catalase, bskat-1 an antioxidant enzyme as a model for...

Catalase, Bskat-1 an antioxidant enzyme as a model for astrobiology isolated from

Bacillus stratosphericus

29

5.1. Introduction

5.1.1. Radiation induced enzyme alternation in astrobiological important

microorganisms

Evidence for the possible interplanetary transfer of biological materials began

with experiments testing the resistance of microbes to the space environmental

conditions (Horneck, 1993; Wickramasinghe, 2004). In space, microbes would be

subjected to different stresses, including extreme vacuum, desiccation, solar and cosmic

radiation, microgravity, and both extreme hot and cold temperatures (Nicholson et al.,

2000). Of these factors, solar UV is the most harmful (Rampelotto et al., 2009; Horneck

et al., 1994; Nicholson et al., 2000; Roya et al., 2002). However, spaceflight

experiments demonstrate that with minimal UV shielding, several types of microbes can

survive for years at exposures to the harsh environment of space (Roya et al., 2002;

Rettberg et al., 2002). Furthermore, it was estimated that, if shielded by two meters of

meteorite, a substantial number of spores would survive after 25 million years in space

(Horneck et al., 2002). Because of their high resistance to different extreme conditions,

spores of Bacillus subtilis are the most widely used model microorganism for these

studies (Nicholson et al., 2000; Horneck, 1993; Horneck et al., 1994). However, various

other microbes have been used, including vegetative cells of the soil bacteria

Deinococcus spp. and Rhodococcus erythropolis, some halophilic Archaea Halorubrum

spp, Halobacterium spp, Chroococcidiopsis and others (Pabulo, 2010).

It was argued that, these upper atmospheric microorganisms were of cometary

origin and thereby cometary panspermia stood vindicated. Although, it was claimed that

bacteria and fungi can be found over the altitude range 18-39 km, such results were



Page 130

generally dismissed on the basis of contamination and were particularly interest in

astrobiology (Shivaji et al., 2006, Shivaji et al., 2009). In atmosphere, evidence for the

occurrence of microorganisms at altitudes of 17-85 samples have been collected in the

higher atmosphere. These samples were obtained using a meteorological rockets, a

specially designed direct-flow sampler and cryosamplers sent up on a balloon

(Wainwright et al., 2003; Shivaji et al., 2006, Shivaji et al., 2009). The dynamic

chemical and biological interactions were very complex, and these organisms that

survive in this environment must tolerate higher level of UV and ionizing irradiation. At

higher altitude, the ionizing radiation (X-ray, γ-ray), UV light and redox cycling causes

the generation of reactive oxygen species during aerobic respiration of microorganism.

These include, superoxide (O2-1

), peroxide (O2-2

), hydrogen peroxide (H2O2), hydroxyl

(OH)- and hydroperoxyl (HOO)

- radicals were generated (Halliwell and Gutteridge,

1999, Hassan and Fridovich, 1979; Vile and Tyrrell, 1993; Jurkiewicz and Buettner,

1994). These oxidants cause chemical modification of the cellular components, nucleic

acids, proteins, and lipids, resulting in a number of metabolic malfunctioning, aging

process, mutagenesis, and cell death (Halliwell and Gutteridge, 1999). Consequently, it

has been proposed that, these microorganisms operate several defense mechanisms

against reactive oxygen intermediates by the action of scavenging enzymes such as

catalase, peroxidase, superoxide dismutase (SOD) and by a number of antioxidants

occurring in cells (Cha and Kim, 1996 & 1999; Halliwell and Gutteridge, 1999).

5.1.2. Catalase

Bacteria have specific enzymes to detoxify potentially lethal reactive oxygen

species (ROS), including superoxide anion radical (O2), hydrogen peroxide (H2O2) and



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hydroxyl radical (HO-). The effects of ROS result in the oxidation of various cellular

components such as DNA, RNA, proteins and lipids. Thus, bacterial defense

mechanisms against oxidative stress play a very important role in their survival.

Catalase [hydrogen peroxide: hydrogen-peroxide oxidoreductase (EC1.11.1.6)] was one

of the central defense enzymes against oxidative stress, which catalyzes the conversion

of H2O2 to water and molecular oxygen (Reaction 1).

2H2O2 2H2O + O2 (Reaction 1)

Many bacteria possess two types of catalases, typical catalase which catalyzes

H2O2 only and catalase-peroxidase which also has an associated peroxidase activity.

They are distinct from each other in many properties. Typical catalase generally

contains heamin prosthetic groups and shows a broad optimum pH in the range of 5-10.

It was resistant to treatment with ethanol/chloroform (Margoliash et al., 1960).

Catalase-peroxidase, which has an optimum pH at 6-6.5, was relatively more sensitive

to temperature and ethanol/ chloroform than the typical catalases (Goldberg and

Hochman, 1989; Hochman et al., 1992).

5.1.3. Classification and catalytic properties of catalase

Three classes of proteins unrelated based on sequence and structure exhibit

significant catalase activity. The classes that were classified as monofunctional, bi

functional and non-heme or Mn-containing catalases. A typical monofunctional catalase

was subdivided based on large or small subunits. Phylogenetic analyses have

demonstrated the existence of two distinct clades or sub groupings of small subunit

enzymes and one clade of large subunit enzymes among the monofunctional catalases

(Klotz et al., 1997). The second, less widespread class was composed of bifunctional,



Page 132

heme containing catalase-peroxidases that were closely related by sequence and

structure to plant peroxidases. The third class of catalase includes the nonheme or Mn-

containing catalases.

