Lactate Dehydrogenase Involved in Lactate

Available online at www.sciencedirect.com

ScienceDirect Procedia Environmental Sciences 28 (2015) 67 – 71

The 5th Sustainable Future for Human Security (SustaiN 2014)

Lactate Dehydrogenase involved in lactate metabolism of Acetobacter pasteurianus Joji Sato, Mamoru Wakayama*, Kazuyoshi Takagi Graduate school of Life Sciences, Ritumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga, 525-8577, Japan

Abstract Acetobacter pasteurianus which is widely used for commercial brewing of vinegar can grow well with lactate as an energy source. In the utilization processes of lactate in A. pasteurianus, it is first converted to pyruvate, and then converted to final product; acetate via acetaldehyde. In the other pathway, pyruvate, formed from lactate is metabolized through the tricarboxylic acid (TCA) cycle. The enzymes catalyzing reaction from pyruvate to acetaldehyde had been identified, however, the enzyme responsible for the oxidation of lactate to pyruvate in A. pasteurianus have not been identified. In this study, we focused on the enzymes involved in the process and aimed at their characterization. This study will contribute to quality improvement of vinegar which provides human health with a good effect. A. pasteurianus assimilated D-lactate as well as L-lactate, indicating that the enzymes catalyzing the oxidation of D- and L-lactate to pyruvate could be produced in the cell. The presence of the enzyme which catalyzes the oxidation of lactate to pyruvate in A. pasteurianus was confirmed by enzymatic assays using dichloropenolindophenol as redox dye, phenazine methosulfate as electron acceptor, and lactate as substrate. © 2015 Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license

© 2015 The Authors. Published by Elsevier B.V. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of Sustain Society. Peer-review under responsibility of Sustain Society

Keywords:Lactate Dehydrogenase; Lactate Metabolism; Acetobacter pasteurianus

1. Introduction Acetobacter pasteurianus is Gram-negative, strictly aerobic bacterium and used for industrial vinegar production. The organism shows a unique resistance to ethanol and acetic acid even in highly acidic environments. Acetobacter

* Corresponding author. Tel.:+81-077-561-2768; fax:+81-077-561-2659. E-mail address:[email protected]

1878-0296 © 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of Sustain Society doi:10.1016/j.proenv.2015.07.010

68

Joji Sato et al. / Procedia Environmental Sciences 28 (2015) 67 – 71

sp. is capable of getting energy and carbon skeleton from L- and D-lactate. L- and D-Lactate are metabolized to pyruvate, acetaldehyde, and acetate by three enzymes, lactate dehydrogenase (LDH), pyruvate decarboxylase (PDC), and aldehyde dehydrogenase, respectively (ALD)1. After oxidation of lactate to pyruvate by L- and D-specific lactate dehydrogenase2, pyruvate could be decarboxylated to acetaldehyde and ultimately oxidized to acetate. Recently, the presence of D-LDH gene in A. pasteurianus 386B was reported3.However, the biochemical characterization of L- and D-LDH in A. pasteurianus has never been examined.

Fig.1. Proposed lactate metabolism in Acetobacter sp.

In this study, we demonstrated the consumption of L- and D-lactate in culturing A. pasteurianus NBRC3283 and the biochemical evidence of L- and D-lactate-oxidizing activity in A. pasteurianus NBRC3283 using the enzyme activity assay with DCIP. The location of the L- and D-lactate-oxidizing enzymes in A. pasteurianusNBRC3283 has also been studied in this work. 2. Materials and Methods 2.1. Materials DL-lactate, L-lactate, D-lactate, phenazine methosulfate (PMS), dichlorophenol-indophenol (DCIP) were purchased from Wako. All other chemicals were of reagent grade. 2.2. Bacterial strain, culture media, and seed culture conditions A. pasteurianus NBRC3283 was used in this study. YP medium consisted of 5 g of extract yeast, 5 g of polypepton and 1 g of MgSO4͌7H2O in 1 litter of distilled water. A. pasteurianus NBRC3283 was first cultivated in 100 ml YP medium in a 300-ml flask and incubated at 30°Cwith rotary shaking at 100 rpm for 20 h. 2.3. Growthin the mediumcontaining lactate The seed culture was diluted 100-fold with 200 ml YP medium containing 0.5% (v/v) L-lactate or 0.5% (v/v) DLlactate in 500-ml flask having baffles and cultivated at 30°C with rotary shaking at 100 rpm. The samples (1 ml) were harvested in 1.5-ml tube. Density of each sample was measured at 610 nm. 2.4. Preparation of cell-free extract and membrane fraction The seed culture was diluted 100-fold with 800 ml YP medium containing 0.5% DL-lactate in 2-l flask and cultivated at 30°C with shaking 100 rpm for 30 h. The cells of A. pasteurianus NBRC3283 were harvested by centrifugation at 15,650×g for 10 min at 4°C, and then resuspended and washed twice with 10 mM potassium phosphate buffer (pH 7.0). The cells were resuspended at 1 g of wet cells per 10 ml of 10 mM potassium phosphate buffer (pH 7.0). Then, those cells were disrupted by sonication in an ice bath. The disrupted cells were centrifuged at 15,650×g for 10 min at 4°C, and the supernatant was used as cell-free extract. By centrifuging the cell-free extract at 100,000×g for 60 min 4°C, the ultra centrifuged supernatant and the membrane fraction were obtained. The resulting cell-free extract, ultra centrifuged supernatant and membrane fraction were then used for determining the enzyme activity.


