Extremophiles (2014) 18:363–373 DOI 10.1007/s00792-013-0621-x
ORIGINAL PAPER
Biochemical characterization of psychrophilic Mn-superoxide dismutase from newly isolated Exiguobacterium sp. OS-77 Kyoshiro Nonaka • Ki-Seok Yoon • Seiji Ogo
Received: 11 July 2013 / Accepted: 29 December 2013 / Published online: 12 January 2014 Ó Springer Japan 2014
Abstract Many types of superoxide dismutases have been purified and characterized from various bacteria, however, a psychrophilic Mn-superoxide dismutase (MnSOD) has not yet been reported. Here, we describe the purification and the biochemical characterization of the psychrophilic MnSOD from Exiguobacterium sp. strain OS-77 (EgMnSOD). According to 16S rRNA sequence analysis, a newly isolated bacterium strain OS-77 belongs to the genus Exiguobacterium. The optimum growth temperature of the strain OS-77 is 20 °C. The EgMnSOD is a homodimer of 23.5 kDa polypeptides determined by SDSPAGE and gel filtration analysis. UV-Vis spectrum and ICP-MS analysis clearly indicated that the homogeneously purified enzyme contains only a Mn ion as a metal cofactor. The optimal reaction pH and temperature of the enzyme were pH 9.0 and 5 °C, respectively. Notably, the purified EgMnSOD was thermostable up to 45 °C and retained 50 % activity after 21.2 min at 60 °C. The differential scanning calorimetry also indicated that the Communicated by A. Driessen. K. Nonaka K.-S. Yoon S. Ogo (&) International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan e-mail: [email protected]
EgMnSOD is thermostable, exhibiting two protein denaturation peaks at 65 and 84 °C. The statistical analysis of amino acid sequence and composition of the EgMnSOD suggests that the enzyme retains psychrophilic characteristics. Keywords Manganese superoxide dismutase Exiguobacterium Purification Psychrophilic Thermostability Genome sequence analysis Abbreviations EgMnSOD Mn-superoxide dismutase from Exiguobacterium sp. OS-77 EcMnSOD Mn-superoxide dismutase from Escherichia coli TtMnSOD Mn-superoxide dismutase from Thermus thermophilus PhFeSOD Fe-superoxide dismutase from Pseudoalteromonas haloplanktis AsFeSOD Fe-superoxide dismutase from Aliivibrio salmonicida SDS-PAGE Sodium dodecyl sulfate–polyacrylamide gel electrophoresis ICP-MS Inductively coupled plasma mass spectroscopy DSC Differential scanning calorimetry
K. Nonaka S. Ogo Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan
Introduction S. Ogo Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi Center Building, 4-1-8 Honcho, Kawaguchi-shi 332-0012, Saitama, Japan
Microorganisms living in extreme environments such as glacier water and hot springs have developed their enzymes to cope with environmental stress. Revealing the molecular
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basis of their adaptations for the extreme environments gives us helpful knowledge to design new biocatalysts having high activity and stability under desired conditions. Superoxide dismutases (SODs) are one of the enzymes used for the studies on the molecular adaptation. They catalyze the disproportionation of a harmful superoxide (O2-) into O2 and H2O2 in aerobic living matter to defend cell system against oxidative damages (Bafana et al. 2011). SODs are divided into four groups based on their protein structures and on their metal components in the active site: MnSODs, FeSODs, CuZnSODs, and NiSODs. Among them, MnSODs and FeSODs have similar structure to each other and exist typically in bacteria (Perry et al. 2010). Despite the molecular environment around the active site metal being similar between MnSODs and FeSODs, most of them exhibit significant activity with only their native metal cofactor (Ose and Fridovich 1979; Yamakura and Suzuki 1980). On the other hand, the cambialistic Mn- or FeSODs, exceptionally display their high activity with either Mn or Fe binding to the active site (Nakamura et al. 2011). This selectivity of the metal cofactors is probably a result of a difference in redox potential caused by the protein environment (Miller 2008), and it can be predicted from the amino acid sequence using the SODa program available at http://babylone.ulb.ac.be/soda (Kwasigroch et al. 2008). So far, a number of thermophilic MnSODs and FeSODs have been purified from various microorganisms such as Thermus thermophilus and Sulfolobus solfataricus (Gogliettino et al. 2004; Liu et al. 2011; Sato and Nakazawa 1978). The molecular mechanisms of heat adaptation have been studied by comparing properties of thermophilic enzymes and mesophilic enzymes (Ding et al. 2012). While psychrophilic FeSODs have been purified and characterized from Pseudoalteromonas haloplanktis (Castellano et al. 2006; Merlino et al. 2010) and Aliivibrio salmonicida (Pedersen et al. 2009), the studies on the metal binding selectivity of the psychrophilic FeSODs have not yet been reported. So far there has been no report on the purification and characterization of a psychrophilic MnSOD from bacteria. In this study, we report the first purification and characterization of the psychrophilic MnSOD from a newly isolated bacterium Exiguobacterium sp. OS-77 (EgMnSOD). The genus Exiguobacterium are Gram-positive facultative anaerobes that have been isolated from diverse sources of extreme environments in a wide range of temperatures between -10 °C and 55 °C (Vishnivetskaya et al. 2009). Although some enzymes such as catalase, alkaline protease, and ATPase have been purified and characterized from the genus of Exiguobacterium (Hara et al. 2007; Kasana and Yadav 2007; Suga and Koyama 2000), there has been no report on the purification of
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MnSOD. Interestingly, the newly found EgMnSOD has psychrophilic characteristics along with thermostability, which may play an important role as an antioxidant in a wide temperature range.
Materials and methods Isolation and culture of the strain OS-77 A water sample was collected from a tepid spring (41 °C, pH 7.9) in Oguni-tyo, Kumamoto, Japan. Enrichment and isolation were carried out in a medium-A containing the following components (per liter): 1.0 g of yeast extract (ForMedium Ltd.), 0.5 g of MgSO47H2O, 3.0 g of (NH4)2SO4, 2.0 g of K2HPO4, 1.0 g of KH2PO4, 2.0 g of Na2S2O35H2O, 48 lg of NiCl26H2O, 3.0 mg FeSO47H2O, and 1.2 mg of CaCl2. The pH of the medium was adjusted to 7.0 with 1.0 M NaOH. Liquid culture was performed aerobically in 150 mL vials containing 30 mL of the medium on a shaker at 30 °C. After several repetitions of enrichment culture, the culture solution was diluted and then streaked onto an agar plate. The orange-colored colonies were selected and supplied to liquid culture. The colonies were re-streaked more than three times to ensure the purity and named as the strain OS-77. Bacterial culture was performed using medium-B containing the following components (per liter): 10.0 g of polypeptone (Nihon Pharmaceutical Co., Ltd.), 2.0 g of yeast extract, and 1.0 g of MgSO47H2O. This medium was also used to investigate the optimal growth conditions such as a wide range of pHs (5.0–11.0), temperatures (5–45 °C), and NaCl concentrations (0–15 %). Mass culture was conducted using three 500 mL flasks containing 330 mL of medium-B (pH 8.0, 0 % of NaCl) by shaking at 140 rpm under air at 20 °C. After 40 h, the stationary phase cells were harvested by centrifugation (10,000g, 20 min, 4 °C) and the pellet was stored at -80 °C. 16S rRNA gene sequence and phylogenetic analysis Genomic DNA of strain OS-77 was isolated from lateexponential-phase cells using an InstaGene kit (Bio-Rad, Hercules, CA, USA). PCR was performed using PrimeSTAR HS DNA polymerase (Takara Bio) with 25 cycles of denaturation at 96 °C for 10 s, annealing at 50 °C for 5 s, and extension at 60 °C for 4 min. The 16S rRNA gene was sequenced using an ABI Prism 3130 genetic analyzer (applied biosystems). The protocols were carried out according to the manufacturer’s instructions. The sequence (1,487 bp) of the 16S rRNA gene of the strain OS-77 was deposited on the DDBJ/EMBL/GenBank databases under accession number AB753864. The CLUSTAL W program
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(Thompson et al. 1994) was used to align the 16S rRNA gene sequences with related sequences (available in the DDBJ/EMBL/GenBank databases). Phylogenetic trees were constructed by neighbor-joining methods (Saitou and Nei 1987) using Molecular Evolutionary Genetics Analysis (MEGA) version 5.0 (Tamura et al. 2011). The topology of the tree was evaluated by means of bootstrap analysis based on 1,000 replicates (Felsenstein 1985). Phenotypic characterization To investigate the phenotypic character of the strain OS-77, the cell was cultured on nutrient agar plates (Oxoid, Hampshire, England) for 24 h at 30 °C. Cell morphology was investigated under the BX50F4 optical microscopy (Olympus, Tokyo, Japan). Gram staining of the cell wall was conducted using Faber G ‘‘Nissui’’ (Nissui Pharmaceutical Co., Ltd., Tokyo). Gas and acid production from glucose, oxidative-fermentative test, catalase activity, and oxidase activity was determined according to the standard bacteriological methods. Physiological characteristics were determined using API CORYNE (bioMe´rieux, Lyon, France).
