JOURNAL OF CULTURE COLLECTIONS

JOU= RNAL OF CULTURE COLLECTIONS

Volume 4, 2004= -2005, pp. 61-67

Characteristics of bacterial strain,

superoxidE dismutase producer, isolated from Bulga= rian thermal spring

Ivanka Bojadzhieva and Elka Emanuilova*

Institute of Microbiology, Bulgarian Acad= emy of Sciences

Acad. G. Bonchev str., bl. 26, 1113 Sofia, Bulgaria

Summary

Twenty one bacterial strains with superoxide dismutase activity were isolated from samples (flowing spring water, water with soil particles, alga bacterial mad and mud) collected near different thermal Bulgarian springs. = The strains were Gram positive, rod shaped, spore forming and moderate thermophiles. Isolate M 20 showed the highest specific total superoxide dismutase activity of 530 U/mg in shaken flasks cultivation at 55 = ;^oC for 4 hours. An activity of 1300 U/mgwas achieved after selection and optimization of cultivation procedures in fermentor. The opti= mal enzyme activity of cell free extract was at 55 ^oC and pH 8.= 0, and the half-life was 30 min at 90 ^oC. The strain was catal= ase positive, it produced peroxidase and proteinase. As a result of morpho-logi= cal, cultural, physiological and biochemical characteristics the isolate M 20 was determined as a moderate thermophile belonging to the genus Bacillus.<= /o:p>

<= o:p>

Introduction

Superoxide dismutases (SODs;= EC 1.15.1.1.) are a class of metal proteins, catalyzing the dismutation of the superoxide radicals (О₂^-) to oxygen and hydrogen peroxide.

In the recent years, due to = the big practical interest to the microbial SODs the attention was focused to the thermostable ones produced by thermophilic microorganisms. The SOD of the anaerobe bacterial hyperthermophil = Аquifex pyrophilus was with half-life 175 min at 95° C [7]. The most thermostable SOD, described recently in the literature was of archaebacteri= al origin. A strain of Sulfolobus sulfataricus produced Fe-Mn SOD with half-life 2 hours at 100° = ;C [9].

The present work= was undertaken with a view to isolate aerobe bacterial thermophiles producers of SOD from Bulgarian thermal springs.

Materials and Methods

Isolation of strains. 0.5 ml from the samples (flowing spring water, water with soil particles, alga bacterial mad and mud) collected near 16 thermal springs in Bulgaria, were added in 1= 00 ml Erlenmeyer flasks with 25 ml of a medium consisting: Bacto peptone - 5.0 g; yeast extract - 5.0 g; К₂HPO₄- 5.0 g; NaCl - 1.0 g; Mg <= /i>SO₄-10 mg; FeSO₄x 7 H₂O, distilled water = 211; 1000 ml, рН – 7.5. After 24 hours cultivation at 50, 55 or 60 ^oС suitable diluted cultu= res were spread on solid medium to obtain single colonies.

The isolates were cultivated in 500 ml Erlenmeyer flasks with 75 ml nutrient medium. 1 m= l of 18 hours old culture was used for inoculation. Samples for determinati= on of strains growth and SOD activity were taken on the 2^d and 4th hour of cultivation.

Cultivation in fermentor. It was carried out in fermentor Bioflo (New Brunsweek Sci.) with working volume of 1000 ml, magnetic stir with 500 rpm and constant air flow= of 1.0 v/v/m (volume air per volume medium per min). 1 ml of 18 hours old culture was used for inoculation. The percentage of the dissolved oxygen (DO) was registered by sterilized oxygen electrode and DO analyzer (New Brunsweek Sci. Model DO – 50). Samples for determination of= the bacterial growth and enzymes activities were taken every 30 min.=

Bacterial growth was determined by accounting the optical density at 660 nm.

Preparation of cell free extract. Samples of cultures were centrifuged at <= st1:metricconverter ProductID=3D"8000 g" w:st=3D"on">8000 g for 15 min at 5° C. The cells pellet was suspended in 3 ml of 0.05 M potassium – phosphate buffer with pH 7.8. 2.0 g sand were added= and mechanical isintegration for 15 min was applied. After centrifugation = the obtained supernatant was analyzed for enzymes activities.=

SOD assay. The method of Beauchamp и Fridovich was used [1]. Definition of one enzyme unit was the amount of cell free extract which inhibited 50 % the reduction of nitroblue tetrazolium to blue formazan at 55 ^oC and pH 7.8. The total specific enzyme activity was expressed as U/mg protei= n. The enzyme termostability at 90° C was recorded.=

Catalase activity assay. The catalase production was tested with 3 % H₂O_=
2. After 5 min appearance of bubbles was accounted as a positive reaction= .

