expression; France) and analyzed with the database V 3.0 [18].
2.6. Identification of coenzyme requiring of lactate dehydrogenase in strain ZJB-07166 for production of pyruvate
2.7. Cultivation of strain ZJB-07166 for the pyruvate production Nitrogen source including NH4 NO3 , NH4 Cl, (NH4 )2 SO4 , urea, beef extract, peptone and yeast extract were added at 0.3% level (w/v) to nitrogen-free medium for optimization of nitrogen source, respectively. Carbon source (1%, w/v) including glucose, glycerol, starch, lactose, maltose, mannitol, citrate and lactate were added to carbon-free medium for the optimization of carbon source, respectively. In order to achieve the maximum relative activity of lactate dehydrogenase, the cultivation time course of strain ZJB-07166 was investigated and optimized. 250-mL Erlenmeyer flasks with 50 mL fresh culture were inoculated at 30 ◦ C for 45 h, and the samples were removed at different time intervals for assays of cell biomass, conversion rate and pH. 2.8. Biotransformation using strain ZJB-07166 for production of pyruvate 10 mL biotransformation system is consisted of 40 mM dl-lactate, 10 mM EDTA and 12.5 g L−1 wet cell in a 50-mL Erlenmeyer flask. The reactions were carried out on a rotary shaker with a speed of 150 rpm at 30 ◦ C. The reaction sample (1 mL) was centrifuged at 10,000 × g for 10 min at 4 ◦ C, and the resulting supernatant was used for analyzing the concentrations. For optimization of the substrate concentrations in the biotransformation, the reactions were carried out with substrate concentrations varied from 10 to 140 mM at 30 ◦ C for 12 h. To investigate the time course of biotransformation, the reactions were performed with the substrate concentration of 40 mM at 30 ◦ C. To determine the effects of EDTA on the stability of pyruvate, two reactions were performed at 30 ◦ C on a rotary shaker with the shaking speed of 150 rpm, one was with 40 mM pyruvate, 10 mM EDTA, 12.5 g L−1 wet cell, and the other was with 40 mM pyruvate and 12.5 g L−1 wet cell. Both samples were determined every 1 h. To obtain high concentration of pyruvate, we added 60 mM lactate every 8 h to the reaction mixture for continuous four times under the optimum transformation conditions. If not specified, all the transformation reactions were carried out in the water system without adding any buffer. 2.9. Analytical method The lactate and pyruvate concentrations were quantified by HPLC (LC-10AS, Shimdazu, Japan) equipped with a reversed C18 column and a UV absorption spectrophotometer at 215 nm. The mobile phase was 5 mM H2 SO4 and the flow rate was set at 1 mL min−1 . The retention times for lactate and pyruvate are 4.2 min and 5.2 min, respectively. 2.10. Data collection and analysis All experiments were performed in triplicate, and the mean values were taken. Data were collected and analyzed using the Design-Expert package (Version 7.0.2, 2006; Stat-Ease, Minneapolis, MN, USA).
2.5. 16S rDNA sequence determination and phylogenetic analysis The chromosomal DNA was isolated according to the method described by Frederich et al. [19] and the 16S rDNA nucleotide sequence was enzymatically amplified. Amplification was carried out with primers: p16S-8: 5 -aga gtt gat cct ggc tca g-3 and p16S-1541: 5 -aag gag gtg atc cag ccg ca-3 in a thermal cycler (PTC-200, BioRad, USA) under the following conditions: 5 min at 95 ◦ C, 35 cycles of 40 s at 95 ◦ C, 60 s at 53 ◦ C, 2 min at 72 ◦ C and one final step of 10 min at 72 ◦ C. The PCR products were extracted and purified from the agarose gel using High Pure PCR Product Purification Kit (Roche, Germany). The resulting PCR fragment was ligated with pMD18-T
3. Results 3.1. Morphological and physiological characteristics of strain ZJB-07166 Various strains capable of biotransformation of lactate to produce pyruvate have been isolated from soils. The strain ZJB-07166
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on sequence similarities. The detailed results are shown in Fig. 2, which revealed that strain ZJB-07166 was closely clustered with S. marcescens (GeneBank accession no. AB061685), having a sequence identity of 99%. The result of this phylogenetic analysis was consistent with that of the ATB system mentioned above. According to the morphology, physiological tests, ATB system (ID 32 E) and the phylogenetic analysis, the strain, ZJB-07166, was identified as a strain of S. marcescens and named S. marcescens ZJB-07166. 3.3. Effect of carbon and nitrogen source on biomass concentration and pyruvate production
Fig. 1. Electron micrograph of strain ZJB-07166. Bar, 1 m.
with high pyruvate production was selected for further studies and deposited in the China Center for Type Culture Collection (CCTCCM 207139). The morphology of strain ZJB-07166 on the lactate inorganic salt medium plate was white, opaque, smooth and convex. The colonies on the enrichment medium plate were mucous and cardinal red. Cells were short, sporous, rod-like with tufts of 2–3 flagella and with a size of (0.5–0.8) × (0.9–1.4) m (Fig. 1). Physiological characteristics from conventional biochemical tests and ATB system analysis are shown in Table 1. The analysis of the ATB system results showed that strain ZJB-07166 had 99.9% similarity to S. marcescens.