2.5. Quantitative analysis of L- or D-lactate We carried out quantitative analysis of L- or D-lactate using L- or D-lactate dehydrogenase methods as follows. L- or D-lactate concentration was detected at 30°C in 1.110 ml of 100mM glycylglycine (pH 10.0) including 450 mM L-Glutamic acid, 4.8mM β-NAD+, 14 U Glutamic pyruvic transaminase (GPT) and medium sample. The reaction was started by addition of 50 U L- or D-lactate dehydrogenase (LDH), and the rate of β-NAD+ reduction was determined by measuring the absorbance changes at 340 nm. 2.6. Enzyme and protein assays The DCIP assay was applied to monitor enzyme activity according to the method described [4]. The activity of lactate oxidation was detected at 30°C in 0.1ml of enzyme sample mixed with 0.9 ml of 50 mM Tris-HCl (pH 7.5), 0.2 mM PMS, and 0.2 mM DCIP. The reactions were started by adding 50 mM L- or D-lactate and the rate of DCIP reduction was determined by measuring the absorbance change at 600 nm and using an extinction coefficient of 21.3 mM-1cm-1 at pH 7.5. One unit of enzyme activity equals the amount of enzyme producing 1 μmol DCIP reduced per min. Protein concentrations were determined by the BCA method, with albumin as the standard 5. 3. Results and Discussion 3.1. Growth of A. pasteurianus NBRC3283 in the medium supplemented with L- or DL-lactate A. peroxydans oxidized L- and D-lactate to pyruvate6. It has been reported that A. pasteurianusoxidized pyruvate to acetaldehyde and acetaldehyde to acetate in energy acquisition process [1]. However, the pathway of L- and Dlactate utilization in strains of A. pasteurianus has not been fully established. To evaluate the consumption of L-and D-lactate in A. pasteurianus NBRC3283, the strain was cultured in YP medium containing L- or DL-lactate as carbon sources. As shown in Fig.2, in YP medium containing 0.5% L- or 0.5% DL-lactate A. pasteurianus NBRC3283 grew well. Since A. pasteurianus NBRC3283 grew well in YP medium containing 0.5% L- or 0.5% DLlactate than simple YP medium, the consumption of L- and D-lactate by A. pasteurianus NBRC3283 was studiedby monitoring L-and D-lactate concentration in culture medium (Fig. 3). Concentrations of L- and D-lactate were significantly decreased in beginning of the exponential growth of A. pasteurianus NBRC3283 and they reached a stable concentration after 30 h. YP medium

YP medium + L-lactate

YP medium + DL-lactate

1,2 1

OD610

0,8 0,6 0,4 0,2

0 0

10

20

30 Time (h)

40

50

60

Fig. 2. Growth of A. pasteurianus NBRC3283 in different culture conditions.

69


OD610

1,2

L-lactate

0,25

1

0,2

OD610

0,8

0,15

0,6 0,1

0,4

0,05

0,2

0

0 0

b

20

OD610

1,2

Time (h)

40

60

L-lactate

D-lactate

1

0,2

0,8 OD610

0,25

0,15

0,6 0,1

0,4

0,05

0,2 0

L- or D-lactate (g/l)

a

L-lactate (g/l)

70

0 0

20

40

Time (h)

60

Fig. 3. Lactate consumption of A. pasteurianus NBRC3283 in different culture conditions. (a) L-lactate, (b)DL-lactate.