Peroxidase activity assay. The production of purpurogalin after reaction with 33 % solutio= n of pyrogalol and 3 % Н₂О₂ was accounted= as a positive peroxidase reaction.

Proteolytic activity assay. Agar diffusion method (3 % fresh milk agar) was applied, as the fresh milk was centrifuged at 6000 rpm for 10 min to remove lipids and used for the test.

Protein concentration was assayed by the method of Bradford [3].

Gram staining and electron microscopy of thin sections were performed according to standard procedures.=

<= o:p>

Results and Di= scussion

<= o:p>

Twenty one isola= tes from 16 thermal Bulgarian springs were tested (Table 1). All were Gram positive aerobes, producers of intracellular SOD. Six of them: isolates M [3, 6_=
1, 10, 19, 20, 21] expressed relatively highest total specific enzyme activity between 210 and= 530 U/mg.

The cell free ex= tracts of these six isolates were analyzed for SOD thermostability and presence of catalase, peroxidase and proteolytic activity (Table 2). Isolate M 20 showed the highest thermostability – the half-life was 30 min at 90&nbs= p;^oC. The isolate was catalase, peroxidase and proteinase positive. Further studi= es were carried out with isolate M20 because of its relatively high enzyme activity and thermostability.

T= able.1. SOD activity of newly isolated thermophilic strains.

Thermal springs

Isolate

Gram staining

Temperature of the sample (^oC)

pH of the sample

Temperature of cultivation= (^oC) *

Hours of cultivation<= /o:p>

2
4

OD_660nm

SOD U/mg=

OD_660nm

SOD U/mg=

Hisar Spring 1<= /span>

M1

+

46

7.98

50

0.6

134

1.36

163

Hisar Spring 2<= /span>

M2

+

42

7.55

50

0.28

140

1.00

130

Hisar bath Bistrica

M3

+

52

7.82

50

0.50

250

1.10

247

Village Banja (Karlovo)

M6₁<= /span>

+

49

7.45

50

0.57

175

0.96

210

Village Banja (Karlovo)

M6₂<= /span>

+

49

7.45

50

0.60

158

1.00

140

Village Ovoshnic

M8

+

45

7.99

50

0.64

193

1.20

155

Village Jagoda<= /span>

M9

+

42

10.14

50

0.65

219

1.40

136

Village Banja (Sliven)

M10

+

55

8.27

50

0.55

169

1.00

308

Pancharevo

M12

+

49

7.96

50

0.68

109

1.34

94

Separeva banja

C1

+

47

7.5

60

0.40

27

1.14

18

C2

+

33

7.0

60

0.26

34

1.15

14

Haskovski mineral bath

M13

+

57

7.0

55

-

-

0.53

110

River Erma

M14

+

37

7.0

55

-

-

0.55

100

River Erma

M15

+

37

7.5

55

0.70

231

1.13

100

Bedenska banja<= /span>

M16

+

52

7.85

55

0.65

35

1.15

67

Bedenska banja<= /span>

M17

+

71

8.0

55

0.73

200

1.12

92

Bedenska banja<= /span>

M18

+

50

8.0

55

0.60

91

1.20

155

Momin prohod

M19

+

65

7.8

55

0.74

29

1.10

283

Dolna banja

M20

+

62

8.85

55

0.50

79

1.05

533

Pchelin<= /p>

M21

+

72

8.0

55

0.80

82

1.22

350

Varvara<= /p>

M22

+

80

8.0

55

0.90

244

0.45

104

* pH of the medi= um for cultivation of all the isolates was 7.5.