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between 150 and 300 mM at a flow rate of 3 mL/min. The eluted SOD fraction was concentrated using Amicon Ultra15 (30,000 NMWL, Millipore Corp., USA) and subjected to a Superdex 200 (1.6 9 50 cm, GE Healthcare UK Ltd.) with 20 mM Tris–HCl buffer (pH 7.8) at a flow rate of 1 mL/min. The fraction showing SOD activity was collected as the purified enzyme. The molecular mass of the purified native protein was estimated by gel filtration of Superdex 200 (1.6 9 50 cm, GE Healthcare UK Ltd.). The gel filtration standard (Bio-rad Laboratories Inc.) was used for molecular mass standard. Protein purity was established by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 15 % separating gel and 5 % stacking gel. The LMW Calibration Kit (GE Healthcare UK Ltd.) was used for standard molecular marker of SDS-PAGE. Protein bands were stained with Coomassie Brilliant Blue R-250. Protein concentration was measured by established procedures using the Bio-Rad Protein Assay (Bio-Rad Laboratories Inc.) (Bradford 1976). Metal contents of the purified enzyme were analyzed by an inductively coupled plasma mass spectrometer (ICP-MS) using Agilent 7500c (Agilent Technologies Inc., USA). UV– Vis spectrum of the purified enzyme was measured using JASCO V-670 spectrophotometer.
Purification of the EgMnSOD Enzyme assay The frozen cells (wet weight: 8 g) were suspended in 50 mM potassium phosphate (KP) buffer (pH 7.8) and lysed by adding lysozyme (Wako Pure Chemical Industries Ltd.) to 1 mg/mL and stirring for 1 h at 30 °C. The lysate was sonicated (60 W, 10 min) with Ultrasonic Disruptor UD-200 (Tomy Seiko Inc., Japan) and ultracentrifuged (140,000g, 1 h, 4 °C) using Optima L-90 K Ultracentrifuge (Beckman Coulter Inc., USA). The supernatant was collected as a soluble cell extract. The EgMnSOD in the cell extract was fractionated by 50 % (w/v) ammonium sulfate precipitation while stirring the solution on an ice bath for 30 min. The precipitated proteins were removed by centrifugation (100,000g, 30 min, 4 °C) and the supernatant was then directly loaded onto a Phenyl Sepharose high performance (2.6 9 15 cm, GE Healthcare UK Ltd.) preequilibrated with 20 mM Tris–HCl buffer (pH 7.8) containing 2 M ammonium sulfate. Column chromatography was performed at 4 °C using an AKTA-FPLC system (GE Healthcare UK Ltd., Buckinghamshire, UK). The EgMnSOD was eluted around 1.5 M concentration by a linear gradient of ammonium sulfate between 1.7 M and 1.3 M at a flow rate of 8 mL/min. The resulting solution was desalted using Sephadex G-25 column and loaded onto a Q-Sepharose high performance (1.6 9 15 cm, GE Healthcare UK Ltd.) pre-equilibrated with 20 mM Tris– HCl buffer (pH 7.8). The EgMnSOD was eluted around 220 mM concentration by a linear gradient of NaCl
SOD assays were performed according to the general method (Castellano et al. 2006; Gogliettino et al. 2004; Hakamada et al. 1997), which is the modified method of McCord and Fridovich (1969). The reaction mixture in a 3 mL cuvette contained 50 mM KP buffer (pH 7.8), 10 lM cytochrome c (Horse heart, Wako Pure Chemical Industries Ltd.), and 50 lM xanthine. A sufficient amount of xanthine oxidase (Butter milk, Oriental Yeast Co. Ltd., Japan) was then injected to generate O2- at a rate of cytochrome c reduction of 0.028 ± 0.003 absorbance/min measured at 550 nm in each condition. For the optimum pH measurement, the Britton-Robinson (BR) universal buffer (pH 6.0–11.0) was used. The buffer was prepared according to the reported method (Britton and Robinson 1931). One unit of SOD activity was defined as the amount of enzyme that caused 50 % inhibition of the cytochrome c reduction under the assay conditions. Thermostability of the EgMnSOD Thermostability of EgMnSOD was determined according to the measurement of the psychrophilic FeSOD from P. haloplanktis (Castellano et al. 2006). The solution (60 lL) of the purified enzyme (20 lg/mL) in 50 mM KP buffer (pH 7.8) was incubated at 25–75 °C for 10 min. An aliquot of the solution was then subjected to SOD activity assay at
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25 °C to determine the residual activity. The activity value of the non-incubated enzyme was calculated as 100 %. Differential scanning calorimetry (DSC) measurement was performed on a X-DSC7000 system (Hitachi Hitech Inc., Japan) using the built-in software for acquisition and analysis. The Ag sealed vessel containing 15 lL of the purified enzyme in 20 mM Tris–HCl buffer (pH 7.8) was used for the measurement sample, and the vessel containing buffer solution without the enzyme was used as a reference. The measurement was conducted by scanning from 30 °C to 100 °C at a heating rate of 1 K/min. Genome and amino acid sequence analysis Draft genome sequence data were generated with a Roche GS FLX system. The DNA was extracted from OS-77 and the 8 kb mate-paired library was produced. The shotgun data consisted of 312,910 reads and generated 141,354,788 bp. The GS De Novo Assembler (v 2.6) was used to assemble the reads into 5 scaffolds containing 23 contigs (total number of bases: 3,151,479). The largest and the N50 contig sizes were 929,170 and 349,882 bp, respectively. The genome had an overall estimated G?C content of 47 %. This whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under accession number PRJDB1123. The N-terminal amino acid sequence of the purified enzyme was determined by the automated Edman degradation system of ABI protein sequencer 473A (Applied Biosystems Japan, Tokyo). The protein band of the purified enzyme was made using SDS-PAGE, and then blotted onto a polyvinylidene difluoride membrane (Matsudaira 1987).
Results and discussion Bacterial characteristics of the strain OS-77 To isolate new bacteria containing psychrophilic MnSOD, we have collected many aquatic samples from different water springs in the Kyushu area, Japan. From these, we have successfully isolated a strain OS-77 from water sample of a tepid spring at Oguni-tyo, Kumamoto, Japan. The isolated strain OS-77 is a Gram-positive, roundshaped, non-spore-forming, and motile bacterium (Fig. 1). The colony was a yellowish orange with the size of 1–2 mm. The 16S rRNA gene sequence of strain OS-77 comprised 1,487 bp (GenBank accession no. AB753864). No complete identical sequences of strain OS-77 could be found in the NCBI nucleotide database. Based upon the comparisons of the 16S rRNA gene sequences, the strain OS-77 belongs to the genus Exiguobacterium, exhibiting 16S rRNA gene sequence identity with its relative species:
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Fig. 1 Optical microscope image of the Gram-stained strain OS-77
99.8 % (E. indicum HHS 31, NR_042347), 99.6 % (E. acetylicum DSM 20416, NR_043479), 98.3 % (E. sibiricum 255-15, NR_075006), 98.2 % (E. antarcticum DSM 14480, NR_043476), 94.8 % (strain AT1b, NR_074970), 94.8 % (E. marinum TF-80, NR_043006), and 94.2 % (E. aurantiacum DSM 6208, NR_043478). The phylogenetic position of the strain OS-77 is represented in the 16S rRNA gene sequence-based phylogenetic tree (Fig. 2). The Exiguobacterium species comprised two phylogenetically divergent groups (Vishnivetskaya et al. 2009): group-I has mostly been isolated from cold habitats such as permafrost (Rodrigues et al. 2006), while group-II has been isolated from moderately hot or slightly alkaline environments. The species involved in group-II are able to grow at high temperature even above 45 °C, but the species involved in group-I are unable to survive at 45 °C. Notably, the bacteria belonging to group-I can grow readily over a wide temperature range between -3 and 45 °C. The phylogenetic tree suggested that the strain OS-77 is in the group-I with its closest relative to the strain of E. indicum HHS 31 (Chaturvedi and Shivaji 2006). The growth temperature range of the strain OS-77 was between 5 and 40 °C with its optimum growth temperature of 20 °C, supporting the suggested phylogenetic position of the strain OS-77. The growth pH range of the strain OS-77 was shown to be between 6.0 and 10.0 with optimum growth at pH 8.0. The strain OS-77 showed salinity tolerance up to 7.5 % (w/v) NaCl solution. These characteristics are compatible to those of strains in the Exiguobacterium genus. Also, the detailed phenotypic characteristics of the strain OS-77 were investigated. The strain OS-77 was found to be positive for activities of catalase, oxidase, b-galactosidase, and a-glucosidase, and hydrolysis of aesculin, starch, and casein, and negative for nitrate reduction, activities of pyrazine amidase, pyrrolidonyl arylamidase, alkaline phosphatase, b-glucuronidase, N-acetyl-b-glucosaminidase, and
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367 95 Exiguobacterium sp. OS-77 (AB753864) 97
Group-I
Exiguobacterium indicum HHS 31 (NR_042347) Exiguobacterium acetylicum DSM 20416 (NR_043479)
100
Exiguobacterium sibiricum 255-15 (NR_075006) 92 84
Group-II
Exiguobacterium antarcticum DSM 14480 (NR_043476) Exiguobacterium sp. AT1b (NR_074970) Exiguobacterium marinum TF-80 (NR_043006)
100
Exiguobacterium aurantiacum DSM 6208 (NR_043478) Bacillus halodurans DSM 497 (AJ302709)
0.01
Fig. 2 Phylogenetic relationship of the strain OS-77 to other members of the genus Exiguobacterium. The tree was produced based on 16S rRNA gene sequences by a neighbor-joining method. Bacillus halodurans DSM 497 was used as the outgroup. Accession
numbers of the sequences used in this study are shown in parentheses. Bootstrap values were based on 1,000 replicates. The scale bar represents 0.01 changes per nucleotide position
urease, and hydrolysis of gelatin. Acid was produced from glucose, ribose, mannitol, maltose, sucrose, and glycogen, but not from xylose and lactose. The strain utilizes ribose, glucose, mannitol, amygdalin, cellobiose, glycogen, and gluconate, and does not utilize glycerol, erythritol, L-arabinose, D-xylose, L-xylose, adonitol, b-methyl-D-xyloside, galactose, fructose, mannose, sorbose, rhamnose, dulcitol, inositol, sorbitol, a-methyl-D-mannoside, a-methyl-D-glucoside, N-acetylglucosamine, arbutin, aesculin, salicin, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconate, and 5-ketogluconate. As shown in Table 1, the phenotypic characteristics of strain OS-77 are clearly distinguishable from those of other Exiguobacterium species. As compared to E. indicum HHS 31, which was determined as the closest strain by phylogenetic tree shown in Fig. 2, the phenotypic characteristics of strain OS-77 are much closer to those of the E. acetylicum DSM 20416 and E. antarcticum DSM 14480.