The effect of carbon source on biomass concentration and pyruvate production was investigated when the lactate was used as sole carbon source. The maximum pyruvate production reached 38.6 mM when the substrate concentration was about 40 mM. However, no pyruvate was produced although the strain grew in media containing glucose, glycerol, starch, lactose, maltose, mannitol and citrate, respectively. The results further confirmed that lactate played the roles of carbon source and inducer for the NADindependent lactate dehydrogenase. Table 2 shows the effect of nitrogen source on the pyruvate production and biomass concentration. The highest relative activity of lactate dehydrogenase and pyruvate production were obtained at 43.5% and 50.7 mM, respectively, when 3 g L−1 of NH4 Cl was used as nitrogen source. The strain ZJB-07166, however, could not produce pyruvate although it grew well when the beef extract, peptone and yeast extract existed in the media as nitrogen sources. Thus, the lactate and NH4 Cl were selected as the optimal carbon and nitrogen source, respectively, for further studies.
3.2. 16S rDNA sequencing and phylogenetic analysis
3.4. Time courses of S. marcescens ZJB-07166 growth and biotransformation
The partial 16S rDNA sequence of strain ZJB-07166 (1461 bp) was determined and deposited in the GenBank database under the accession no. EU031439. A phylogenetic tree was constructed based
The investigation of time courses of S. marcescens ZJB-07166 growth showed that the lag phase of growth lasted for 8 h. And the biomass concentration of S. marcescens ZJB-07166 increased
Table 1 Biochemical profile and identification of strain ZJB-07166 by the ATB system and conventional biochemical tests. Characteristics
Properties
Characteristics
Properties
Gram staining Oxidase Catalase l-Omithine l-Arginine l-Lysine Urea l-Arabitol Acide galacturonique Potassium 5-cetogluconate 5-Bromo-3-indoxyl-nonanoate Sodium pyruvate 4-Nitrophenyl--D-glucopyranoside d-Mannitol d-Maltose Adonitol Palatinose Acid l-aspartique 4-nitroanilide 4-Nitrophenyl--d-glucuronide Sodium malonate l-Tryptophane 5-Bromo-4-chloro-3-indolyl-N-acetyl-B-d-glucosaminde 4-Nitrophenyl--d-galactopyranoside d-Glucose d-Saccharose l-Arabinose 4-Nitrophenyl--d-glucuronide
− − + + − + − − − + + − + + + + − + − − − − + + + − −
l-Tryptophane 5-Bromo-4-chloro-3-indolyl-N-acetyl--d-glucosaminde 4-Nitrophenyl--d-galactopyranoside d-Glucose d-Saccharose l-Arabinose d-Arabitol 4-Nitrophenyl-␣-d-glucopyranoside 4-Nitrophenyl-␣-d-galactopyranoside d-Trehalose l-Rhamnose Inositol d-Cellobiose d-Sorbitol 4-Nitrophenyl-␣-d-maltopyranoside d-Arabitol 4-Nitrophenyl-␣-d-glucopyranoside 4-Nitrophenyl-␣-d-galactopyranoside d-Trehalose l-Rhamnose Inositol d-Cellobiose d-Sorbitol 4-Nitrophenyl-␣-d-maltopyranoside Acide l-aspartique 4-nitroanilide l-Tryptophane
− − + + + − − − − + − + − + − − − − + − + − + − + −
Note: +, positive; −, negative.
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Fig. 2. The phylogenetic dendrogram for S. marcescens ZJB-07166 and related strains based on the 16S rDNA sequence. Numbers in parentheses are accession numbers of published sequences. Bootstrap values were based on 1000 replicates. Plesiomonas shigelloides was used as the outgroup. Table 2 Effects of nitrogen source on biomass concentration and relative activity. Nitrogen source
Biomass (g L−1 )
Relative activity (%)
NH4 NO3 NH4 Cl (NH4 )2 SO4 Urea Beef extract Peptone Yeast extract
0.6 1.2 1.7 0.5 8.2 8.9 9.3
54 94 36 48 0 0 0
from 8 h of incubation to 20 h and reached its maximum at about 28 h. Then the cell biomass concentration decreased slightly and later became plateaued. The transformation process started at the beginning of the exponential phase of growth (8 h), and reached its maximum after about 17 h. The high transformation rate was maintained from 25 h to 30 h, and then started to decrease continuously. The pH varied from 6.2 to 8.1 during the cultivation, and increased greatly after 28 h from 7.1 to 8.1 as the biomass decreased, which could be attributed to cell lysis (Fig. 3).
Fig. 3. The time course of S. marcescens ZJB-07166.
3.5. Effects of substrate concentration on the biotransformation rate Different substrate concentrations ranging from 10 to 140 mM were used to test the effect of substrate concentration on the biotransformation rate. S. marcescens ZJB-07166 can convert dl-lactate and produced pyruvate in 10 h with conversion rate of about 90% when the substrate concentration was lower than 30 mM. However, when the dl-lactate concentration was higher than 60 mM, the biotransformation rate dramatically decreased from 90% to 10%, indicating that the biotransformation ability of S. marcescens ZJB-07166 was significantly inhibited (Fig. 4). Thus, the substrate concentration of 40 mM was selected for further studies. In addition, the results showed that the enzyme from this strain does not have stereospecificities. 3.6. The effect of EDTA on pyruvate stability When the initial substrate concentration was 40 mM, almost 100% of dl-lactate was converted by free cell of S. marcescens ZJB-07166. However, it is interesting to find that about 20% pyruvate was metabolized. In order to inhibit the metabolization
Fig. 4. The biotransformation with substrate concentrations varied from 10 to 140 mM at 30 ◦ C.