T= able 2. Enzyme characteristics* of new isolated thermophilic producers of SOD.=

Isolate<= /p>
Half-life (t _½=)
at 90 ^{oC
(min)}

Proteolytic activity<= /p>

Catalase activity

Peroxydase activity

C*

CFE

C

CFE

C

CFE**

М3

15

-

+

+

+

-

-

М6

<5

+

+

+

+

-

-

М10

5

+

+

-

-

+

+

M19

5

-

+

-

-

+

+

М20

30

+

+

+

+

+

+

М21

<5

+

+

+

+

+

+

* Cultural super= natant (C); cell free extract (CFE).

Isolate M 20 was= Gram positive aerobe, rod shaped, sporulated bacterium (Fig. 1). Electron microscopic photographs of thin sections showed typical morphology and ultra structure for the Gram positive, rod shape bacterial representatives. Some = of the M20 properties are presented in Table 3. It could be described as a moderate thermophile from the genus Bacillus. The strain Bacillus sp. M20 was deposited in the National Bank for Industrial Microorganisms and Ce= ll Cultures, Sofia, Bulgaria (No. 8389).

Fig. 1. Ultra thin sections= of 3 hours old vegetative cells of Bacil= lus sp. isolate M 20, cultivated in shake flasks: A. 6 000 x; B. 3 250 x (CM - cell membrane, CW - cell wall).=

Table 3. Morphological, physiological and cultural characteristics of isolate M20.

Characteristics

Isolate M20

Colony morphology on nutrient agar on t= he 18^th hour at 55 ^oC

size

1.0-1.5 mm

form

circular

color

beige

Morphology of cells on nutrient agar on= the 18^th hour at 55 ^oC

form

rods

size

3.0 – 7.0 x 0.8 – 1.3 &micr= o;m

motility

+

spores

ellipsoidal, terminal=

Temperatures for growth (^oC)=

minimal

36

optimal

35

maximal

65

pH ranges for growth<= /p>

5.5 – 8.5

Growth in nutrient broth
turbid without sediment and pellicle

Anaerobic growth

-

SOD from thermop= hilic strains of genus Bacillus was s= tudied at the species B. stearothermophilu= s. The investigations were focused mainly on clarifying the crystal enzyme structure [8], the enzyme amino acids composition [5] and the gene cloning in Saccharomyces cerevisiae [2] and Escherichia = coli [4].

There are scarce data in the literature about the optimal conditions for the cultivat= ion of B. stearothermоphilus = strains for maximal SOD production as well as about the enzyme thermo stability in = cell free extract.

Gligic et al. isolated from mineral waters a strain of B. stearothermоphilus that produced up to 500 U/mg<= sup> SOD for 8 hours cultivation on a shaker at 60° C [6]. F= rom hot spring waters in Thailand, a strain B. stearothermоphilus TL S33, also producing SOD, was isolated = with maximal activity of 130 U/mg at 48 hours and 65° C cultivation temperature [10].

Isolate M 20 cul= tivated in fermentor had a maximal SOD activity of 1300 U/mg for a relatively short period (half an hour) in the middle of the exponential phase of growth when the residual oxygen was 70 %. When the residual oxygen percentage decreased at the end of the exponential growth the SOD activity also decrea= sed (Fig. 2).

As it can be see= n from the results in Fig. 3 the optimal SOD activity of the isolate cell free ext= ract was at temperature 55 ^oC and pH 8.0.

Fig. 2. Specif= ic SOD activity (А), dissol= ve oxygen concentration (В)= and bacterial growth (С),dur= ing cultivation of Bacillus sp. iso= late М 20 in fermentor.<= /span>

Fig. 3. Effect of the temperature (A) = and pH (B) on the SOD activity of Ba= cillus sp. isolate M.

The relatively h= igh SOD activity and thermostability of Bac= illus sp. isolate M 20 give a reason for next investigations, related to the optimization of culture conditions for SOD production as well as to the characterization of the purified enzyme properties.

References

1.     Beauchamp, C. O., I. Fridovi= ch, 1971. Anal. Biochem., 44, 276-287.

2.     Bowler, C., L. V. Kaer, W. M= . Casmp, M. V. Montague, D. Inze, P. Dhaese, 1990. J. Bacteriol., 127, 1539-1546.

3.     Bradford, M., 1976. J. Biochem., 72, 248-254.

4.     Breham, J. K., S. P. Chember= s, K. J. Brow, T. Atkinson, N. P. Minton, 1991. Appl. Microbiol. Technol., 36, 3= 58- 363.