molecular mass of the EgMnSOD is consistent with those of other Fe- and MnSODs (Bafana et al. 2011). The purified SOD is a purple-colored protein having a broad absorption band with a maximum at 478 nm (Fig. 4), which is known as a typical characteristic of MnSOD (Whittaker and Whittaker 1991). The molar absorption coefficient value at 478 nm was 1,104 M-1 cm-1. ICP-MS analysis of the SOD showed that the enzyme contains Mn (0.57 atom/chain), but not Fe (0.01 atom/chain), indicating that the purified SOD from Exiguobacterium sp. OS-77 belongs to the group of MnSODs.
Purification of the EgMnSOD The EgMnSOD was successfully purified from the strain OS-77 through sequential purification steps using columns of phenyl sepharose high performance, Q-sepharose high performance, and Superdex 200 (Table 2). The EgMnSOD was purified to 50-fold with a yield of 41 % and a specific activity of 10,500 U/mg. The molecular mass of the native enzyme was estimated to be approximately 50 kDa by Superdex 200 gel filtration column. The EgMnSOD is a homodimer of polypeptides determined as a molecular mass of approximately 23.5 ± 1.0 kDa in SDS-PAGE (Fig. 3), corresponding to the molecular mass of 22,532 Da calculated from the deduced amino acid sequence. The
Activity and thermostability of the EgMnSOD The effect of pH on the activity of the EgMnSOD was measured in 50 mM BR buffers with pH ranging from 6.0 to 11.0. The EgMnSOD remained at least 70 % of activity under all the measured pH conditions compared with the activity at optimum pH of 9.0 (Fig. 5a). Outside this range, the measurement could not be performed because of rapid denaturation of xanthine oxidase and cytochrome c. The effect of temperature on the activity of the EgMnSOD was also measured in 50 mM KP buffer at pH 7.8. The optimum temperature of the EgMnSOD was around 5 °C (Fig. 5b), indicating that the EgMnSOD has a psychrophilic property. The activity at 25 °C achieved approximately 80 % of the optimum activity. These results showing a slight loss of the activity under various pH and temperature conditions are compatible to the PhFeSOD (Castellano et al. 2006). The catalytic activity of the EgMnSOD (10,500 U/mg at 25 °C) is higher than those of the mesophilic MnSOD from Escherichia coli (EcMnSOD, 7,300 U/mg at 25 °C) (Whittaker and Whittaker 1991) and the thermophilic MnSOD from Thermus thermophilus (TtMnSOD, 1,400 U/mg at 25 °C) (Liu et al. 2011). The
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368 Table 1 Differential phenotypic characteristics of the strain OS-77 and its related strains of the genus Exiguobacterium
Extremophiles (2014) 18:363–373
Characteristics
1
2
3
4
5
Habitat
Spring water
Glacial water
Creamery waste
Permafrost
Lake water
Size (mm)
1–2
2–4
2–5
3.5–4
2–3
Shape
Round
Round
Irregular
Round
Round
Color
Yellowish orange
Yellowish orange
Yellowish orange
Bright orange
Orange
? ?
? –
– w
? ?
– ?
40
30
37
40
41
–
–
?
?
?
Colony morphology
Growth temperature (°C) 5 37 Maximum temperature of growth (°C) Hydrolysis of Gelatin Aesculin
?
–
?
–
?
Starch
?
–
?
ND
?
Casein
?
–
?
ND
?
Nitrate reduction
–
?
–
–
–
Acid production from glucose, ribose, maltose, sucrose
?
–
?
ND
?
Sucrose, glycerol Fructose, mannose, trehalose
– –
? –
? ?
? ?
? ?
Utilization of
Ribose
?
–
?
?
?
Raffinose
–
?
–
?
?
Galactose
–
?
–
?
–
L-Xylose
–
?
–
ND
–
–
?
–
±
– –
D-Xylose,
Strains: 1, strain OS-77; 2, E. indicum HHS 31; 3, E. acetylicum DSM 20416; 4, E. sibiricum 255-15; 5, E. antarcticum DSM 14480. Data for all strains except OS-77 are from the report by Chaturvedi and Shivaji (2006) ? positive, - negative, ND no data available, ± variable reaction, w weakly positive a
Estimated from the genome sequence
rhamnose, melibiose
Melezitose
–
–
?
±
L-Fucose, xylitol
–
–
?
–
–
Mannitol
?
–
?
?
–
Erythritol
–
?
–
ND
–
Sorbitol, dulcitol, inositol, inulin
–
?
–
–
–
Arbutin
–
?
–
?
?
Salicin
–
–
?
?
–
Amygdalin
?
–
–
?
?
a-Methyl-D-mannoside
–
–
?
–
?
a-Methyl-D-glucoside N-Acetylglucosamine
– –
– –
? –
– ?
– –
Starch
–
–
?
?
?
Glycogen
?
?
–
ND
?
Gluconate
?
?
–
–
–
5-Ketogluconate
–
?
–
–
–
DNA G?C content (mol%)
47a
48
47
47.7
47
16S rRNA gene similarity (%)
100
99.8
99.6
98.3
98.2
result is compatible to the general feature of psychrophilic enzymes that is the improved catalytic efficiency at low temperature when compared to those of mesophilic and thermophilic enzymes (Georlette et al. 2004).