5.1.4. Radiation induced catalase regulation in bacteria

The ROS hypothesis for UV damage has been studied using catalases, which

destroy peroxide and other ROS (Eisenstark, 1989). Because wild type E. coli strains

were able to resist complete inactivation by two known catalases in E. coli are

suspected to provide protection (Eisenstark, 1989). The 84 kDa catalase,

hydroperoxidase-I (HPI), was membrane bound, encoded by katG and has catalase and

peroxidase activity (Triggs-Raine et al., 1990). HPI was expressed only during

anaerobic growth was predicted to repair after UVA damage by increasing cell

permeability to antioxidants (Leven et al., 1990). The 93 kDa catalase,

hydroperoxidase-II (HPII), was cytoplasmic, encoded by katE, and has only catalase

activity (Van Ossowski et al., 1991). HPI was expressed during aerobic exponential

growth (Mulvey et al., 1990) but its role in UVA damage remains unclear due to

contradicting evidence. The katG, katE double knockout E. coli strain, UM2, has

previously shown increased sensitivity to UVA consistent with a role in UVA resistance

(Amiri et al., 2007). However, the katG knockout strain, UM197, did not suffer after

UVA exposure compared to its isogenic parent, suggesting that only katE was required

for protection (Amiri et al., 2007). However, the UM2 strain complemented with katE

also failed to protect against UVA (Cheng et al., 2009) speculatively because of over-

expression of HPII by the pBAD24 construct (Banerjee et al., 2009). Lack of protection

was thought to be because of the photosensitizing properties of HPII at high



Page 133

concentrations (Cheng et al., 2009). Another study successfully created an alternative

catalase double knockout of the UM197 strain, CKLW09W2, using antisense katE

DNA (akatE) in the pBAD24 vector (Chan et al., 2010); however, the authors did not

test the strain’s ability to resist UVA damage. To test for HPII-mediated UVA

protection at different HPII concentrations, this study controlled expression of the akatE

message in CKLW09W2 using the inducible araC promoter on the pBAD24 construct.

The aim of this study was to control expression levels of HPII in HPI knockout cells to

elucidate whether HPII alone has protective properties against UVA damage at lower

concentrations and determine the expression level above which photosensitizing

properties of HPII take over.

5.1.5. Stability of catalase

The unusual resistance to denaturation and proteolysis supports the catalases

with large subunits. Resistance to pH and thermal denaturation was noted early in the

study of catalases from E. coli and other enteric bacteria used as a diagnostic test to

differentiate among catalases in crude extracts (Meir and Yagil, 1984; Goldberg and

Hochman, 1989). Recent research on thermal stability of catalase revealed that, there

was actually a small increase in activity above 60°C and that activity began to drop only

above 80°C, with a Tm of 83°C (Switala et al., 1989). Boiling in buffer, or alternatively,

heating to 65°C in 5.6 M urea was required to dissociate the dimer. While not as stable

as HPII, small subunit catalases such as bovine liver catalase (BLC) still exhibited

enhanced stability for an enzyme from an organism normally growing at 37°C, with a

Tm for loss of activity of 56°C. The thermal stability of HPII has also been utilized as a

purification step (Switala and Loewen, 2002). Catalases, generally, were also resistant



Page 134

to treatment with an ethanol: chloroform mixture, another property that could be

exploited as a tool for catalase purification (Switala and Loewen, 2002). Both thermal

stability and resistance of the majority of the protein to proteolysis can be explained in

terms of a very rigid, stable structure that resists unfolding, thereby preventing access to

the protease active site. Resistance to proteolysis was an advantage. Because, it was

expressed in stationary phase, a period of rapid protein turnover and elevated protease

levels. There would have been a strong selective pressure to retain this property, but it

was not clear whether resistance to proteolysis evolved in E. coli and thermal stability

was an inadvertent outcome. Alternatively, the catalase could have originated in a

thermophilic organism and been horizontally transferred to E. coli, where protease

resistance has been the selective pressure for retention of thermal resistance.

5.2. Materials and Methods

5.2.1. Chemicals

All the chemicals used for the experiments were of analytical grade and

chemicals were of the highest possible purity. For the preparation of culture media, the

chemicals used such as, Bacto peptone, Beef extract, Tryptone, Starch, yeast extract and

agar were purchased from Himedia laboratory (Mumbai, India). Other Inorganic salts

such as, NaCl, MgSO4, ferrous sulphate, magnesium chlorides, KH2PO4, NaHCO3,

Na2CO3 were purchased from Merck chemicals (Mumbai, India). Certain trace element

such as, Na2EDTA; ZnSO4 MnCl2, H3BO3, CaCl2, CuCl2, NiCl2, Na2MoO4, Na2SO3, and

FeSO4 purchased from Sigma-Aldrich (Mumbai, India) and Himedia laboratory

(Mumbai, India). (NH4)2SO4 was purchased from Himedia laboratory (Mumbai, India).

Phenyl Sepharose High Performance column was purchased from Pharmacia (Uppsala,



Page 135

Sweden) Gel filtration chromatographic resin such as Superdex 200 HR was purchased

from Pharmacia (Uppsala, Sweden)

5.2.2. Bacterial strains

Bacillus stratosphericus was adopted or purchased from Microbial Type Culture

Collection (MTCC) Chandigarh, India and Center for Cellular and Molecular Biology

(CCMB) Hyderabad, India. This strain was deposited previously in MTCC under

accession number 7305 and is publically available. The strain was previously isolated

from the stratosphere (approximately 41 km high altitude) using cryogenic tubes for air

sampling experiments from high altitudes. The physicochemical properties of the strain

are found to be grow in between 8 and 37ºC, but not at 40ºC. Growth occurs between

pH 6-10, but not at pH 5 or pH 11. The strain has extreme resistant to UV radiation and

other ionizing radiations (Shivaji et al., 2006).

5.2.3. Reference strains

Dienococcus radiodurans R1 and E.Coli cells were used as reference strains.