5.     Brock, C. J., J. E. Walker, = 1980. Biochemistry, 24, 2873-2882

6.     Gligic, L., Z. Radovic, G. Z= avisic, 2000. Enzyme Microb. Technol., 27, 798-792

7.     Lim, J.-H., K. Y. Hwang, J. = Choi, D. Y. Lee, B. Y. Ahn, Y. Cho, K. S. Kim, Y. S. Han, 2001. Biochem. Biophys. Res. Commun., 288, 263-268

8.     Parker, M. W., C. C. C. F. B= lake, 1988. J. Mol. Biol., 199, 649-661.
9.     Russo, A. D., R. Ruilo, G. N= itti, M. Masuilo, V. Bochini, 1997. Biochim. Biophys. Acta ,1343, 23-30=

10.     Sookkheo, B., S. Sinchaikul,= H. Thanan, O. Thonngprason, S. Phutrakul, S.-T. Chen, 2002. Proteomics, 2, 1311-1315.

Thermal springs	Isolate	Gram staining	Temperature of the sample (^oC)	pH of the sample	Temperature of cultivation= (^oC) *	Hours of cultivation<= /o:p>
2	4
OD_660nm	SOD U/mg=	OD_660nm	SOD U/mg=
Hisar Spring 1<= /span>	M1	+	46	7.98	50	0.6	134	1.36	163
Hisar Spring 2<= /span>	M2	+	42	7.55	50	0.28	140	1.00	130
Hisar bath Bistrica	M3	+	52	7.82	50	0.50	250	1.10	247
Village Banja (Karlovo)	M6₁<= /span>	+	49	7.45	50	0.57	175	0.96	210
Village Banja (Karlovo)	M6₂<= /span>	+	49	7.45	50	0.60	158	1.00	140
Village Ovoshnic	M8	+	45	7.99	50	0.64	193	1.20	155
Village Jagoda<= /span>	M9	+	42	10.14	50	0.65	219	1.40	136
Village Banja (Sliven)	M10	+	55	8.27	50	0.55	169	1.00	308
Pancharevo	M12	+	49	7.96	50	0.68	109	1.34	94
Separeva banja	C1	+	47	7.5	60	0.40	27	1.14	18
C2	+	33	7.0	60	0.26	34	1.15	14
Haskovski mineral bath	M13	+	57	7.0	55	-	-	0.53	110
River Erma	M14	+	37	7.0	55	-	-	0.55	100
River Erma	M15	+	37	7.5	55	0.70	231	1.13	100
Bedenska banja<= /span>	M16	+	52	7.85	55	0.65	35	1.15	67
Bedenska banja<= /span>	M17	+	71	8.0	55	0.73	200	1.12	92
Bedenska banja<= /span>	M18	+	50	8.0	55	0.60	91	1.20	155
Momin prohod	M19	+	65	7.8	55	0.74	29	1.10	283
Dolna banja	M20	+	62	8.85	55	0.50	79	1.05	533
Pchelin<= /p>	M21	+	72	8.0	55	0.80	82	1.22	350
Varvara<= /p>	M22	+	80	8.0	55	0.90	244	0.45	104

Isolate<= /p>	Half-life (t _½=) at 90 ^{oC (min)}	Proteolytic activity<= /p>	Catalase activity	Peroxydase activity
C*	CFE	C	CFE	C	CFE**
М3	15	-	+	+	+	-	-
М6	<5	+	+	+	+	-	-
М10	5	+	+	-	-	+	+
M19	5	-	+	-	-	+	+
М20	30	+	+	+	+	+	+
М21	<5	+	+	+	+	+	+

Characteristics	Isolate M20
Colony morphology on nutrient agar on t= he 18^th hour at 55 ^oC	size	1.0-1.5 mm
form	circular
color	beige
Morphology of cells on nutrient agar on= the 18^th hour at 55 ^oC	form	rods
size	3.0 – 7.0 x 0.8 – 1.3 &micr= o;m
motility	+
spores	ellipsoidal, terminal=
Temperatures for growth (^oC)=	minimal	36
optimal	35
maximal	65
pH ranges for growth<= /p>	5.5 – 8.5
Growth in nutrient broth	turbid without sediment and pellicle
Anaerobic growth	-