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Fig. 5. The time course of biotransformation of dl-lactate to pyruvate by S. marcescens ZJB-07166.
of pyruvate, the inhibition effect of different reagents including ethylenediamine tetraacetic acid (EDTA) were investigated. The results showed that EDTA has an apparent inhibition effect on pyruvate degradation, which might be explained by the fact that EDTA inhibits the activities of the pyruvate-degrading enzymes by complexing ions. The detailed results in Fig. 5 showed that a small amount of pyruvate was degraded with the addition of EDTA (10 mM). In further biotransformation process, EDTA is required for the preparation of pyruvate from lactate in this study. 3.7. Confirmation of lactate dehydrogenase type from S. marcescens ZJB-07166 The enzyme involved in the pyruvate production by S. marcescens ZJB-07166 did not belong to lactate oxidase or glycolate oxidase as no H2 O2 was detected during the biotransformation process. As coenzyme, FMN/FAD combines closely to the apoenzyme, but not to NAD+ /NADH. Therefore, the activity of NAD-linked lactate dehydrogenase will lose completely after dialysis. In this study, the dialysis of crude enzyme from S. marcescens ZJB-07166 did not lose its biotransformation ability compared to that of untreated enzyme, suggesting that S. marcescens ZJB-07166 lactate dehydrogenase is NAD-independent enzyme. 4. Discussion It is very important to construct a more efficient selection or screening method for isolation of microorganisms for the given products [26,27]. In this study, a relative rapid screening method was established to screen the pyruvate producing strain based on the properties of pyruvate reacted with DNPH to form reddish brown 2,4-dinitrophenylhydrazone. The strains with reaction mixture of deep color were selected for further studies. This efficient screening procedure, which allows the testing of a large set of strains and minimizing the effort of finding ones with lactate dehydrogenases activities, was used to explore potential strains for this biocatalysis process. The strain ZJB-07166 with highest biotransformation ability of converting lactate to pyruvate was isolated and identified as a strain of S. marcescens using morphological characteristics combined with 16S rDNA sequence, a very efficient and widely used method in identification of strains [28,29]. This strain could utilize dl-lactate as the sole carbon source for growth under certain incubation conditions. To the best of our knowledge, S. marcescens ZJB-07166 is a novel strain used for producing pyruvate by biotransforming dl-lactate. The transformation reactions were carried out in the water system without adding any buffer, which was beneficial for the separation process [8]. The catalyst
was removed by filtration or centrifugation, leaving an aqueous solution of a relatively pure pyruvate, which makes the isolation process easier to perform the production of pyruvate in industrial scale. In addition, we found that EDTA had an obvious stabilizing effect on pyruvate in biotransformation, and this can be contributed to the EDTA can be chelate with ions in the reaction system, which inhibit the activities of enzymes related with further degradation of pyruvate. We found that S. marcescens ZJB-07166 isolated in this study did not produce NAD-linked lactate dehydrogenase, lactate oxidase, or glycolate oxidase. The enzyme from S. marcescens ZJB-07166 was induced by lactate, which is the same with the NAD-independent lactate dehydrogenases found in Butyribacterium rettgeri, Pseudomonas aeruginosa and Enterobacteriaceae [10]. As reported by Garivé, the strain Serratia sp. could produce NAD-independent lactate dehydrogenase without reverse reactions in the catalysis process [10]. The pyruvate is located at a key junction point of metabolism for assimilatory and dissimilatory reactions, respiratory dissimilation and alcoholic fermentation. Thus it can be easily used by different metabolic pathways [30,31]. It is necessary to control fermentation and biotransformation conditions to inhibit the activities of enzymes that are responsible for the degradation of pyruvate in order to obtain a high yield. In this study, we found that the simple inorganic fermentation medium was useful for improve the production of pyruvate and restrict the synthesis of the enzymes in the downstream of pyruvate. Mutation breeding of the S. marcescens ZJB-07166 to get some auxotrophic strains, such as vitamin or lipoic acid auxotrophs would be also useful tools for improvement the activity of lactate dehydrogenase and pyruvate production. Further studies focusing on learning about lactate dehydrogenase produced by S. marcescens ZJB-07166 and increasing the pyruvate production are under going. 5. Conclusion In this study, we isolated and characterized a strain that was capable of converting dl-lactate to pyruvate. Based on the morphology and 16S rDNA gene sequence, ZJB-07166 was identified as a strain of S. marcescens and named S. marcescens ZJB-07166. The NAD-independent lactate dehydrogenase was found in S. marcescens ZJB-07166. The optimum nitrogen source and carbon source were NH4 Cl and dl-lactate, respectively. And the optimum substrate concentration for biotransformation was 40 mM. Under the optimum conditions, we achieved the highest pyruvate production of 210 mM. The results showed that S. marcescens ZJB-07166 is a promising strain for the industrial applications. This study paved the foundation for the production of pyruvate in the industrial scale. Acknowledgements This work was supported by the Major Basic Research Development Program of China (No. 2007CB714306) and the Natural Scientific Foundation of Zhejiang (No. Z4090612). References [1] Ma CQ, Xu P, Dou YM, Qu YB. Highly efficient conversion of lactate to pyruvate using whole cells of Acinetobacter sp. Biotechnol Prog 2003;19(6):1672–6. [2] Kalman D, Colker CM, Wilets I, Roufs JB, Antonio J. The effects of pyruvate supplementation on body composition in overweight individuals. Nutrition 1999;15(5):337–40. [3] Mallet RT. Pyruvate: metabolic protector of cardiac performance. Proc Soc Exp Biol Med 2000;223(2):136–48. [4] Mallet RT, Sun J, Knott EM, Sharma AB, Olivencia-Yurvati AH. Metabolic cardioprotection by pyruvate: recent progress. Exp Biol Med 2005;230(7):435–43. [5] Enei H, Nakazawa H, Matsui H, Okumura S, Yamada H. Enzymic preparation of l-tyrosine or 3, 4-dihydroxyphenyl-l-alanine from pyruvate, ammonia and indole. FEBS Lett 1972;21:39–41.