Because Dienococcus radiodurans R1 is currently known as extreme radiation resistant

microorganism on earth

5.3.3. Culture conditions for Bacillus stratosphericus

The lyophilized Bacillus stratosphericus cells were initially cultivated

aerobically on nutrient agar medium of pH 7.0 at 40ºC for two days. Further,

B.stratosphericus was sub cultured in a medium containing 8 g/l–1

yeast extract, 8 gl-

1extract from meat and 1 g/l

–1 KH2PO4 at 40ºC and pH 7.0, buffered with 50 mM

NaHCO3/Na2CO3. Then, 1% (w/w) of a trace element solution (2.5 gl–1

Na2EDTA; 0.1

g/l–1

ZnSO4·7H2O; 0.030 g/l–1

MnCl2·4H2O; 0.3g/l–1

H3BO3; 0.2 g/l–1

CaCl2·6H2O;



Page 136

0.01g/l–1

CuCl2·2H2O; 0.02g/l–1

NiCl2·6H2O; 0.9g/l–1

Na2MoO4·2H2O; 0.03 g/l–

1Na2SO3·5H2O, and 1g/l

–1FeSO4·7H2O) was added to the incubation mixture. For

reference strains Dienococcus radiodurans R1 was cultured in a TYGM medium (1%

tryptone 0.5% yeast extract, 0.2% glucose, and 0.2% L-methionine) at 30ºC with

continuous aeration at 150 rpm (Soung and Lee, 2000; Yun and Lee, 2001). E.Coli cells

were grown in nutrient broth medium of pH 7.0 at 37ºC

5.2.4. Radiation experiments

B. stratosphericus cells were cultured in the media as described earlier and

treated with different doses of UVA and γ- radiations (1 kGy to 10 kGy).

Ultraviolet-A exposure

Late log phase cells of B. stratosphericus were serially diluted with normal

saline and plated. Cells were exposed to different doses of UVA radiation using Xenon

lamp (15 W; Philips UV lamp, India) and 300 nm cut off band pass filter. The plates

were wrapped with aluminum foil and incubated at 32ºC for 48h and the number of

colonies appeared on plates were recorded. For control, D. radiodurans and E.coli cells

were cultured and exposed different dose of UVA light and the percent survivility was

calculated.

γ-radiation exposure

The cells of B. stratosphericus and reference strains were suspended in sterile

phosphate buffered saline (PBS) and exposed to different doses (1 kGy to 10 kGy) of γ-

radiation (radiation dose 4.87 kGy/h) using Cobalt 60

irradiator. Appropriate dilution of

treated cells was placed on media and number of colony forming units was counted

after 48h of incubation at 37ºC and the percent survivility was calculated.



Page 137

5.2.5. Catalase induction by UV and γ -irradiation on bacterial cells

For induction experiment, the bacterial cells was cultured in TYGM (1%

tryptone, 0.5% yeast extract, 0.2% glucose, and 0.2% methionine) with continuous

shaking condition (150 rpm) at 40ºC of pH 7.0 for 48 hours. Catalase was induced by

pre-incubating the bacterial suspension with continuous UV (UVA) irradiation (200-

400 nm) (15 W; Philips UV lamp, India) for an hour. The UV dose was measured using

VLX-3W radiometer (Radiometer, France). For γ-irradiation, cell suspensions were

exposed to different doses (1 kGy to 10 kGy) of γ-radiation (radiation dose 4.87 kGy/h)

using Co60

irradiator of strain were irradiated at room temperature for 1 h. Then, the cell

suspension was cultured on TYGM liquid media for 48 hours. The growth of bacterial

strain in both cases was monitored at 600 nm (Schmazdu UV-Visible

spectrophotometer, Japan). The cells were harvested and suspended in 50 mM

potassium phosphate buffer, pH 7.0 were used for preparation of cell free extracts.

5.2.6. Preparation of cell extraction

UV and γ-irradiated culture broth of B stratosphericus was centrifuged at 10,000

X g for 20 min at 4ºC to separate the cells from the broth. The pellet containing cells

were suspended in minimum amount of 50 mM potassium phosphate buffer (pH 7.0)

and kept at 4ºC for an hour. After incubation with appropriate buffer, the protein was

extracted by sonication for 10 min in ice-bath and can be used as a crude extract. The

protein was concentrated to several folds by Ultrafiltration (PM-10 membrane, Amicon,

Beverly, MA, USA).



Page 138

5.2.7. Activity profile of catalase in crude extract

The ultra filtration extract were resolved on 8.5% gel by PAGE in Tris-glycine

buffer (Hedrick and Smith, 1968; Wayne and Diaz, 1986). Electrophoresis was carried

out at 75 volts through the stacking gel, and at 100-150 volts through the resolving gel,

using vertical mini slab gel unit (BIORAD, USA) filled with running buffer.

Visualization of catalase bands resolved by the treatment of gels with horseradish

peroxidase prior to incubating in 5 mM H2O2 solution followed by diaminobenzidine. In

the latter staining method, gels were incubated initially with 5 mM hydrogen peroxide

and followed by a freshly prepared mixture of 2% ferric chloride and 2% potassium

ferric cyanide.

5.2.8. Catalase activity assay

Catalase activity in sonic extract of cells was measured by

spectrophotometrically by monitoring the decrease in absorbance at 240 nm due to

decomposition of hydrogen peroxide in 50 mM potassium phosphate buffer, pH 7.0 at

25ºC. One unit of Catalase was defined as the disappearance of one µmol of hydrogen

peroxide per minute (ε = 43.6 M-1

cm-1

) (Nak and Young, 2000).

5.2.9. Estimation of protein concentration

Protein concentration was measured by the standard procedure of Lowry et al.,

(1953). A standard plot was developed using Bovine Serum Albumin (BSA) of different

concentration and the protein concentration of test samples was determined for crude

preparation and each steps of the purification.