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The thermostability of the EgMnSOD was examined by pre-incubating the enzyme in the 50 mM KP buffer at pH 7.8 under various temperatures (25–75 °C) for 10 min. The EgMnSOD was stable up to 45 °C and was gradually
Extremophiles (2014) 18:363–373
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Table 2 Purification of the EgMnSOD from Exiguobacterium sp. OS-77 Step
Activity (U)
Protein (mg)
Specific activity (U/mg)
Yield (%)
Purification (fold) 1.0
Soluble cell extract
61,100
294
208
100
Ammonium sulfate precipitation
60,100
190
316
98
1.5
Phenyl sepharose HP
48,100
7.00
6,870
79
33
Q-sepharose HP
37,400
4.01
9,330
61
45
Superdex 200
25,300
2.42
10,500
41
50
(kDa)
M
1
97 66 45
30 23.5 kDa 20.1
14.4 Fig. 3 SDS-PAGE of the purified MnSOD. Lane M molecular marker, lane 1 the EgMnSOD
of the PhFeSOD at 50 °C was estimated to be 14.2 min. The EgMnSOD shows high thermal stability compared to the maximal temperature (40 °C) for growth of the strain OS-77. Such properties are also found in other psychrophilic enzymes from various microorganisms (Castellano et al. 2006; Pedersen et al. 2009). The thermal stability of the EgMnSOD was supported by the results of DSC measurement showing two transition points with midpoint transition temperature (Tm) at 65 and 84 °C (Fig. 6). The first transition point was almost same as the half-inactivation temperature of the EgMnSOD. The similar result has been reported in the DSC measurement of mesophilic EcMnSOD, displaying two transition curves detected at 71 and 89 °C (Mizuno et al. 2004; Whittaker 2010). In the papers, the two transitions at low and high Tm are discussed as being due to the unfolding of the reduced Mn(II)-state and the oxidized Mn(III)-state of the enzyme, respectively. Conformational structure of the oxidized Mn(III)-state enzyme seems to be more stable than that of the reduced Mn(II)-state enzyme, correlating with higher Tm value. The results suggest that the EgMnSOD also has a similar structural organization of unfolding process to those of other mesophilic MnSODs purified from different organisms. Genome analysis of the strain OS-77 and amino acid sequence of the EgMnSOD
Fig. 4 UV-Vis absorption spectrum of the purified EgMnSOD. The enzyme solution (16.2 mg/mL) in 20 mM Tris–HCl buffer (pH 7.8) was used for the measurement
inactivated above the temperature (Fig. 5c). The halfinactivation temperature of the EgMnSOD was calculated as 63.2 °C. The half-life of activity at 60 °C was estimated to be 21.2 min (Fig. 5d). As shown in Table 3, the EgMnSOD showed the enhanced thermostability compared to the PhFeSOD. The half-inactivation temperature of the PhFeSOD was 54.2 °C under the thermal incubation for 10 min (Castellano et al. 2006). Also, the half-life activity
The amino acid sequence of the EgMnSOD was revealed by the combination of whole genome shotgun sequencing and N-terminal amino acid analysis. The draft genome sequence of the strain OS-77 was obtained using de novo assembly method. A gene coding an MnSOD (sodA) has been found using the BLAST program with reference to the sodA gene (Exig_0858) of E. sibiricum 255-15 (Rodrigues et al. 2008). The N-terminal amino acid sequence analysis of the purified EgMnSOD provided the sequence of SKFELPE, which is identical to the amino acid sequence translated from the nucleic acid sequence of the sodA gene of the strain OS-77. The complete amino acid sequence of the EgMnSOD consists of 202 amino acid residues (Fig. 7) with a calculated molecular mass of 22,532 Da. The amino acid sequence alignment of the EgMnSOD with the
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Extremophiles (2014) 18:363–373
A
B
C
D
Fig. 5 Activity and thermostability of the EgMnSOD. a The effect of pH on the activity measured in 50 mM BR buffer at 25 °C. b The effect of temperature on the activity. c Thermostability under various temperature conditions. d Heat-inactivation profile during incubation. For the heat-inactivation profile, a solution of the EgMnSOD (20 lg/
mL) in 50 mM KP buffer (pH 7.8) was incubated at 60 °C for 5–40 min. An aliquot of the solution was then subjected to SOD activity assay at 25 °C to determine the residual activity. The activity value of the non-incubated enzyme was calculated as 100 %. The data are from three independent experiments per condition
Table 3 Effects of temperature on the activity of the SODs Protein
Half-inactivation temperature (°C)a
Half-life of activity (min)b
References
PhFeSOD
54.2 (10 min)
14.2 (50 °C)
Castellano et al. (2006)
EgMnSOD
63.2 (10 min)
21.2 (60 °C)
This work
EcMnSOD
ND
TtMnSOD
105 (60 min)
9.0 (50 °C) 12.0 (110 °C)
Hunter et al. (2002) Liu et al. (2011)
ND no data available a
The values in the parentheses are the incubation time
b
The values in the parentheses are the incubation temperature
mesophilic EcMnSOD and the thermophilic TtMnSOD showed the amino acid identity of 60 and 62 %, respectively. Typical residues that are involved in the active site (H28, Y36, H82, Q149, D164, and H168) are completely conserved in the EgMnSOD. Recently, Vendittis et al. (2008) proposed that average parameters related to amino
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Fig. 6 Thermal unfolding profiles of the EgMnSOD measured by DSC. The molecular weight of 48,000 was used for calculation of molar heat capacity (Cp)
acid composition of the SODs such as mass, volume, accessible surface area, and hydrophobicity are linearly correlated with the optimum growth temperature of the
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Fig. 7 Multiple alignment of the amino acid sequences of EgMnSOD, EcMnSOD, and TtMnSOD. The sequence of the EgMnSOD (GenBank ID: GAD04318) was translated from the nucleic acid sequence of sodA gene. The accession numbers of the amino acid sequences of the EcMnSOD and TtMnSOD are NP_418344 and YP_004164, respectively. The multiple alignment was conducted
using constraint-based multiple alignment tool (COBALT, http:// www.ncbi.nlm.nih.gov/tools/cobalt/) in the National Center for Biotechnology Information (NCBI). The residues conserved in all sequences are represented in yellow background, and the residues typically involved in the active site are represented in pale rose background
Table 4 Parameters related to the amino acid compositions of PhFeSOD, EgMnSOD, EcMnSOD, and TtMnSOD Protein
Mass (Da)
˚ 3) Volume (A
˚ 2) Accessible surface area (A
Hydrophobicity (kJ/mol)
Optimum growth temperature (°C)
PhFeSOD EgMnSOD
110.7 (111.0) 111.6 (111.3)
133.5 (133.6) 134.3 (134.1)
159.8 (160.5) 161.7 (161.0)
4.83 (4.69) 4.59 (4.72)
15 20
EcMnSOD
112.1 (112.2)
135.7 (135.6)
162.9 (162.6)
4.71 (4.83)
37
TtMnSOD
113.9 (114.0)
139.0 (138.3)
165.7 (165.5)
5.14 (5.01)
68
The parameters related to the amino acid compositions of EgMnSOD, EcMnSOD, and TtMnSOD were determined according to the method described by Vendittis et al. (2008). The values in parentheses were derived from the linear fitting equations for SODs in the report: for mass, y = 110.15 ? 0.05608x; for volume, y = 132.3 ? 0.08818x; for accessible surface area, y = 159.1 ? 0.09383x; for hydrophobicity, y = 4.60 ? 0.006087x. (y each parameter, x optimum growth temperature). The parameter data for PhFeSOD were obtained from the report studied by Castellano et al. (2006)
host microorganisms. Among these parameters, the former three parameters are related to the ratio of bulky residues, and the average hydrophobicity reflects the ratio of hydrophobic residues in the enzyme. The low ratio of bulky and hydrophobic residues results in an increased number of cavities and less hydrophobic interactions in the protein core, respectively. These factors can cause high molecular flexibility that is hypothesized as the reason of psychrophilicity of the enzymes. As shown in Table 4, the values of all average parameters of the EgMnSOD were lower than those of the mesophilic EcMnSOD and thermophilic TtMnSOD. The differences of the parameters among EgMnSOD, EcMnSOD, and TtMnSOD are matched to the values calculated using linear fitting equation reported by Vendittis et al. (2008). This result indicates the structural adaptation of the EgMnSOD toward low-temperature conditions. Although the hydrophobicity value of the PhFeSOD appears to be slightly higher than that of the EgMnSOD, however, all of the other parameters related to
the ratio of bulky residues of the EgMnSOD were higher than those of the PhFeSOD, which may affect the high thermostability of the EgMnSOD. Further structure-based studies of the EgMnSOD will help to understand how the enzyme achieves its psychrophilicity along with thermostability.
Conclusions This report presents biochemical characterization of the psychrophilic MnSOD from a newly isolated bacterium Exiguobacterium sp. OS-77. We determined the draft genome sequence of Exiguobacterium sp. OS-77 and then assigned the gene encoding EgMnSOD. The calculated value of the parameters related to the amino acid composition suggested structural cold-adaptation of the EgMnSOD. The most significant finding of this study is that the EgMnSOD has psychrophilic character along with
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thermostability. The present results suggest that the EgMnSOD might be a promising material that may contribute to revealing the molecular mechanisms lying in psychrophilic enzymes. Acknowledgments This work was supported by the World Premier International Research Center Initiative (WPI), Grants-in-Aid: 23655053, 25620047, 25248017, and 24109016 (Scientific Research on Innovative Areas ‘‘Stimuli-responsive Chemical Species’’) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan and the Basic Research Programs CREST Type, ‘‘Development of the Foundation for Nano-Interface Technology’’ from JST, Japan.