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Biotransformation of dl-lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166夽 Zhi-Qiang Liu, Li-Zhuang Jia, Yu-Guo Zheng ∗ Institute of Bioengineering, Zhejiang University of Technology, No. 18, Chaowang Road, Hangzhou 310014, Zhejiang Province, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 24 February 2010 Received in revised form 4 June 2010 Accepted 17 June 2010 Keywords: Serratia marcescens dl-Lactate Pyruvate Lactate dehydrogenase Identification Biotransformation
a b s t r a c t The production of pyruvate using biotransformation from dl-lactate has been recently drawn more and more attentions due to the wide applications of pyruvate in chemicals, drugs, and agrochemicals industries. In the current study, a strain ZJB-07166, which was capable of converting dl-lactate to pyruvate, was newly isolated and characterized and later identified as Serratia marcescens based on the morphology, physiological tests, ATB system and its 16S rDNA sequence. The strain S. marcescens ZJB-07166 was applied in biotransformation of dl-lactate to pyruvate and the detailed time courses for cultivation and biotransformation were investigated. The optimum nitrogen source and carbon source in the microorganism culture for production of lactate dehydrogenase were NH4 Cl and dl-lactate, respectively. The optimum substrate concentration for biotransformation was around 40 mM and EDTA had an obvious stabilizing effect on pyruvate in biotransformation process. The pyruvate production concentration of 210 mM was achieved under the optimum conditions. These results demonstrated that the newly isolated S. marcescens ZJB-07166 was a promising strain for pyruvate production in industrial scale. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction The commercial demand for pyruvate is expanding since it has been widely used in chemicals, drugs, and agrochemicals industries [1]. It is commonly known that pyruvate is regarded as a valuable drug due to its applications in weight reduction and cardioprotection [2–4] and a relatively new supplement to the athletic and sports nutrition. In addition, pyruvate is a useful precursor for synthesis of various chemicals [1]. As an important substrate, it has been widely used in enzymatic production of amino acids such as l-tyrosine, l-dihydroxyphenylalanine, and l-tryptophan [5,6]. Pyruvate is currently prepared by chemical synthesis, fermentation [7], and biocatalysis [8–13]. Due to the increasing concerns on environment-friendly processes, there is growing interest in the pyruvate production by biotechnological methods [14]. However, the difficulty of separating pyruvate from the fermentation broths results in a high-cost pyruvate production [13]. Recently, biocatalysis for pyruvate production has been attracted a considerable interest due to its advantages including improvement the conversion rate, the mild reaction conditions and easier separa-
夽 Note: Nucleotide sequence data reported in this study are available in the GenBank databases under the accession number EU031439. ∗ Corresponding author. Tel.: +86 571 88320614, fax: +86 571 88320630. E-mail addresses: [email protected], [email protected] (Y.-G. Zheng). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.06.012
tion process of product from reaction broth, as well as possible low cost for commercial production. dl-Lactate is a suitable substrate for producing pyruvate in the process of biocatalysis [8–12]. Biocatalysis for production of pyruvate is competitive and attractive for its low-cost and feasible applications in industrial scale, which is regarded as the most promising process for pyruvate production [12]. It is reported that lactate can be converted to pyruvate by lactate dehydrogenase [10], lactate oxidase [12], and glycolate oxidase [8]. Lactate dehydrogenase is classified into two categories: NAD-linked lactate dehydrogenase and NAD-independent lactate dehydrogenase. NAD-linked lactate dehydrogenase converts lactate to pyruvate requiring NAD+ as coenzyme; while NADindependent lactate dehydrogenase also catalyzes the reaction, but in most cases, the natural hydrogen acceptor is unknown [10]. Lactate oxidase and glycolate oxidase catalyze the transformation from lactate to pyruvate requiring FAD/FMN as coenzyme and produce H2 O2 [10]. By far, several strains were isolated and reported for preparing pyruvate from lactate. For instances, Edwardsiella tarda and Pseudomonas putida SM-6 could oxidize dl-lactate to prepare pyruvate using lactate oxidase [12,15]. Acinetobacter sp. was reported to produce pyruvate from dl-lactate without producing H2 O2 [1]. Hansenula polymorpha and Pichia pastoris also can express glycolate oxidase for converting l-lactate to pyruvate [8]. Recently, Pseudomonas stutzeri was reported to prepare pyruvate with both NAD-linked lactate dehydrogenase and NAD-independent lactate dehydrogenase [16]. However, there are no reports related to the
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stains that have been applied into industrial-scale production of pyruvate. Thus it is of vital necessity to screen new promising strains with industrially potential for biotransformation of lactate to produce pyruvate. In this present study, we investigated the morphology and biochemical characteristics of a new strain for the production of pyruvate using dl-lactate as substrate. This strain, later named Serratia sp. ZJB-07166, was isolated from soil samples and its 16S ribosomal DNA (rDNA) was sequenced for classification. Cultivation conditions and biotransformation conditions for the pyruvate production were optimized as well. In addition, the type of the lactate dehydrogenase from S. marcescens ZJB-07166 were studied and confirmed.