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5.2.10. Purification of Catalase-BsKat-1

The cell lysate was subjected to fractionation by ammonium sulfate precipitation

at 40% saturation to remove impurities, followed by 70% saturation in a second step to

precipitate the Bskat. The precipitated proteins were collected by centrifugation, and the

pellet was dissolved in phosphate buffer (50 mM, pH 7.0) with 1.0 M (NH4)2SO4. The

solution was applied to a Phenyl Sepharose High Performance column (Pharmacia,

Uppsala, Sweden) equilibrated with 1.0 M (NH4)2SO4 in phosphate buffer. Fractions

containing protein were eluted with a linear decreasing gradient of 1.0-0 M (NH4)2SO4.

Catalase active fractions were pooled and concentrated in an ultrafiltration cell

(Sartorius, Epsom, UK) using a membrane with a 100 kDa cutoff. The concentrated

enzyme solution was subjected to a Superdex 200 HR 10/30 column (Pharmacia,

Uppsala, Sweden) previously equilibrated with phosphate buffer (50 mM, pH 7.0)

containing 0.15 M NaCl. The column fractions were eluted with the same buffer. The

active fractions were collected and stored -20ºC until further use.

5.2.11. Molecular mass determination

The molecular weight of the enzyme analyzed using 12.5% non-denaturating

polyacrylamide gels (BIORAD, USA) essentially as described by Laemmli, (1970).

After electrophoresis, gels were stained for 1 h or until clear visualization of protein

bands with the Colloidal protein strainer (Aristogene Biosciences, Bangalore, India).

The apparent molecular weight of the purified enzyme was determined by comparing

with Rf values of standard proteins (Bio-Rad Laboratories, Hercules, CA). The native

molecular mass of catalase was estimated by gel filtration using Sephacryl 200 HR

using Myosin (205 kDa), Phosphorylase B (97 kDa), Ovalbumin (45 kDa), Trypsinogen



Page 140

(24 kDa) and Aprotinin (6.5 kDa) as a molecular weight standards. pI of the native

enzyme was determined by the method described by Thompson et al., (2003), except

for perioxidase staining. The native pI was determined from the standards range from

4.45 to 9.6 (Biorad, Hercules, CA).

5.2.12. Effect of temperature, pH and stability

To determine the influence of temperature on enzymatic activity, enzyme

samples were incubated at temperatures range from 15 to 60⁰C. Thermostability was

investigated by pre incubating the samples at temperatures range of 45-75⁰C. After

various time intervals (0.5 to 3 hours), samples were withdrawn and residual activity

was determined at the standard assay conditions. The pH optimum of catalase activity

was determined in various buffers from pH 4.0 to pH 12.0. The buffers used were

Formic acid buffer (pH 3.3 to 4.3); Trimethylamine/acetic acid buffer (pH 4.3 to 5.3);

Trimethylamine/carbonate buffer (pH 5.9 to 6.9) and Phosphate buffer (pH 7 to 12) at

optimum temperature.

5.2.13. Effects of inhibitors, metal ions and spectral studies

Effects of inhibitors on catalase activity were tested using 3-amino-1, 2, 4-

triazol, sodium azide, hydroxylamine hydrochloride and sodium cyanide at a

concentration of 0.1 to 1.0 mM. The enzyme (1 mg/ml) was assayed by pre incubated

with the inhibitor for five min prior to assay at room temperature. The absorption

spectra of native and reduced enzyme were measured on Schmidzu UV-Visible

Spectrophotometer (Schmidzu, Japan). For this, the native enzyme was reduced with 1

mM sodium dithionite, and enzyme treated with 10 mM KCN were pre incubated for 5

min at 45⁰C in phosphate buffer (50 mM, pH 10.0). The reaction was monitored



Page 141

spectrophotometrically as described previously (Hochman and Goldberg, 1991). The

protoheme type and content of the enzyme was determined through the formation of a

pyridine hemochrome in absorption spectra. The molar absorption coefficient for the

pyridine hemochrome was assumed as 191.5 mM-1

cm-1

(Falk, 1964). Effect of some

metal ions on enzyme activity was determined by pre incubating at a final concentration

of 1 mM Zn2+

,Fe2+

,Ca2+

,Mg2+

,Cu2+

(Chloride form) 10 mM EDTA and histidine at 45

°C at pH 10. The enzyme activity was determined after incubation with the substrate for

30 min.

5.3. Results

5.3.1. B. stratosphericus showed differential response to UV-A and γ radiation

compared to D radiodurans and E.coli

The late log phase cells of B. stratosphericus were exposed to different doses of

γ and UVA radiation and cell survival was monitored on TYGM agar (described

earlier). Survival of all three strains (including reference strains) were determined

following γ-irradiation at doses up to 12 kGy. The results of γ-radiation exposure

showed the differential surveillance of B. stratosphericus cells/spores. When compared

to the reference strains, B. stratosphericus had shown nearly two log cycles loss in γ-

radiation beyond 4 kGy (34%). In contrast to D.radiodurans, which is currently known

as highest radiation resistance organism on earth, shown resistant beyond 8 kGy

exposure of γ-radiation (68%). However, the other mesophilic control strain E.coli cells

did not appeared much resistant for γ-radiation above 2 kGy (6%) (Fig.5.1)



2

Figure 5.1: Survival curves for B. stratosphericus (■), Deinococcus radiodurans R1 (●)

and Escherichia coli (▲) following exposure to 1 to 12 kGy (radiation dose 4.87

kGy/h) using Co60

irradiator. Values are the mean ± standard deviation of triplicate

experiments.



Page 143

Figure 5.2: Survival curves for B. stratosphericus (●), Deinococcus radiodurans R1 (■)

and Escherichia coli (▲) following exposure to UVA (A) (1.5 kJ.m2). Values are the

mean ± standard deviation of triplicate experiments.