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ORIGINAL PAPER
Biochemical characterization of psychrophilic Mn-superoxide dismutase from newly isolated Exiguobacterium sp. OS-77 Kyoshiro Nonaka • Ki-Seok Yoon • Seiji Ogo
Received: 11 July 2013 / Accepted: 29 December 2013 / Published online: 12 January 2014 Ó Springer Japan 2014
Abstract Many types of superoxide dismutases have been purified and characterized from various bacteria, however, a psychrophilic Mn-superoxide dismutase (MnSOD) has not yet been reported. Here, we describe the purification and the biochemical characterization of the psychrophilic MnSOD from Exiguobacterium sp. strain OS-77 (EgMnSOD). According to 16S rRNA sequence analysis, a newly isolated bacterium strain OS-77 belongs to the genus Exiguobacterium. The optimum growth temperature of the strain OS-77 is 20 °C. The EgMnSOD is a homodimer of 23.5 kDa polypeptides determined by SDSPAGE and gel filtration analysis. UV-Vis spectrum and ICP-MS analysis clearly indicated that the homogeneously purified enzyme contains only a Mn ion as a metal cofactor. The optimal reaction pH and temperature of the enzyme were pH 9.0 and 5 °C, respectively. Notably, the purified EgMnSOD was thermostable up to 45 °C and retained 50 % activity after 21.2 min at 60 °C. The differential scanning calorimetry also indicated that the Communicated by A. Driessen. K. Nonaka K.-S. Yoon S. Ogo (&) International Institute for Carbon-Neutral Energy Research (WPI-I2CNER), Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan e-mail: [email protected]
EgMnSOD is thermostable, exhibiting two protein denaturation peaks at 65 and 84 °C. The statistical analysis of amino acid sequence and composition of the EgMnSOD suggests that the enzyme retains psychrophilic characteristics. Keywords Manganese superoxide dismutase Exiguobacterium Purification Psychrophilic Thermostability Genome sequence analysis Abbreviations EgMnSOD Mn-superoxide dismutase from Exiguobacterium sp. OS-77 EcMnSOD Mn-superoxide dismutase from Escherichia coli TtMnSOD Mn-superoxide dismutase from Thermus thermophilus PhFeSOD Fe-superoxide dismutase from Pseudoalteromonas haloplanktis AsFeSOD Fe-superoxide dismutase from Aliivibrio salmonicida SDS-PAGE Sodium dodecyl sulfate–polyacrylamide gel electrophoresis ICP-MS Inductively coupled plasma mass spectroscopy DSC Differential scanning calorimetry
K. Nonaka S. Ogo Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan
Introduction S. Ogo Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi Center Building, 4-1-8 Honcho, Kawaguchi-shi 332-0012, Saitama, Japan
Microorganisms living in extreme environments such as glacier water and hot springs have developed their enzymes to cope with environmental stress. Revealing the molecular
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basis of their adaptations for the extreme environments gives us helpful knowledge to design new biocatalysts having high activity and stability under desired conditions. Superoxide dismutases (SODs) are one of the enzymes used for the studies on the molecular adaptation. They catalyze the disproportionation of a harmful superoxide (O2-) into O2 and H2O2 in aerobic living matter to defend cell system against oxidative damages (Bafana et al. 2011). SODs are divided into four groups based on their protein structures and on their metal components in the active site: MnSODs, FeSODs, CuZnSODs, and NiSODs. Among them, MnSODs and FeSODs have similar structure to each other and exist typically in bacteria (Perry et al. 2010). Despite the molecular environment around the active site metal being similar between MnSODs and FeSODs, most of them exhibit significant activity with only their native metal cofactor (Ose and Fridovich 1979; Yamakura and Suzuki 1980). On the other hand, the cambialistic Mn- or FeSODs, exceptionally display their high activity with either Mn or Fe binding to the active site (Nakamura et al. 2011). This selectivity of the metal cofactors is probably a result of a difference in redox potential caused by the protein environment (Miller 2008), and it can be predicted from the amino acid sequence using the SODa program available at http://babylone.ulb.ac.be/soda (Kwasigroch et al. 2008). So far, a number of thermophilic MnSODs and FeSODs have been purified from various microorganisms such as Thermus thermophilus and Sulfolobus solfataricus (Gogliettino et al. 2004; Liu et al. 2011; Sato and Nakazawa 1978). The molecular mechanisms of heat adaptation have been studied by comparing properties of thermophilic enzymes and mesophilic enzymes (Ding et al. 2012). While psychrophilic FeSODs have been purified and characterized from Pseudoalteromonas haloplanktis (Castellano et al. 2006; Merlino et al. 2010) and Aliivibrio salmonicida (Pedersen et al. 2009), the studies on the metal binding selectivity of the psychrophilic FeSODs have not yet been reported. So far there has been no report on the purification and characterization of a psychrophilic MnSOD from bacteria. In this study, we report the first purification and characterization of the psychrophilic MnSOD from a newly isolated bacterium Exiguobacterium sp. OS-77 (EgMnSOD). The genus Exiguobacterium are Gram-positive facultative anaerobes that have been isolated from diverse sources of extreme environments in a wide range of temperatures between -10 °C and 55 °C (Vishnivetskaya et al. 2009). Although some enzymes such as catalase, alkaline protease, and ATPase have been purified and characterized from the genus of Exiguobacterium (Hara et al. 2007; Kasana and Yadav 2007; Suga and Koyama 2000), there has been no report on the purification of
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MnSOD. Interestingly, the newly found EgMnSOD has psychrophilic characteristics along with thermostability, which may play an important role as an antioxidant in a wide temperature range.
Materials and methods Isolation and culture of the strain OS-77 A water sample was collected from a tepid spring (41 °C, pH 7.9) in Oguni-tyo, Kumamoto, Japan. Enrichment and isolation were carried out in a medium-A containing the following components (per liter): 1.0 g of yeast extract (ForMedium Ltd.), 0.5 g of MgSO47H2O, 3.0 g of (NH4)2SO4, 2.0 g of K2HPO4, 1.0 g of KH2PO4, 2.0 g of Na2S2O35H2O, 48 lg of NiCl26H2O, 3.0 mg FeSO47H2O, and 1.2 mg of CaCl2. The pH of the medium was adjusted to 7.0 with 1.0 M NaOH. Liquid culture was performed aerobically in 150 mL vials containing 30 mL of the medium on a shaker at 30 °C. After several repetitions of enrichment culture, the culture solution was diluted and then streaked onto an agar plate. The orange-colored colonies were selected and supplied to liquid culture. The colonies were re-streaked more than three times to ensure the purity and named as the strain OS-77. Bacterial culture was performed using medium-B containing the following components (per liter): 10.0 g of polypeptone (Nihon Pharmaceutical Co., Ltd.), 2.0 g of yeast extract, and 1.0 g of MgSO47H2O. This medium was also used to investigate the optimal growth conditions such as a wide range of pHs (5.0–11.0), temperatures (5–45 °C), and NaCl concentrations (0–15 %). Mass culture was conducted using three 500 mL flasks containing 330 mL of medium-B (pH 8.0, 0 % of NaCl) by shaking at 140 rpm under air at 20 °C. After 40 h, the stationary phase cells were harvested by centrifugation (10,000g, 20 min, 4 °C) and the pellet was stored at -80 °C. 16S rRNA gene sequence and phylogenetic analysis Genomic DNA of strain OS-77 was isolated from lateexponential-phase cells using an InstaGene kit (Bio-Rad, Hercules, CA, USA). PCR was performed using PrimeSTAR HS DNA polymerase (Takara Bio) with 25 cycles of denaturation at 96 °C for 10 s, annealing at 50 °C for 5 s, and extension at 60 °C for 4 min. The 16S rRNA gene was sequenced using an ABI Prism 3130 genetic analyzer (applied biosystems). The protocols were carried out according to the manufacturer’s instructions. The sequence (1,487 bp) of the 16S rRNA gene of the strain OS-77 was deposited on the DDBJ/EMBL/GenBank databases under accession number AB753864. The CLUSTAL W program
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(Thompson et al. 1994) was used to align the 16S rRNA gene sequences with related sequences (available in the DDBJ/EMBL/GenBank databases). Phylogenetic trees were constructed by neighbor-joining methods (Saitou and Nei 1987) using Molecular Evolutionary Genetics Analysis (MEGA) version 5.0 (Tamura et al. 2011). The topology of the tree was evaluated by means of bootstrap analysis based on 1,000 replicates (Felsenstein 1985). Phenotypic characterization To investigate the phenotypic character of the strain OS-77, the cell was cultured on nutrient agar plates (Oxoid, Hampshire, England) for 24 h at 30 °C. Cell morphology was investigated under the BX50F4 optical microscopy (Olympus, Tokyo, Japan). Gram staining of the cell wall was conducted using Faber G ‘‘Nissui’’ (Nissui Pharmaceutical Co., Ltd., Tokyo). Gas and acid production from glucose, oxidative-fermentative test, catalase activity, and oxidase activity was determined according to the standard bacteriological methods. Physiological characteristics were determined using API CORYNE (bioMe´rieux, Lyon, France).