(Takara, Japan) by using the T/A cloning procedure [20,21]. The constructed vector was transferred into the competent cell Escherichia coli JM109 according to the method of Chung et al. [22], and then spread on the LB plate containing the X-gal (5-bromo-4-chloro-3-indolyl--d-galactoside), IPTG (isopropyl-1-thio--dgalactoside) and ampicillin (50 g mL−1 ). Subsequently a positive clone, designated E. coli JM109/pMD18-T-ZJB-07166, was obtained. DNA was sequenced on both strands with an Applied Biosystems Model 377 Bautomatic DNA sequencer, and a dye-labeled terminator sequencing kit (Applied Biosystems, Foster, CA, USA). The sequences obtained were compiled and compared with sequences in the GenBank databases using BLAST program [23]. The sequences were aligned using multiple sequence alignment software, CLUSTAL W ver. 1.81 [24]. A phylogenetic tree was constructed with MegAlign software (DNASTAR Inc., Madison, WI, USA) based on the partial 16S rDNA sequences of 15 strains similar to strain ZJB-07166.
2. Materials and methods
To determine whether the lactate dehydrogenase produced in strain ZJB-07166 requires NAD+ as coenzyme when it functioned, the crude enzyme was dialyzed overnight to extract NAD+ or NADH, and the biotransformation ability of lactate dehydrogenase was compared before and after dialysis. H2 O2 in the biotransformation mixture was detected based on the color reaction of H2 O2 with KI in the presence of starch [25].
2.1. Materials dl-Lactate, pyruvate and 2,4-dinitrophenylhydrazine (DNPH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The soil samples used for isolation were collected from different regions in Hangzhou city of Zhejiang province, China. All the other chemicals were of reagent grade and commercially available. 2.2. Media and culture conditions The lactate inorganic salt medium used as selective enrichment medium for isolation was composed of the following components: dl-lactate 1%, NH4 Cl 0.3%, KH2 PO4 0.1% and K2 HPO4 0.1%. The initial pH of the medium was adjusted to 7.0 with 1 mM NaOH. The enrichment medium plates consisted of dl-lactate 1%, peptone 0.15%, yeast extract 0.15%, agar 2%, and pH was adjusted to 7.0. The enrichment liquid medium used for strain isolation consisted of dl-lactate 1%, NH4 Cl 0.3%, KH2 PO4 0.1%, K2 HPO4 0.1% and MgSO4 ·7H2 O 0.0003%. Nitrogen-free medium consisted of dl-lactate 1%, KH2 PO4 0.1%, K2 HPO4 0.1% and MgSO4 ·7H2 O 0.0003%. Carbon-free medium consisted of NH4 Cl 0.3%, KH2 PO4 0.1%, K2 HPO4 0.1% and MgSO4 ·7H2 O 0.0003%. Fermentation was carried out at 30 ◦ C for 25 h on a rotary shaker with a shaking speed of 200 rpm. 2.3. Screening and isolation of strains for production of pyruvate Soil samples (1 g) was inoculated into 49 mL of the lactate inorganic salt medium, and incubated at 30 ◦ C for 48 h. Then 1.0 mL of the resulting fermentation broth was transferred into the same isolation medium and incubated for 48 h under the same conditions. After enrichment, the fermentation broth was spread onto the enrichment medium plate, and incubated at 30 ◦ C for 24 h to get the single colonies. The rapid screening method was based on the detection of pyruvate amount by the DNPH method [17]. The single colonies were transferred to the lactate inorganic salt medium and incubated at 30 ◦ C for 36 h. The fermentation broth was then centrifuged at 10,000 × g for 10 min under 4 ◦ C, and 0.1 mL of supernatant was mixed with 1 mL DNPH solution (0.5 g L−1 DNPH in 1 M HCl). The reaction was conducted at 37 ◦ C for 10 min in a water bath, and 10 mL 0.4 M NaOH was added to stop the reaction. DNPH can react with keto acid and forms reddish brown 2,4dinitrophenylhydrazone and the production of pyruvate was judged by the color change. These strains with high yield of pyruvate were further studied for biotransformation of lactate to produce pyruvate. The lactate inorganic salt medium was also tested by the DNPH method, and there is no color change. 2.4. Phenotypic and biochemical characterization Morphology of strain ZJB-07166 was observed with a light microscope (Leica DM4000 B, Germany) and transmission electron microscope (JEM-1200EX, Japan). The oxidase activity was assayed with oxidase reagent (bioMerieux, MarcyI’Etoile, France). The carbon source utilization was examined by a standardized micromethod, with ID32 E (bioMerieux, arcy-I’Etoile, France). The results were obtained using an automated reader (bio-Merieux ATB Expression; France) and analyzed with the database V 3.0 [18].