In addition, cells of B.stratosphericus and D.radiodurans cells were showed less

sensitive to high doses (>1.0 kJm−2

) of UVA (84 and 56 % respectively). However,

E.Coli cells was appeared to be very sensitivity to high dose of UVA radiation (>1.0



Page 144

kJm−2

) (less than 10%). The percent surveillance of all three strains under radiation

exposure was determined by mean SD values of colony forming units (Fig. 5.2).

5.3.2. Enhanced production of catalase (Bskat-1) activity of Bacillus stratosphericus

after exposure to UV and γ- radiation

The exposure of UV (Fig.5.3) and γ-radiation (Fig 5.4) on cells yielded

significant changes in activities of the antioxidant enzyme (catalase). γ-radiation

induces gradual increase in catalase activity in B. stratosphericus and D. radiodurans at

an exposure of 2 to 6 kGy respectively. The maximum induction of catalase was

determined at an exposure of 6 kGy (about 98%), however its activity was lowered to

80% beyond 6 kGy in control strain D radiodurans. In B. stratosphericus, while

absorbed 96% increase in activity at 4 kGy. However, dose beyond 4 kGy resulted in

drastic fall in activity (10%) from initial. On the contrary, pattern of change in catalase

activity was different in case of gamma irradiated E.coli. This strain, when irradiated at

2 kGy experienced less than one fold decrease in catalase.



Page 145

Figure 5.3: Catalase activity of B. stratosphericus, D. radiodurans R1 and E. coli at 1

to 12 kGy (radiation dose 4.87 kGy/h). The highest residual activity of B.

stratosphericus was observed at 4 kGy. Values were the mean ± standard deviation of

triplicate experiments.



Page 146

Figure 5.4: Catalase activity curves for B. stratosphericus (circle), Deinococcus

radiodurans R1 (box) and Escherichia coli (triangle) after following exposure to UVA

for one hour. Values were the mean ± standard deviation of triplicate experiments.

At an exposure of UV (>1.0 kJm−2) was also experienced the drastic changes in

enzyme activity in both B. stratosphericus and control group. It was noted that,

irradiation at >1.0 kJm−2 significant increased in the activity was observed (63.1% fold)

in B. stratosphericus and 60.23% in D radiodurans. Whereas, in E.coli, the effect of

UV does not affect much on the enzyme activity (8.23%). These results attributed that,

UV and γ-radiation induce catalase activity in B. stratosphericus and this may be

involve in protection from the oxidative stress generated from radiations.



Page 147

5.3.3. Purification and PAGE pattern of radiation induced catalase Bskat-1

Two chromatographic purification steps were employed for catalase Bskat-1.

The purification by initial HIC column, using decreasing gradient of 1.0-0 M

(NH4)2SO4, resulted in two peaks eluted at 800 and 420-440 mM (NH4)2SO4

respectively (Fig 5.5). At this stage, the fractions were pooled and enzyme activity was

determined to be 20,900 U/mg with a 6.76 fold, which was higher than the crude

extract. Further, the purification fold was enhanced to 9.77 fold with specific activity of

30,200 U/mg by concentrated on ultra filtration. The purification strategy for Bskat-1

was determined to highest in superdex 200 HR gel filtration chromatographic column.

This step was resulted in single enzyme active peak (Fig 5.6). At this final purification

steps, the activity of the purified enzyme was determined to be 45,250 U/mg and 14.67

fold. The detailed series of purification steps and corresponding enzyme activity were

summarized in Table 5.1. Upon chromatographic elution from gel filtration, the

molecular mass of the purified enzyme was determined by 12.5% denaturating PAGE.

The electrophoresis pattern of the purified enzyme showed a single dark massive band

corresponding to 28 kDa (Fig 5.7). However, exact molecular weight of the enzyme was

determined by Sephacryl 200 HR gel filtration chromatography using standard markers

(described in material and methods). The exact molecular weight of the enzyme was

corresponds to 120 kDa (Fig. 5.8), which was indication of an enzyme with four

identical subunits. Since, catalase was a homo tetramer, containing four identical

subunits, which corresponds to the molecular weight of 120 kDa (characteristic of

catalase). The activity staining for catalase was determined by 8% native PAGE



Page 148

(materials and methods), resulted in appearance of single white fluorescent colored

band in dark background (Fig. 5.7).

.

Figure 5.5: Bskat-1 was purified from phenyl sepharose high performance HIC

chromatography. The column was previously equilibrated with 1.0M (NH4)2SO4 in

phosphate buffer and fractions were eluted with a linear decreasing gradient of 1.0–0 M

(NH4)2SO4 at a flow rate of 30 ml/hour. The enzyme active fraction was eluted at 420

mM concentration (NH4)2SO4 (dotted lines).



Page 149

Figure 5.6: Chromatogram of Superdex 200 HR 10/30 gel filtration chromatography.

The column was equilibrated and enzyme active fractions was eluted using phosphate

buffer (50 mM, pH 7.0) containing 0.15 M NaCl at a flow rate of 30 ml/hour.

Cata

lase

, B

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llu

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P

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e 1

50

T

ota

l

act

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y

(U)

Tota

l

pro

tein

(mg)

Sp

ecif

ic

act

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y

(U/m

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Yie

ld

(%)

Pu

rifi

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(fold

)

Cel

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Ph

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Sep

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(HIC

)

Ult

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Su

per

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220

X10

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234

X10

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160

X10

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54.3

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712

112

53

10

3090

20,9

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30,2

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45,2

50

100

106

72.7

24.6

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14.6

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On

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s d

efin

ed, as

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equ

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des

troyin

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µm

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per

min

Tab

le 5

.1:

Puri

fica

tion s

tage

pat

tern

of

cata

lase

(BsK

at-1)

from

the B. Stratosp

hericus



Page 151

Figure 5.7: Non-reducing 12.5% PAGE pattern of purified Bskat-1 from B.

stratosphericus. Lane A, standard protein molecular weight markers. Lane B, untreated

native Bskat showing single band corresponding to 28 kDa. Lane C, activity profile of

catalase band was resolved on gel by incubating the gels in 5 mM H2O2, 2% ferric

chloride and 2% potassium ferric cyanide. The catlase band showing colored band in

dark background.