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between 150 and 300 mM at a flow rate of 3 mL/min. The eluted SOD fraction was concentrated using Amicon Ultra15 (30,000 NMWL, Millipore Corp., USA) and subjected to a Superdex 200 (1.6 9 50 cm, GE Healthcare UK Ltd.) with 20 mM Tris–HCl buffer (pH 7.8) at a flow rate of 1 mL/min. The fraction showing SOD activity was collected as the purified enzyme. The molecular mass of the purified native protein was estimated by gel filtration of Superdex 200 (1.6 9 50 cm, GE Healthcare UK Ltd.). The gel filtration standard (Bio-rad Laboratories Inc.) was used for molecular mass standard. Protein purity was established by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 15 % separating gel and 5 % stacking gel. The LMW Calibration Kit (GE Healthcare UK Ltd.) was used for standard molecular marker of SDS-PAGE. Protein bands were stained with Coomassie Brilliant Blue R-250. Protein concentration was measured by established procedures using the Bio-Rad Protein Assay (Bio-Rad Laboratories Inc.) (Bradford 1976). Metal contents of the purified enzyme were analyzed by an inductively coupled plasma mass spectrometer (ICP-MS) using Agilent 7500c (Agilent Technologies Inc., USA). UV– Vis spectrum of the purified enzyme was measured using JASCO V-670 spectrophotometer.
Purification of the EgMnSOD Enzyme assay The frozen cells (wet weight: 8 g) were suspended in 50 mM potassium phosphate (KP) buffer (pH 7.8) and lysed by adding lysozyme (Wako Pure Chemical Industries Ltd.) to 1 mg/mL and stirring for 1 h at 30 °C. The lysate was sonicated (60 W, 10 min) with Ultrasonic Disruptor UD-200 (Tomy Seiko Inc., Japan) and ultracentrifuged (140,000g, 1 h, 4 °C) using Optima L-90 K Ultracentrifuge (Beckman Coulter Inc., USA). The supernatant was collected as a soluble cell extract. The EgMnSOD in the cell extract was fractionated by 50 % (w/v) ammonium sulfate precipitation while stirring the solution on an ice bath for 30 min. The precipitated proteins were removed by centrifugation (100,000g, 30 min, 4 °C) and the supernatant was then directly loaded onto a Phenyl Sepharose high performance (2.6 9 15 cm, GE Healthcare UK Ltd.) preequilibrated with 20 mM Tris–HCl buffer (pH 7.8) containing 2 M ammonium sulfate. Column chromatography was performed at 4 °C using an AKTA-FPLC system (GE Healthcare UK Ltd., Buckinghamshire, UK). The EgMnSOD was eluted around 1.5 M concentration by a linear gradient of ammonium sulfate between 1.7 M and 1.3 M at a flow rate of 8 mL/min. The resulting solution was desalted using Sephadex G-25 column and loaded onto a Q-Sepharose high performance (1.6 9 15 cm, GE Healthcare UK Ltd.) pre-equilibrated with 20 mM Tris– HCl buffer (pH 7.8). The EgMnSOD was eluted around 220 mM concentration by a linear gradient of NaCl
SOD assays were performed according to the general method (Castellano et al. 2006; Gogliettino et al. 2004; Hakamada et al. 1997), which is the modified method of McCord and Fridovich (1969). The reaction mixture in a 3 mL cuvette contained 50 mM KP buffer (pH 7.8), 10 lM cytochrome c (Horse heart, Wako Pure Chemical Industries Ltd.), and 50 lM xanthine. A sufficient amount of xanthine oxidase (Butter milk, Oriental Yeast Co. Ltd., Japan) was then injected to generate O2- at a rate of cytochrome c reduction of 0.028 ± 0.003 absorbance/min measured at 550 nm in each condition. For the optimum pH measurement, the Britton-Robinson (BR) universal buffer (pH 6.0–11.0) was used. The buffer was prepared according to the reported method (Britton and Robinson 1931). One unit of SOD activity was defined as the amount of enzyme that caused 50 % inhibition of the cytochrome c reduction under the assay conditions. Thermostability of the EgMnSOD Thermostability of EgMnSOD was determined according to the measurement of the psychrophilic FeSOD from P. haloplanktis (Castellano et al. 2006). The solution (60 lL) of the purified enzyme (20 lg/mL) in 50 mM KP buffer (pH 7.8) was incubated at 25–75 °C for 10 min. An aliquot of the solution was then subjected to SOD activity assay at
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25 °C to determine the residual activity. The activity value of the non-incubated enzyme was calculated as 100 %. Differential scanning calorimetry (DSC) measurement was performed on a X-DSC7000 system (Hitachi Hitech Inc., Japan) using the built-in software for acquisition and analysis. The Ag sealed vessel containing 15 lL of the purified enzyme in 20 mM Tris–HCl buffer (pH 7.8) was used for the measurement sample, and the vessel containing buffer solution without the enzyme was used as a reference. The measurement was conducted by scanning from 30 °C to 100 °C at a heating rate of 1 K/min. Genome and amino acid sequence analysis Draft genome sequence data were generated with a Roche GS FLX system. The DNA was extracted from OS-77 and the 8 kb mate-paired library was produced. The shotgun data consisted of 312,910 reads and generated 141,354,788 bp. The GS De Novo Assembler (v 2.6) was used to assemble the reads into 5 scaffolds containing 23 contigs (total number of bases: 3,151,479). The largest and the N50 contig sizes were 929,170 and 349,882 bp, respectively. The genome had an overall estimated G?C content of 47 %. This whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under accession number PRJDB1123. The N-terminal amino acid sequence of the purified enzyme was determined by the automated Edman degradation system of ABI protein sequencer 473A (Applied Biosystems Japan, Tokyo). The protein band of the purified enzyme was made using SDS-PAGE, and then blotted onto a polyvinylidene difluoride membrane (Matsudaira 1987).
Results and discussion Bacterial characteristics of the strain OS-77 To isolate new bacteria containing psychrophilic MnSOD, we have collected many aquatic samples from different water springs in the Kyushu area, Japan. From these, we have successfully isolated a strain OS-77 from water sample of a tepid spring at Oguni-tyo, Kumamoto, Japan. The isolated strain OS-77 is a Gram-positive, roundshaped, non-spore-forming, and motile bacterium (Fig. 1). The colony was a yellowish orange with the size of 1–2 mm. The 16S rRNA gene sequence of strain OS-77 comprised 1,487 bp (GenBank accession no. AB753864). No complete identical sequences of strain OS-77 could be found in the NCBI nucleotide database. Based upon the comparisons of the 16S rRNA gene sequences, the strain OS-77 belongs to the genus Exiguobacterium, exhibiting 16S rRNA gene sequence identity with its relative species:
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Fig. 1 Optical microscope image of the Gram-stained strain OS-77
99.8 % (E. indicum HHS 31, NR_042347), 99.6 % (E. acetylicum DSM 20416, NR_043479), 98.3 % (E. sibiricum 255-15, NR_075006), 98.2 % (E. antarcticum DSM 14480, NR_043476), 94.8 % (strain AT1b, NR_074970), 94.8 % (E. marinum TF-80, NR_043006), and 94.2 % (E. aurantiacum DSM 6208, NR_043478). The phylogenetic position of the strain OS-77 is represented in the 16S rRNA gene sequence-based phylogenetic tree (Fig. 2). The Exiguobacterium species comprised two phylogenetically divergent groups (Vishnivetskaya et al. 2009): group-I has mostly been isolated from cold habitats such as permafrost (Rodrigues et al. 2006), while group-II has been isolated from moderately hot or slightly alkaline environments. The species involved in group-II are able to grow at high temperature even above 45 °C, but the species involved in group-I are unable to survive at 45 °C. Notably, the bacteria belonging to group-I can grow readily over a wide temperature range between -3 and 45 °C. The phylogenetic tree suggested that the strain OS-77 is in the group-I with its closest relative to the strain of E. indicum HHS 31 (Chaturvedi and Shivaji 2006). The growth temperature range of the strain OS-77 was between 5 and 40 °C with its optimum growth temperature of 20 °C, supporting the suggested phylogenetic position of the strain OS-77. The growth pH range of the strain OS-77 was shown to be between 6.0 and 10.0 with optimum growth at pH 8.0. The strain OS-77 showed salinity tolerance up to 7.5 % (w/v) NaCl solution. These characteristics are compatible to those of strains in the Exiguobacterium genus. Also, the detailed phenotypic characteristics of the strain OS-77 were investigated. The strain OS-77 was found to be positive for activities of catalase, oxidase, b-galactosidase, and a-glucosidase, and hydrolysis of aesculin, starch, and casein, and negative for nitrate reduction, activities of pyrazine amidase, pyrrolidonyl arylamidase, alkaline phosphatase, b-glucuronidase, N-acetyl-b-glucosaminidase, and
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367 95 Exiguobacterium sp. OS-77 (AB753864) 97
Group-I
Exiguobacterium indicum HHS 31 (NR_042347) Exiguobacterium acetylicum DSM 20416 (NR_043479)
100
Exiguobacterium sibiricum 255-15 (NR_075006) 92 84
Group-II
Exiguobacterium antarcticum DSM 14480 (NR_043476) Exiguobacterium sp. AT1b (NR_074970) Exiguobacterium marinum TF-80 (NR_043006)
100
Exiguobacterium aurantiacum DSM 6208 (NR_043478) Bacillus halodurans DSM 497 (AJ302709)
0.01
Fig. 2 Phylogenetic relationship of the strain OS-77 to other members of the genus Exiguobacterium. The tree was produced based on 16S rRNA gene sequences by a neighbor-joining method. Bacillus halodurans DSM 497 was used as the outgroup. Accession
numbers of the sequences used in this study are shown in parentheses. Bootstrap values were based on 1,000 replicates. The scale bar represents 0.01 changes per nucleotide position
urease, and hydrolysis of gelatin. Acid was produced from glucose, ribose, mannitol, maltose, sucrose, and glycogen, but not from xylose and lactose. The strain utilizes ribose, glucose, mannitol, amygdalin, cellobiose, glycogen, and gluconate, and does not utilize glycerol, erythritol, L-arabinose, D-xylose, L-xylose, adonitol, b-methyl-D-xyloside, galactose, fructose, mannose, sorbose, rhamnose, dulcitol, inositol, sorbitol, a-methyl-D-mannoside, a-methyl-D-glucoside, N-acetylglucosamine, arbutin, aesculin, salicin, lactose, melibiose, sucrose, trehalose, inulin, melezitose, raffinose, starch, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-ketogluconate, and 5-ketogluconate. As shown in Table 1, the phenotypic characteristics of strain OS-77 are clearly distinguishable from those of other Exiguobacterium species. As compared to E. indicum HHS 31, which was determined as the closest strain by phylogenetic tree shown in Fig. 2, the phenotypic characteristics of strain OS-77 are much closer to those of the E. acetylicum DSM 20416 and E. antarcticum DSM 14480.