2.6. Identification of coenzyme requiring of lactate dehydrogenase in strain ZJB-07166 for production of pyruvate
2.7. Cultivation of strain ZJB-07166 for the pyruvate production Nitrogen source including NH4 NO3 , NH4 Cl, (NH4 )2 SO4 , urea, beef extract, peptone and yeast extract were added at 0.3% level (w/v) to nitrogen-free medium for optimization of nitrogen source, respectively. Carbon source (1%, w/v) including glucose, glycerol, starch, lactose, maltose, mannitol, citrate and lactate were added to carbon-free medium for the optimization of carbon source, respectively. In order to achieve the maximum relative activity of lactate dehydrogenase, the cultivation time course of strain ZJB-07166 was investigated and optimized. 250-mL Erlenmeyer flasks with 50 mL fresh culture were inoculated at 30 ◦ C for 45 h, and the samples were removed at different time intervals for assays of cell biomass, conversion rate and pH. 2.8. Biotransformation using strain ZJB-07166 for production of pyruvate 10 mL biotransformation system is consisted of 40 mM dl-lactate, 10 mM EDTA and 12.5 g L−1 wet cell in a 50-mL Erlenmeyer flask. The reactions were carried out on a rotary shaker with a speed of 150 rpm at 30 ◦ C. The reaction sample (1 mL) was centrifuged at 10,000 × g for 10 min at 4 ◦ C, and the resulting supernatant was used for analyzing the concentrations. For optimization of the substrate concentrations in the biotransformation, the reactions were carried out with substrate concentrations varied from 10 to 140 mM at 30 ◦ C for 12 h. To investigate the time course of biotransformation, the reactions were performed with the substrate concentration of 40 mM at 30 ◦ C. To determine the effects of EDTA on the stability of pyruvate, two reactions were performed at 30 ◦ C on a rotary shaker with the shaking speed of 150 rpm, one was with 40 mM pyruvate, 10 mM EDTA, 12.5 g L−1 wet cell, and the other was with 40 mM pyruvate and 12.5 g L−1 wet cell. Both samples were determined every 1 h. To obtain high concentration of pyruvate, we added 60 mM lactate every 8 h to the reaction mixture for continuous four times under the optimum transformation conditions. If not specified, all the transformation reactions were carried out in the water system without adding any buffer. 2.9. Analytical method The lactate and pyruvate concentrations were quantified by HPLC (LC-10AS, Shimdazu, Japan) equipped with a reversed C18 column and a UV absorption spectrophotometer at 215 nm. The mobile phase was 5 mM H2 SO4 and the flow rate was set at 1 mL min−1 . The retention times for lactate and pyruvate are 4.2 min and 5.2 min, respectively. 2.10. Data collection and analysis All experiments were performed in triplicate, and the mean values were taken. Data were collected and analyzed using the Design-Expert package (Version 7.0.2, 2006; Stat-Ease, Minneapolis, MN, USA).
2.5. 16S rDNA sequence determination and phylogenetic analysis The chromosomal DNA was isolated according to the method described by Frederich et al. [19] and the 16S rDNA nucleotide sequence was enzymatically amplified. Amplification was carried out with primers: p16S-8: 5 -aga gtt gat cct ggc tca g-3 and p16S-1541: 5 -aag gag gtg atc cag ccg ca-3 in a thermal cycler (PTC-200, BioRad, USA) under the following conditions: 5 min at 95 ◦ C, 35 cycles of 40 s at 95 ◦ C, 60 s at 53 ◦ C, 2 min at 72 ◦ C and one final step of 10 min at 72 ◦ C. The PCR products were extracted and purified from the agarose gel using High Pure PCR Product Purification Kit (Roche, Germany). The resulting PCR fragment was ligated with pMD18-T
3. Results 3.1. Morphological and physiological characteristics of strain ZJB-07166 Various strains capable of biotransformation of lactate to produce pyruvate have been isolated from soils. The strain ZJB-07166
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on sequence similarities. The detailed results are shown in Fig. 2, which revealed that strain ZJB-07166 was closely clustered with S. marcescens (GeneBank accession no. AB061685), having a sequence identity of 99%. The result of this phylogenetic analysis was consistent with that of the ATB system mentioned above. According to the morphology, physiological tests, ATB system (ID 32 E) and the phylogenetic analysis, the strain, ZJB-07166, was identified as a strain of S. marcescens and named S. marcescens ZJB-07166. 3.3. Effect of carbon and nitrogen source on biomass concentration and pyruvate production
Fig. 1. Electron micrograph of strain ZJB-07166. Bar, 1 m.
with high pyruvate production was selected for further studies and deposited in the China Center for Type Culture Collection (CCTCCM 207139). The morphology of strain ZJB-07166 on the lactate inorganic salt medium plate was white, opaque, smooth and convex. The colonies on the enrichment medium plate were mucous and cardinal red. Cells were short, sporous, rod-like with tufts of 2–3 flagella and with a size of (0.5–0.8) × (0.9–1.4) m (Fig. 1). Physiological characteristics from conventional biochemical tests and ATB system analysis are shown in Table 1. The analysis of the ATB system results showed that strain ZJB-07166 had 99.9% similarity to S. marcescens.
The effect of carbon source on biomass concentration and pyruvate production was investigated when the lactate was used as sole carbon source. The maximum pyruvate production reached 38.6 mM when the substrate concentration was about 40 mM. However, no pyruvate was produced although the strain grew in media containing glucose, glycerol, starch, lactose, maltose, mannitol and citrate, respectively. The results further confirmed that lactate played the roles of carbon source and inducer for the NADindependent lactate dehydrogenase. Table 2 shows the effect of nitrogen source on the pyruvate production and biomass concentration. The highest relative activity of lactate dehydrogenase and pyruvate production were obtained at 43.5% and 50.7 mM, respectively, when 3 g L−1 of NH4 Cl was used as nitrogen source. The strain ZJB-07166, however, could not produce pyruvate although it grew well when the beef extract, peptone and yeast extract existed in the media as nitrogen sources. Thus, the lactate and NH4 Cl were selected as the optimal carbon and nitrogen source, respectively, for further studies.