Page 152

Figure 5.8: Native molecular weight of Bskat-1 was determined using Sephacryl 200

HR gel filtration column chromatography. The column was previously equilibrated with

50 mM phosphate buffer, pH 7.0. The standards used were Myosin (205 kDa),

Phosphorylase B (97 kDa), Ovalbumin (45 kDa), Trypsinogen (24 kDa) and Aprotinin

(6.5 kDa) as a molecular weight standards. The molecular weight of Bskat-1 was 120

kDa (indicated on marked lines)

5.3.4. Effect of temperature and pH of Bskat-1

The activity of Bskat-1 as a function of temperature and pH was shown in

Figures 5.8 and 5.9. The enzyme had virtually no activity at below 15°C, and activity

increased with increasing temperature up to the maximum activity at 55°C. In contrast,

it had activity over the range of 20-55°C with an optimum temperature for activity at

45°C (Fig. 5.8a). This was slightly thermostable when compared to other mesophilic

catalase.



Page 153

(a)

(b)

Figure 5.8: (a) & (b) Effect of temperature and thermostability on Bskat-1 from B.

stratosphericus. The optimum temperature of enzymes activity was determined by

incubated for 30 min at 15 to 60⁰C prior to initiation of the reaction. The thermostability

was assayed at 45 to 75⁰C at different interval of time. Data represents the mean of at

least three independent experiments.



Page 154

The stability of the Bskat-1 was observed approximately 13% increase in

activity observed at 45°C for 1 h of incubation. At 55°C, the residual activity was 80%,

which was higher than that of initial 30 min of incubation time (60%). Post hours of

incubation, the residual activity was drastically lowered. When the enzyme was

incubated at more elevated temperatures (65-75°C), the enzyme activity appears to

cease more rapidly (Fig. 5.8b). It was determined that, Bskat-1 had activity over a broad

pH range of 4-12, with the optimal activity at pH 10. This pH range of activity was

comparable to typical monofunctional catalases (Fig. 5.9). The typical pI of the native

enzyme was determined to be 7.8.

Figure 5.9: Bskat-1 activity as a function of pH. Various buffers from pH 4.0 to pH

12.0 [Formic acid buffer (pH 3.3 to 4.3); Trimethylamine / acetic acid buffer (pH 4.3 to

5.3); Trimethylamine / carbonate buffer (pH 5.9 to 6.9) and Phosphate buffer (pH 7 to

12)] were used at optimum temperature. Data represents the mean of three independent

experiments



Page 155

5.3.5. Effect of metal ions, inhibitors and spectral studies

Metal ions (1 mM) such as Na+, Mg

2+, K

+, and Mn

2+ did not shown effect on

catalase activity. However, Co2+

and Ba2+

showed a partial inhibition on the enzyme

(Table 5.2). Histidine and EDTA at a concentration of 10 mM reduced activity of 19%

and 27%, respectively. Catalase activity was strongly inhibited by hydroxylamine

hydrochloride, sodium cyanide and sodium azide at a concentration of (0.1 to 1 mM).

Catalase specific inhibitor like, 3-Amino-1, 2, 4-triazole had minimum inhibitory action

on Bskat-1 (Table 5.3). Inhibitory values of these chemical were corresponds to the

residual activity of the enzyme.

Table 5.2: Effect of inhibitors on BsKat-1 activity

Inhibitor Final concentration(mM) % inhibition

Sodium azide 0.1 89

1.0 97

Sodium cyanide 0.1 75

1.0 99

3-Amino-1,2,4-triazole 0.1 23

1.0 29

Hydroxylamine hydrochloride 0.1 82

1.0 99



Page 156

Table 5.3: Effect of metal ions on BsKat-1 activity

Like a typical heme containing catalase, Bskat-1 showed a strong Soret peaks at

410 nm (Fig. 5.10a). This provides evidence that the Bskat-1 was a heme catalase rather

than a Mn-catalase. The peak was shifted from 410 to 422 nm on following the addition

of 10 mM sodium cyanide (Fig. 5.10a). This indicates that, binding of cyanide to the

heme moiety of the Bskat-1. However, spectra remained unchanged after the treatment

of the enzyme with 1 mM sodium dithionite. This characteristic was consistent with the

above observation that Bskat-1 lacks peroxidase activity. Treatment of the enzyme with

pyridine/NaOH and sodium dithionite produced pyridine hemochrome whose major

absorption peaks shifted from 410 to 420, 522 and 560 nm (Fig. 5.10b). This indicates

that Bskat-1 contains protoheme IX as its heme group. The protoheme content of this

enzyme was determined from the absorption of its pyridine hemochromogen at 420 nm.

There are 3.1 molecules of protoheme IX per tetrameric molecule of Bskat-1. The heme

content as well as the RZ (Reinheitszahl) A410/A280 ratio of 0.54 again indicates that

Bskat-1 shares properties of typical monofunctional catalases.

Control None 100

NaCl 1mM 89

KCl - 86

MnCl2 - 99

CoCl2 - 54

MgCl2 - 91

BaCl2 - 51

Histidine 10 mM 19

EDTA - 27

Effectors Concentration * Residual Activity (%)



Page 157

(a)

(b)

Figure 5.10: (a) Absorption spectrum of native Bskat-1 and changes in spectra after the

treatment of 10 mM cyanide and 1 mM dithionite.