molecular mass of the EgMnSOD is consistent with those of other Fe- and MnSODs (Bafana et al. 2011). The purified SOD is a purple-colored protein having a broad absorption band with a maximum at 478 nm (Fig. 4), which is known as a typical characteristic of MnSOD (Whittaker and Whittaker 1991). The molar absorption coefficient value at 478 nm was 1,104 M-1 cm-1. ICP-MS analysis of the SOD showed that the enzyme contains Mn (0.57 atom/chain), but not Fe (0.01 atom/chain), indicating that the purified SOD from Exiguobacterium sp. OS-77 belongs to the group of MnSODs.
Purification of the EgMnSOD The EgMnSOD was successfully purified from the strain OS-77 through sequential purification steps using columns of phenyl sepharose high performance, Q-sepharose high performance, and Superdex 200 (Table 2). The EgMnSOD was purified to 50-fold with a yield of 41 % and a specific activity of 10,500 U/mg. The molecular mass of the native enzyme was estimated to be approximately 50 kDa by Superdex 200 gel filtration column. The EgMnSOD is a homodimer of polypeptides determined as a molecular mass of approximately 23.5 ± 1.0 kDa in SDS-PAGE (Fig. 3), corresponding to the molecular mass of 22,532 Da calculated from the deduced amino acid sequence. The
Activity and thermostability of the EgMnSOD The effect of pH on the activity of the EgMnSOD was measured in 50 mM BR buffers with pH ranging from 6.0 to 11.0. The EgMnSOD remained at least 70 % of activity under all the measured pH conditions compared with the activity at optimum pH of 9.0 (Fig. 5a). Outside this range, the measurement could not be performed because of rapid denaturation of xanthine oxidase and cytochrome c. The effect of temperature on the activity of the EgMnSOD was also measured in 50 mM KP buffer at pH 7.8. The optimum temperature of the EgMnSOD was around 5 °C (Fig. 5b), indicating that the EgMnSOD has a psychrophilic property. The activity at 25 °C achieved approximately 80 % of the optimum activity. These results showing a slight loss of the activity under various pH and temperature conditions are compatible to the PhFeSOD (Castellano et al. 2006). The catalytic activity of the EgMnSOD (10,500 U/mg at 25 °C) is higher than those of the mesophilic MnSOD from Escherichia coli (EcMnSOD, 7,300 U/mg at 25 °C) (Whittaker and Whittaker 1991) and the thermophilic MnSOD from Thermus thermophilus (TtMnSOD, 1,400 U/mg at 25 °C) (Liu et al. 2011). The
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368 Table 1 Differential phenotypic characteristics of the strain OS-77 and its related strains of the genus Exiguobacterium
Extremophiles (2014) 18:363–373
Characteristics
1
2
3
4
5
Habitat
Spring water
Glacial water
Creamery waste
Permafrost
Lake water
Size (mm)
1–2
2–4
2–5
3.5–4
2–3
Shape
Round
Round
Irregular
Round
Round
Color
Yellowish orange
Yellowish orange
Yellowish orange
Bright orange
Orange
? ?
? –
– w
? ?
– ?
40
30
37
40
41
–
–
?
?
?
Colony morphology
Growth temperature (°C) 5 37 Maximum temperature of growth (°C) Hydrolysis of Gelatin Aesculin
?
–
?
–
?
Starch
?
–
?
ND
?
Casein
?
–
?
ND
?
Nitrate reduction
–
?
–
–
–
Acid production from glucose, ribose, maltose, sucrose
?
–
?
ND
?
Sucrose, glycerol Fructose, mannose, trehalose
– –
? –
? ?
? ?
? ?
Utilization of
Ribose
?
–
?
?
?
Raffinose
–
?
–
?
?
Galactose
–
?
–
?
–
L-Xylose
–
?
–
ND
–
–
?
–
±
– –
D-Xylose,
Strains: 1, strain OS-77; 2, E. indicum HHS 31; 3, E. acetylicum DSM 20416; 4, E. sibiricum 255-15; 5, E. antarcticum DSM 14480. Data for all strains except OS-77 are from the report by Chaturvedi and Shivaji (2006) ? positive, - negative, ND no data available, ± variable reaction, w weakly positive a
Estimated from the genome sequence
rhamnose, melibiose
Melezitose
–
–
?
±
L-Fucose, xylitol
–
–
?
–
–
Mannitol
?
–
?
?
–
Erythritol
–
?
–
ND
–
Sorbitol, dulcitol, inositol, inulin
–
?
–
–
–
Arbutin
–
?
–
?
?
Salicin
–
–
?
?
–
Amygdalin
?
–
–
?
?
a-Methyl-D-mannoside
–
–
?
–
?
a-Methyl-D-glucoside N-Acetylglucosamine
– –
– –
? –
– ?
– –
Starch
–
–
?
?
?
Glycogen
?
?
–
ND
?
Gluconate
?
?
–
–
–
5-Ketogluconate
–
?
–
–
–
DNA G?C content (mol%)
47a
48
47
47.7
47
16S rRNA gene similarity (%)
100
99.8
99.6
98.3
98.2
result is compatible to the general feature of psychrophilic enzymes that is the improved catalytic efficiency at low temperature when compared to those of mesophilic and thermophilic enzymes (Georlette et al. 2004).