3.2. 16S rDNA sequencing and phylogenetic analysis
3.4. Time courses of S. marcescens ZJB-07166 growth and biotransformation
The partial 16S rDNA sequence of strain ZJB-07166 (1461 bp) was determined and deposited in the GenBank database under the accession no. EU031439. A phylogenetic tree was constructed based
The investigation of time courses of S. marcescens ZJB-07166 growth showed that the lag phase of growth lasted for 8 h. And the biomass concentration of S. marcescens ZJB-07166 increased
Table 1 Biochemical profile and identification of strain ZJB-07166 by the ATB system and conventional biochemical tests. Characteristics
Properties
Characteristics
Properties
Gram staining Oxidase Catalase l-Omithine l-Arginine l-Lysine Urea l-Arabitol Acide galacturonique Potassium 5-cetogluconate 5-Bromo-3-indoxyl-nonanoate Sodium pyruvate 4-Nitrophenyl--D-glucopyranoside d-Mannitol d-Maltose Adonitol Palatinose Acid l-aspartique 4-nitroanilide 4-Nitrophenyl--d-glucuronide Sodium malonate l-Tryptophane 5-Bromo-4-chloro-3-indolyl-N-acetyl-B-d-glucosaminde 4-Nitrophenyl--d-galactopyranoside d-Glucose d-Saccharose l-Arabinose 4-Nitrophenyl--d-glucuronide
− − + + − + − − − + + − + + + + − + − − − − + + + − −
l-Tryptophane 5-Bromo-4-chloro-3-indolyl-N-acetyl--d-glucosaminde 4-Nitrophenyl--d-galactopyranoside d-Glucose d-Saccharose l-Arabinose d-Arabitol 4-Nitrophenyl-␣-d-glucopyranoside 4-Nitrophenyl-␣-d-galactopyranoside d-Trehalose l-Rhamnose Inositol d-Cellobiose d-Sorbitol 4-Nitrophenyl-␣-d-maltopyranoside d-Arabitol 4-Nitrophenyl-␣-d-glucopyranoside 4-Nitrophenyl-␣-d-galactopyranoside d-Trehalose l-Rhamnose Inositol d-Cellobiose d-Sorbitol 4-Nitrophenyl-␣-d-maltopyranoside Acide l-aspartique 4-nitroanilide l-Tryptophane
− − + + + − − − − + − + − + − − − − + − + − + − + −
Note: +, positive; −, negative.
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Fig. 2. The phylogenetic dendrogram for S. marcescens ZJB-07166 and related strains based on the 16S rDNA sequence. Numbers in parentheses are accession numbers of published sequences. Bootstrap values were based on 1000 replicates. Plesiomonas shigelloides was used as the outgroup. Table 2 Effects of nitrogen source on biomass concentration and relative activity. Nitrogen source
Biomass (g L−1 )
Relative activity (%)
NH4 NO3 NH4 Cl (NH4 )2 SO4 Urea Beef extract Peptone Yeast extract
0.6 1.2 1.7 0.5 8.2 8.9 9.3
54 94 36 48 0 0 0
from 8 h of incubation to 20 h and reached its maximum at about 28 h. Then the cell biomass concentration decreased slightly and later became plateaued. The transformation process started at the beginning of the exponential phase of growth (8 h), and reached its maximum after about 17 h. The high transformation rate was maintained from 25 h to 30 h, and then started to decrease continuously. The pH varied from 6.2 to 8.1 during the cultivation, and increased greatly after 28 h from 7.1 to 8.1 as the biomass decreased, which could be attributed to cell lysis (Fig. 3).
Fig. 3. The time course of S. marcescens ZJB-07166.
3.5. Effects of substrate concentration on the biotransformation rate Different substrate concentrations ranging from 10 to 140 mM were used to test the effect of substrate concentration on the biotransformation rate. S. marcescens ZJB-07166 can convert dl-lactate and produced pyruvate in 10 h with conversion rate of about 90% when the substrate concentration was lower than 30 mM. However, when the dl-lactate concentration was higher than 60 mM, the biotransformation rate dramatically decreased from 90% to 10%, indicating that the biotransformation ability of S. marcescens ZJB-07166 was significantly inhibited (Fig. 4). Thus, the substrate concentration of 40 mM was selected for further studies. In addition, the results showed that the enzyme from this strain does not have stereospecificities. 3.6. The effect of EDTA on pyruvate stability When the initial substrate concentration was 40 mM, almost 100% of dl-lactate was converted by free cell of S. marcescens ZJB-07166. However, it is interesting to find that about 20% pyruvate was metabolized. In order to inhibit the metabolization
Fig. 4. The biotransformation with substrate concentrations varied from 10 to 140 mM at 30 ◦ C.