(b) Absorption spectrum of pyridine hemochrome of Bskat-1



Page 158

5.4. Discussion

It was generally accepted that, the bacteria and fungi are present in the

stratosphere unlikely to survive the high exposure to ultraviolet and other types of

ionizing radiation (Yinjie et al., 2008). It is not surprising that, the stratospheric

microbes exhibit potentially UV and ionizing resistant physiology. However, study on

differential mechanism of radio-protective nature of microorganism was explained. B.

simplex cells, which were relatively resistant to such radiation, whose vegetative cells

tend to clump together; essentially because the outer cells provide a UV barrier

(Wainwright et al., 2003). Another studies on Bacillus spores revealed that, the survival

of nearly 6 years in space when shielded from UV radiation. Whereas, when exposed to

the full range of space conditions, only a small proportion of the spores from the

innermost part of the samples were able to survive (Roya et al., 2002). Another

mechanism of Sarcina lutea, which forms UV protective packets of cells, is 100 times

more resistant to UV than E coli, while B. subtilis and Staphylococcus aureus were

respectively three and eight times more resistant (Whisler, 1940). It was known that,

genus Deinococcus were extraordinarily resistant to UV and ionizing radiation and the

mechanism has been studied on repairing the damaged DNA. There have been,

however, very few published studies on radiation surveillance in terrestrial bacterial

species Deinococcus spp. and Bacillus spores have been used as the model for space

radiation experiment (Wayne et al., 2000). Some evidence suggests that, radiation

resistance in D. radiodurans involves the use of RecBC and scavenging enzyme

systems against oxidative DNA damage (Nivedita et al., 2008). Basal levels of catalase

and SOD activities in cell extracts of D. radiodurans cultures pretreated with 10 mM



Page 159

H2O2 were more resistant to the lethal effect of γ-irradiation. Changes in the

concentration and activities of scavenging enzymes, such as catalase (kat) and

superoxide dismutase (SOD) were valid as biomarkers in stratospheric bacterial species.

The DNA damage was due to radiation induced reactive oxygen intermediates, which

leads to the impairment of SOD and catalase, the two important components of the

antioxidants defense system (Imam et al., 2010).

In the present research work, the cells of B. stratosphericus showed the

differential surveillance in both UVA and γ-radiations. It had shown nearly two log

cycles loss in γ-radiation beyond 4 kGy. However, the data from the present and

previous studies, the resistance of D. radiodurans and E. coli was 8 kGy and 2 kGy of

γ-radiation respectively (Issei et al., 2006). Moreover, Roya et al., (2002) exposed

thirteen isolates to 5.5 kGy of γ-radiation with D37 (dose for 37% survival) and all

isolates were shown resistant up to 3.5 kGy. It was also determined that, only 7%

survival at the very high dose of 5.5 kGy. In comparison, the D37 for E. coli was lower

than 100 Gy (Roya et al., 2002). Although, B. stratosphericus and D.radiodurans cells

were showed less sensitive to high doses (>1.0 kJm−2

) of UVA radiation. Whereas,

E.coli cells was appeared to be very sensitivity to high dose (Issei et al., 2006).

Catalase Bskat-1 from B. stratosphericus was purified using two

chromatographic steps. However, it was reported that, the requirement of various

chromatographic steps were varied in different bacterial catalases. After, the complete

purification by Superdex 200 HR gel filtration chromatography, the specific activity of

purified Bskat-1 was determined to be 48,000 U/mg (Table 5.1). However, this was not

particularly interesting when compared with the catalase activity of other radioresistant



Page 160

and other bacterial species. It was reported that, after four chromatographic purification

steps, the specific activity of catalase (KatA) from D. radiodurans was determined to be

68,800 U/mg (Issei et al., 2006). The highest catalase activity was observed in D.

radiophilus, which is 3-fold higher than that of D. radiopugnans, which showed the

lowest activity. Although, Thompson et al., (2003) purified catalase from thermophilic

Thermus brockianus using the three chromatographic steps, that resulted in 65-fold

purification to a specific activity of 5300 U/mg. However, Xunlong et al., (1998) over

expressed and purified the recombinant secretary catalase from B. subtils requires only

two chromatographic steps. In his study, the simple purification steps yielded nearly

homogeneous catalase, with ~70% recovery. The purified recombinant catalase has a

specific activity of 34,600 U/mg under optimal conditions. The purified Bskat-1 was

shown at single band at 28 kDa in SDS–PAGE analysis and the gel filtration results

showed an approximate native protein molecular mass of 120 kDa, indicative of an

enzyme with four identical subunits. This was slightly lower than those reported for

other tetrameric catalase enzymes. i.e., Bacillus sp. with 70.5 and 282 kDa, E. coli with

84.3 and 337 kDa, Rhodobacter capsulatus with 59 and 236 kDa, Neurospora crassa

with 80 and 320 kDa and T. brockianus with 42.5 and 178 kDa subunit and native

molecular mass, respectively (Yumoto et al., 1990; Thompson et al., 2003). With

respect to Bskat-1, comparable size of the catalases has been observed in Streptomyces

venezuelae (Knoch et al., 1989), Mycobacterium tuberculosis (Wayne and Diaz, 1986),

and K. pneumoniae (Goldberg and Hochman, 1989). However, all of these catalases

were homodimers. A manganese-containing catalase from the obligate thermophile

Thermoleophilum album (Allgood and Perry, 1986) consists of four identical subunits



Page 161

of 34 kDa. The presence of two different subunits was known only in the case of the

catalase-peroxidase of Vitreoscilla (Abrams and Webster, 1990). Loewen and Switala,

(1986) have explained the appearance of the E. coli HPII catalase in the form of two

subunits of 90 and 92 kDa with proteolytic degradation. In comparision, the molecular

weight of the catalases from radiation resistant Deinococcus spp. was varied from 650

kDa in D. proteolyticus to 155 kDa in D.radiophilus. In comparision, catalase KatA

from D. radiodurans 65 kDa by SDS-PAGE analysis and 240 kDa by gel filtration,

suggesting that KatA forms a same homotetramer in solution. The isoelectric point of

Bskat-1 was 7.8. However, based on the available literature, pI of Bskat-1 was slightly

above when compared to those reported for catalases and catalase-peroxides from

Halobacterium halobium (4.0), Thermoascus aurantiacus (4.5), Vitreoscilla sp. of (5.0

and 5.2), and Anacystis nidulans of (4.7). (Obinger et al., 1997; Brown et al., 1995;

Abrams and Webster, 1990; Wang et al., 1998).