3.2. Location of the enzymes To assess the location of enzymes, we measured enzyme activity of cell fractions prepared by sonication and differential centrifugation. As shown in Table 1, high specific activity was observed in the ultra centrifuged supernatant of the cell-free extract treated with detergent. All most of the enzymes activities in cell-free extract were obtained in the membrane fraction after ultracentrifugation of cell-free extract. Hence, the enzymes in A. pasteurianus NBRC3283 responsible for L- and D-lactate-oxidation were identified as membrane-bound enzymes. Table 1. Location of L- and D-lactate oxidative activities. Enzyme source

Activity(Units/mg) L-lactate

D-lactate

0.60×10

-3

0.44×10-3

Ultracentrifugation supernatant

0.19×10

-3

0.21×10-3

Membrane fraction

1.32×10-3

0.79×10-3

Cell-free extract


3.3. Solubilization of enzymes The extraction of enzymes was done by treating cell-free extract with several detergents at the concentration of 2% (w/v). Two enzymes could be solubilized with different detergents (Table 2). Treatment with no detergent was detected low L- and D-lactate-oxidizing enzyme activities (0.19×10-3Units/mg and 0.21×10-3 Units/mg). Treatment with Triton X-10 yielded a maximal solubilization of L-lactate-oxidizing enzyme specific activities, it was 0.91×10-3(Units/mg). On the other hand, treatment with Triton X-100 yielded a maximal solubilization of D-lactate-oxidizing enzyme specific activities, it was 0.73×10-3(Units/mg). In addition, subsequent biochemical characterization of the L-lactate-oxidizing enzyme and the D-lactate-oxidizing enzyme in A. pasteurianus NBRC3283 would beperformed with X-10 extraction and with X-100 extraction respectively. Table 2. Effect of different detergents on solubilization of lactate-oxidizing enzymes. Detergent

L-lactate-oxidizing enzyme (Units/mg)

D-lactate-oxidizing enzyme (Units/mg)

No detergent

0.19×10-3

0.21×10-3

Triton X-10

0.91×10-3

0.60×10-3

Triton X-100

0.86×10

-3

0.73×10-3

Tween 20

0.73×10-3

0.48×10-3

Tween 80

-3

0.54×10-3

0.72×10

4. Conclusion In this study, we demonstrated A. pasteurianus NBRC3283 was grown in the medium including L- or DL-lactate. Then L- and D-lactate was utilized by A. pasteurianus NBRC3283. The enzymes that catalyzed the oxidization of lactate in A. pasteurianusNBRC3283 have been confirmed using enzyme assay with redox dye. L- and D-lactateoxidizing enzymes were identified as membrane-bound enzymes. For extraction for L- and D-lactate-oxidizing enzymes, two kinds of detergents could be available. L-Lactate-oxidizing enzyme could be extracted with Triton X10 and D-lactate-oxidizing enzyme with Triton X-100. Acknowledgements I would like to express my sincere gratitude to Mr. Asep A. Prihanto for his continuous support and encouragement throughout my research. References 1. Chandra Raj. K, Ingram LO, Maupin-Furlow JA. Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus. Arch Microbiol 2001;176:443-51. 2. Deley J, Schel J. Studies on the metabolism of Acetobacter peroxydans ІІ. The enzymatic mechanism of lactate metabolism. Biochim Biophys Acta 1959;35:154-65. 3. Illeghems K, De Vuyst L, Weckx S. Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain welladapted to the cocoa bean fermentation ecosystem. BMC Genomics 2013;14:526. 4. Ma C, Gao C, Qiu J, Hao J, Liu W, Wang A, Zhang Y, Wang M, Xu P. Membrane-bound L-and D-lactate dehydrogenase activities of a newly isolated Pseudomonas stuezeri strain. Appl Microbiol Biotechol 2007;77:91-8. 5. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. A,al Biochem 1985;150:76-85. 6. Cheldelin VH, Kawasaki EH, King TE. Tricarboxylic acid cycle activity in Acetobacter pasteurianus.J. Bacteriol 1956;72:418-21.

71