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Fig. 5. The time course of biotransformation of dl-lactate to pyruvate by S. marcescens ZJB-07166.
of pyruvate, the inhibition effect of different reagents including ethylenediamine tetraacetic acid (EDTA) were investigated. The results showed that EDTA has an apparent inhibition effect on pyruvate degradation, which might be explained by the fact that EDTA inhibits the activities of the pyruvate-degrading enzymes by complexing ions. The detailed results in Fig. 5 showed that a small amount of pyruvate was degraded with the addition of EDTA (10 mM). In further biotransformation process, EDTA is required for the preparation of pyruvate from lactate in this study. 3.7. Confirmation of lactate dehydrogenase type from S. marcescens ZJB-07166 The enzyme involved in the pyruvate production by S. marcescens ZJB-07166 did not belong to lactate oxidase or glycolate oxidase as no H2 O2 was detected during the biotransformation process. As coenzyme, FMN/FAD combines closely to the apoenzyme, but not to NAD+ /NADH. Therefore, the activity of NAD-linked lactate dehydrogenase will lose completely after dialysis. In this study, the dialysis of crude enzyme from S. marcescens ZJB-07166 did not lose its biotransformation ability compared to that of untreated enzyme, suggesting that S. marcescens ZJB-07166 lactate dehydrogenase is NAD-independent enzyme. 4. Discussion It is very important to construct a more efficient selection or screening method for isolation of microorganisms for the given products [26,27]. In this study, a relative rapid screening method was established to screen the pyruvate producing strain based on the properties of pyruvate reacted with DNPH to form reddish brown 2,4-dinitrophenylhydrazone. The strains with reaction mixture of deep color were selected for further studies. This efficient screening procedure, which allows the testing of a large set of strains and minimizing the effort of finding ones with lactate dehydrogenases activities, was used to explore potential strains for this biocatalysis process. The strain ZJB-07166 with highest biotransformation ability of converting lactate to pyruvate was isolated and identified as a strain of S. marcescens using morphological characteristics combined with 16S rDNA sequence, a very efficient and widely used method in identification of strains [28,29]. This strain could utilize dl-lactate as the sole carbon source for growth under certain incubation conditions. To the best of our knowledge, S. marcescens ZJB-07166 is a novel strain used for producing pyruvate by biotransforming dl-lactate. The transformation reactions were carried out in the water system without adding any buffer, which was beneficial for the separation process [8]. The catalyst
was removed by filtration or centrifugation, leaving an aqueous solution of a relatively pure pyruvate, which makes the isolation process easier to perform the production of pyruvate in industrial scale. In addition, we found that EDTA had an obvious stabilizing effect on pyruvate in biotransformation, and this can be contributed to the EDTA can be chelate with ions in the reaction system, which inhibit the activities of enzymes related with further degradation of pyruvate. We found that S. marcescens ZJB-07166 isolated in this study did not produce NAD-linked lactate dehydrogenase, lactate oxidase, or glycolate oxidase. The enzyme from S. marcescens ZJB-07166 was induced by lactate, which is the same with the NAD-independent lactate dehydrogenases found in Butyribacterium rettgeri, Pseudomonas aeruginosa and Enterobacteriaceae [10]. As reported by Garivé, the strain Serratia sp. could produce NAD-independent lactate dehydrogenase without reverse reactions in the catalysis process [10]. The pyruvate is located at a key junction point of metabolism for assimilatory and dissimilatory reactions, respiratory dissimilation and alcoholic fermentation. Thus it can be easily used by different metabolic pathways [30,31]. It is necessary to control fermentation and biotransformation conditions to inhibit the activities of enzymes that are responsible for the degradation of pyruvate in order to obtain a high yield. In this study, we found that the simple inorganic fermentation medium was useful for improve the production of pyruvate and restrict the synthesis of the enzymes in the downstream of pyruvate. Mutation breeding of the S. marcescens ZJB-07166 to get some auxotrophic strains, such as vitamin or lipoic acid auxotrophs would be also useful tools for improvement the activity of lactate dehydrogenase and pyruvate production. Further studies focusing on learning about lactate dehydrogenase produced by S. marcescens ZJB-07166 and increasing the pyruvate production are under going. 5. Conclusion In this study, we isolated and characterized a strain that was capable of converting dl-lactate to pyruvate. Based on the morphology and 16S rDNA gene sequence, ZJB-07166 was identified as a strain of S. marcescens and named S. marcescens ZJB-07166. The NAD-independent lactate dehydrogenase was found in S. marcescens ZJB-07166. The optimum nitrogen source and carbon source were NH4 Cl and dl-lactate, respectively. And the optimum substrate concentration for biotransformation was 40 mM. Under the optimum conditions, we achieved the highest pyruvate production of 210 mM. The results showed that S. marcescens ZJB-07166 is a promising strain for the industrial applications. This study paved the foundation for the production of pyruvate in the industrial scale. Acknowledgements This work was supported by the Major Basic Research Development Program of China (No. 2007CB714306) and the Natural Scientific Foundation of Zhejiang (No. Z4090612). References [1] Ma CQ, Xu P, Dou YM, Qu YB. Highly efficient conversion of lactate to pyruvate using whole cells of Acinetobacter sp. Biotechnol Prog 2003;19(6):1672–6. [2] Kalman D, Colker CM, Wilets I, Roufs JB, Antonio J. The effects of pyruvate supplementation on body composition in overweight individuals. Nutrition 1999;15(5):337–40. [3] Mallet RT. Pyruvate: metabolic protector of cardiac performance. Proc Soc Exp Biol Med 2000;223(2):136–48. [4] Mallet RT, Sun J, Knott EM, Sharma AB, Olivencia-Yurvati AH. Metabolic cardioprotection by pyruvate: recent progress. Exp Biol Med 2005;230(7):435–43. [5] Enei H, Nakazawa H, Matsui H, Okumura S, Yamada H. Enzymic preparation of l-tyrosine or 3, 4-dihydroxyphenyl-l-alanine from pyruvate, ammonia and indole. FEBS Lett 1972;21:39–41.
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