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The thermostability of the EgMnSOD was examined by pre-incubating the enzyme in the 50 mM KP buffer at pH 7.8 under various temperatures (25–75 °C) for 10 min. The EgMnSOD was stable up to 45 °C and was gradually
Extremophiles (2014) 18:363–373
369
Table 2 Purification of the EgMnSOD from Exiguobacterium sp. OS-77 Step
Activity (U)
Protein (mg)
Specific activity (U/mg)
Yield (%)
Purification (fold) 1.0
Soluble cell extract
61,100
294
208
100
Ammonium sulfate precipitation
60,100
190
316
98
1.5
Phenyl sepharose HP
48,100
7.00
6,870
79
33
Q-sepharose HP
37,400
4.01
9,330
61
45
Superdex 200
25,300
2.42
10,500
41
50
(kDa)
M
1
97 66 45
30 23.5 kDa 20.1
14.4 Fig. 3 SDS-PAGE of the purified MnSOD. Lane M molecular marker, lane 1 the EgMnSOD
of the PhFeSOD at 50 °C was estimated to be 14.2 min. The EgMnSOD shows high thermal stability compared to the maximal temperature (40 °C) for growth of the strain OS-77. Such properties are also found in other psychrophilic enzymes from various microorganisms (Castellano et al. 2006; Pedersen et al. 2009). The thermal stability of the EgMnSOD was supported by the results of DSC measurement showing two transition points with midpoint transition temperature (Tm) at 65 and 84 °C (Fig. 6). The first transition point was almost same as the half-inactivation temperature of the EgMnSOD. The similar result has been reported in the DSC measurement of mesophilic EcMnSOD, displaying two transition curves detected at 71 and 89 °C (Mizuno et al. 2004; Whittaker 2010). In the papers, the two transitions at low and high Tm are discussed as being due to the unfolding of the reduced Mn(II)-state and the oxidized Mn(III)-state of the enzyme, respectively. Conformational structure of the oxidized Mn(III)-state enzyme seems to be more stable than that of the reduced Mn(II)-state enzyme, correlating with higher Tm value. The results suggest that the EgMnSOD also has a similar structural organization of unfolding process to those of other mesophilic MnSODs purified from different organisms. Genome analysis of the strain OS-77 and amino acid sequence of the EgMnSOD
Fig. 4 UV-Vis absorption spectrum of the purified EgMnSOD. The enzyme solution (16.2 mg/mL) in 20 mM Tris–HCl buffer (pH 7.8) was used for the measurement
inactivated above the temperature (Fig. 5c). The halfinactivation temperature of the EgMnSOD was calculated as 63.2 °C. The half-life of activity at 60 °C was estimated to be 21.2 min (Fig. 5d). As shown in Table 3, the EgMnSOD showed the enhanced thermostability compared to the PhFeSOD. The half-inactivation temperature of the PhFeSOD was 54.2 °C under the thermal incubation for 10 min (Castellano et al. 2006). Also, the half-life activity
The amino acid sequence of the EgMnSOD was revealed by the combination of whole genome shotgun sequencing and N-terminal amino acid analysis. The draft genome sequence of the strain OS-77 was obtained using de novo assembly method. A gene coding an MnSOD (sodA) has been found using the BLAST program with reference to the sodA gene (Exig_0858) of E. sibiricum 255-15 (Rodrigues et al. 2008). The N-terminal amino acid sequence analysis of the purified EgMnSOD provided the sequence of SKFELPE, which is identical to the amino acid sequence translated from the nucleic acid sequence of the sodA gene of the strain OS-77. The complete amino acid sequence of the EgMnSOD consists of 202 amino acid residues (Fig. 7) with a calculated molecular mass of 22,532 Da. The amino acid sequence alignment of the EgMnSOD with the
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370
Extremophiles (2014) 18:363–373
A
B
C
D
Fig. 5 Activity and thermostability of the EgMnSOD. a The effect of pH on the activity measured in 50 mM BR buffer at 25 °C. b The effect of temperature on the activity. c Thermostability under various temperature conditions. d Heat-inactivation profile during incubation. For the heat-inactivation profile, a solution of the EgMnSOD (20 lg/
mL) in 50 mM KP buffer (pH 7.8) was incubated at 60 °C for 5–40 min. An aliquot of the solution was then subjected to SOD activity assay at 25 °C to determine the residual activity. The activity value of the non-incubated enzyme was calculated as 100 %. The data are from three independent experiments per condition
Table 3 Effects of temperature on the activity of the SODs Protein
Half-inactivation temperature (°C)a
Half-life of activity (min)b
References
PhFeSOD
54.2 (10 min)
14.2 (50 °C)
Castellano et al. (2006)
EgMnSOD
63.2 (10 min)
21.2 (60 °C)
This work
EcMnSOD
ND
TtMnSOD
105 (60 min)
9.0 (50 °C) 12.0 (110 °C)
Hunter et al. (2002) Liu et al. (2011)
ND no data available a
The values in the parentheses are the incubation time
b
The values in the parentheses are the incubation temperature
mesophilic EcMnSOD and the thermophilic TtMnSOD showed the amino acid identity of 60 and 62 %, respectively. Typical residues that are involved in the active site (H28, Y36, H82, Q149, D164, and H168) are completely conserved in the EgMnSOD. Recently, Vendittis et al. (2008) proposed that average parameters related to amino
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Fig. 6 Thermal unfolding profiles of the EgMnSOD measured by DSC. The molecular weight of 48,000 was used for calculation of molar heat capacity (Cp)
acid composition of the SODs such as mass, volume, accessible surface area, and hydrophobicity are linearly correlated with the optimum growth temperature of the
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371
Fig. 7 Multiple alignment of the amino acid sequences of EgMnSOD, EcMnSOD, and TtMnSOD. The sequence of the EgMnSOD (GenBank ID: GAD04318) was translated from the nucleic acid sequence of sodA gene. The accession numbers of the amino acid sequences of the EcMnSOD and TtMnSOD are NP_418344 and YP_004164, respectively. The multiple alignment was conducted
using constraint-based multiple alignment tool (COBALT, http:// www.ncbi.nlm.nih.gov/tools/cobalt/) in the National Center for Biotechnology Information (NCBI). The residues conserved in all sequences are represented in yellow background, and the residues typically involved in the active site are represented in pale rose background
Table 4 Parameters related to the amino acid compositions of PhFeSOD, EgMnSOD, EcMnSOD, and TtMnSOD Protein
Mass (Da)
˚ 3) Volume (A
˚ 2) Accessible surface area (A
Hydrophobicity (kJ/mol)
Optimum growth temperature (°C)
PhFeSOD EgMnSOD
110.7 (111.0) 111.6 (111.3)
133.5 (133.6) 134.3 (134.1)
159.8 (160.5) 161.7 (161.0)
4.83 (4.69) 4.59 (4.72)
15 20
EcMnSOD
112.1 (112.2)
135.7 (135.6)
162.9 (162.6)
4.71 (4.83)
37
TtMnSOD
113.9 (114.0)
139.0 (138.3)
165.7 (165.5)
5.14 (5.01)
68
The parameters related to the amino acid compositions of EgMnSOD, EcMnSOD, and TtMnSOD were determined according to the method described by Vendittis et al. (2008). The values in parentheses were derived from the linear fitting equations for SODs in the report: for mass, y = 110.15 ? 0.05608x; for volume, y = 132.3 ? 0.08818x; for accessible surface area, y = 159.1 ? 0.09383x; for hydrophobicity, y = 4.60 ? 0.006087x. (y each parameter, x optimum growth temperature). The parameter data for PhFeSOD were obtained from the report studied by Castellano et al. (2006)
host microorganisms. Among these parameters, the former three parameters are related to the ratio of bulky residues, and the average hydrophobicity reflects the ratio of hydrophobic residues in the enzyme. The low ratio of bulky and hydrophobic residues results in an increased number of cavities and less hydrophobic interactions in the protein core, respectively. These factors can cause high molecular flexibility that is hypothesized as the reason of psychrophilicity of the enzymes. As shown in Table 4, the values of all average parameters of the EgMnSOD were lower than those of the mesophilic EcMnSOD and thermophilic TtMnSOD. The differences of the parameters among EgMnSOD, EcMnSOD, and TtMnSOD are matched to the values calculated using linear fitting equation reported by Vendittis et al. (2008). This result indicates the structural adaptation of the EgMnSOD toward low-temperature conditions. Although the hydrophobicity value of the PhFeSOD appears to be slightly higher than that of the EgMnSOD, however, all of the other parameters related to
the ratio of bulky residues of the EgMnSOD were higher than those of the PhFeSOD, which may affect the high thermostability of the EgMnSOD. Further structure-based studies of the EgMnSOD will help to understand how the enzyme achieves its psychrophilicity along with thermostability.
Conclusions This report presents biochemical characterization of the psychrophilic MnSOD from a newly isolated bacterium Exiguobacterium sp. OS-77. We determined the draft genome sequence of Exiguobacterium sp. OS-77 and then assigned the gene encoding EgMnSOD. The calculated value of the parameters related to the amino acid composition suggested structural cold-adaptation of the EgMnSOD. The most significant finding of this study is that the EgMnSOD has psychrophilic character along with
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thermostability. The present results suggest that the EgMnSOD might be a promising material that may contribute to revealing the molecular mechanisms lying in psychrophilic enzymes. Acknowledgments This work was supported by the World Premier International Research Center Initiative (WPI), Grants-in-Aid: 23655053, 25620047, 25248017, and 24109016 (Scientific Research on Innovative Areas ‘‘Stimuli-responsive Chemical Species’’) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan and the Basic Research Programs CREST Type, ‘‘Development of the Foundation for Nano-Interface Technology’’ from JST, Japan.
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