The temperature optimum of the Bskat-1 was slightly moderate thermostable. It

was shown activity between 25-55ºC, with temperature optimum of 45ºC. It was slight

high when compared to the radiation resistant Deinococcus spp. and other mesophilic

bacterial catalases. At 55-60ºC, the residual activity of Bskat-1 was reduced to 70%.

However, catalase (KatA) from D. radiodurans was active over a temperature range

from 20°C to 70°C, with optimum activity occurring at 30°C. Also, other commercially

available catalase enzyme from bovine liver and A. niger, the optimum assay

temperature demonstrating the enzymatic activity of catalases was 40°C and 50°C to

60°C, respectively. At 70°C, the activity of A. niger, bovine liver and D. radiodurans

catalases was reduced to 82%, 45% and 20%, respectively, of each activity at the



Page 162

optimum assay temperature. It was reported that, psycrophilic bacteria V. rumoiensis S-

1T secrete catalase, which was shown optimum at 40°C and completely suppressed by

incubation at 60°C. While, the M. luteus catalase activity was suppressed at 70°C (Isao

et al., 2000). Like strain S-1T

, at specific time of incubation, Bskat-1 loses its activity

above 60°C by 70% residual activity. At 90°C, the Bskat-1 residual activity was

completely suppressed to 10% for 15 min. These results suggested that the

thermostability of Bskat-1 is similar to the strain S-1T

catalase and lower than those of

the A. niger, M. luteus and bovine liver catalases (Isao et al., 2000). The pH stability of

the Bskat-1 was active over broad pH range of 4-12 with the optimal activity at pH 10,

which was closely related catalase-peroxidase from thermophilic B. stearothermophilus.

These results indicate the activity of Bskat-1 was more likely to alkaline environment.

However, catalases from B. bacillus and KN25 was shown lower pH stability than

Bskat-1 (Bol and Yasbin, 1991). In comparison, KN25 and bovine liver catalases was

shown highest activity between pH 5.0 and 8.0, with an optimal pH was determined at

6.0 (Xunlong et al., 1998). Similar to Bskat-1, strain S-1T

catalase exhibit same pH range

for the activity (pH 6-10) (Isao et al., 2000). Indicative of results from thermal stability

and pH, it can confine that the Bskat-1 was similar to the strain S-1T

catalase.

Like other catalase and catalase-perioxidase from Bacillus SF (Marinka et al.,

2001); D. radiodurans (Issei et al., 2006); V. rumoiensis (Isao et al., 2000);

hydroxylamine, azide, cyanide and 3-amino-1, 2, 4-triazole (Marcinkeviciene et al.,,

1995) had inhibitory effects on Bskat-1. It has been reported that, inhibitory action of

hydroxylamine on nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH

2.4.1 catalase. It had cyanide, azide, and hydroxylamine decreased its activity by 72.5,



Page 163

76.4, and 85.1%, respectively (Detlef and Michael, 1998). It was also confirmed that, 2-

Mercaptoethanol and sodium dithionite at a concentration of was a less-potent inhibitor.

Other various metal ions like Na+, Mg

2+, K

+, and Mn

2+ had a stimulatory effect on

catalase activity. However, in case of Bskat-1, there was a partial inhibition was

observed by Co2+

and Ba2+

metal ions. Nevertheless, its inhibitory effect could not be

quantitated by the catalase assay because of interference with the oxygen electrode

(Detlef and Michael, 1998). In relation to catalase from Thermus brockianu, Bskat-1 is a

typical heme containing catalase (Thompson et al., 2003). Bskat-1 had shown a strong

Soret peaks at 410 nm. This provides evidence that the Bskat-1 was a heme catalase

rather than a Mn-catalase as have been described in Thermus species (Kagawa et al.,

1999). It was described that, typical Mn-catalases lacks the Soret peak (Kagawa et al.,

1999; Yumoto et al., 1990; Brown et al., 1995; Abrams and Webster, 1990). Although,

KatG from Mycobacterium tuberculosis had a Soret peak at 408 nm (Johnsson et al.,

1997). The Soret band was shifted to 422 nm upon the addition of CN- . Similar shifts

have also been described for other catalases but varying degree (Hochman and

Shemesh, 1987; Goldberg and Hochman, 1989; Hyoung-pyo et al., 1994; Isao et al.,

2000). However, sodium dithionite did not alter any spectrum of the native enzyme

indicating the negative effect on Bskat-1. Major absorption at 522 and 560 nm for

Bskat-1(with respect to range), which was typical for protoheme IX for other catalases

(Detlef and Michael, 1998; Hyoung-pyo et al., 1994; Isao et al., 2000). The Bskat-1 has

similar absorption for typical heme containing catlase and share common spectral

attribute among currently known catalases.



Page 164

Results from this investigation revealed that, a typical mono functional, moderate

temperature and alkali stable catalase (Bskat-1) was a potent antioxidant enzyme system

for radiation induced oxidative stress in B. stratosphericus. These extreme properties of

the novel enzyme could be a potential candidate for studying the mechanism of

protecting radiation-induced stress in space radiation experiments.



Page 165

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